MM-GBSA calculations were run leading to -116 also.8 kcal/mol for DB03208 and -97.8 kcal/mol for strictinin. to assess CK1 binding after strictinin treatment.(TIF) pone.0217789.s002.tif (439K) GUID:?2585BEC2-C3FC-415E-AE03-6ED5927B0965 S3 Fig: Strictinin will not affect nonmalignant MCF-10A cell motility. Wound curing assay looking into strictinin influence on MCF-10A migration (* = p.worth 0.05, n = 3).(TIF) pone.0217789.s003.tif (125K) GUID:?6745E744-B667-4141-9E7B-5D1201B2B120 Data Availability StatementAll relevant data are inside the manuscirpt and its own Helping informaiton files. Abstract Triple Adverse Breast Tumor (TNBC), probably the most intense subtype of breasts cancer, can be seen as a the lack of hormone receptors targeted by hormone therapies like Tamoxifen usually. Because therapy success and achievement prices for TNBC lag significantly behind additional breasts tumor subtypes, there is certainly significant fascination with developing novel anti-TNBC real estate agents that may target TNBC particularly, with minimal results on nonmalignant cells. To the aim, our research identifies the anti-TNBC aftereffect of strictinin, an ellagitanin previously isolated from docking evaluation proteins and Ligand framework 3D constructions from the ligands, DB03208 and strictinin, had been built-in Schrodingers Maestro. For every ligand, the tautomeric areas had been produced at pH = 7 using Maestros Epik [18,19]. The cheapest tautomeric condition was chosen and then minimized to the most energetically beneficial structure. Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1) has an extracellular website, a transmembrane website, and an intracellular website. We modeled a truncated version of ROR1 (tROR1) that is identical with the intracellular, C-terminal region of ROR1 but does not contain the transmembrane nor the extracellular domains. The sequence for tROR1 (“type”:”entrez-protein”,”attrs”:”text”:”AAC50714.1″,”term_id”:”1589740″,”term_text”:”AAC50714.1″AAC50714.1) was retrieved from your NCBI protein database. The 3D structure of tROR1 (Fig 1) was then expected using I-TASSER [20C22]. The model for tROR1 was then preprocessed, optimized, and minimized using Maestros Protein Preparation Wizard [23]. We then mapped for active sites using Maestros SiteMap [24,25]. Open in a separate windows Fig 1 Strictinin interacts with ROR1 in TNBC.a) Homology Model of tROR1. Whereas N-terminus is definitely indicated by a reddish ball, C-terminus is definitely indicated by a blue ball. b, c) Strictinin docked to tROR1 and ligand relationships diagram. Whereas N-terminus is definitely indicated by a reddish ball, C-terminus is definitely indicated Rabbit Polyclonal to GANP by a blue ball. d, e) Immunoblot and immunofluorescence investigating basal ROR1 manifestation in two TNBC lines and non-malignant breast epithelial collection, MCF-10A. f) MTT assessing effect of ROR1 siRNA knockdown on strictinin cytotoxicity in TNBC. g) Immunoblot showing effect of siROR1 knockdown and strictinin on ROR1 manifestation in MDA-MB-231. h) Co-immunoprecipitation of ROR1 receptor showing inhibition of Wnt-5a binding to ROR1 after strictinin treatment. (* = p.value 0.05, n = 3). Ligand docking The active site (S1A Fig) we selected was the one closest to the residues indicated inside a earlier docking of DB03208 to tROR1 by Nath et Al [7]. A receptor grid was generated around the active site of tROR1. DB03208 and Strictinin were separately docked to tROR1 using Glide docking followed by Induced Match Docking [26C31]. To check the validity of our docking, we compared our ligand connection diagram to that of Nath et Al [7]. Our DB03208 ligand relationships showed many related residue relationships to theirs (S1B and S1C Fig). The binding site of strictinin is very close to the binding site of DB03208 (S1D Fig), suggesting these two ligands might have related drug action against tROR1. Immunoblotting Following numerous treatments, the cells were washed once in 1X PBS and lysed with RIPA buffer (150 mM NaCl, 1.0% Triton-X-100, 0.5% Sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) with 1 mM PMSF. Protein concentrations were determined by Bradford Assay using Pierce 660 nm Protein Assay reagent (thermofisher, Waltham, MA, USA). Following SDS-PAGE, Proteins were transferred to nitrocellulose membranes, which were clogged for 1-hour in 5% milk with mild agitation. Membranes were incubated over night at 4C with numerous antibodies then washed in TBS-T (20mM Tris, 150mM NaCl, 0.1% Tween 20), and incubated for 1-hour at.The rationale for this was an abundance of previous work emphasizing GSK3?s key part in regulating cell spreading and motility [41,43C45]. analyzed and match (lower panel) using NanoAnalyze (TA Devices). Binding guidelines, included as an inset (top panel), show an enthalpically driven 1-to-1 binding connection.(TIFF) pone.0217789.s001.tiff (13M) GUID:?67FEC7A8-CA41-4BE8-A461-8CFB7C28BB22 S2 Fig: Strictinin inhibits AKT phosphorylation and downstream GSK3? phosphorylation self-employed of CK1. a). FOXO-luciferase assay assessing strictinin effect on P13K/AKT activity in BT-549 after 24h. (* = p.value 0.05, n = 3) b) Co-Immunoprecipitation of ROR1 to assess CK1 binding after strictinin treatment.(TIF) pone.0217789.s002.tif (439K) GUID:?2585BEC2-C3FC-415E-AE03-6ED5927B0965 S3 Fig: Strictinin does not affect non-malignant MCF-10A cell motility. Wound healing assay investigating strictinin effect on MCF-10A migration (* = p.value 0.05, n = 3).(TIF) pone.0217789.s003.tif (125K) GUID:?6745E744-B667-4141-9E7B-5D1201B2B120 Data Availability StatementAll relevant data are within the manuscirpt and its Supporting informaiton files. Abstract Triple Bad Breast Malignancy (TNBC), probably the most aggressive subtype of breast cancer, is definitely characterized by the absence of hormone receptors usually targeted by hormone therapies like Tamoxifen. Because therapy success and survival rates for TNBC lag much behind other breast cancer subtypes, there is significant desire for developing novel anti-TNBC providers that can target TNBC specifically, with minimal effects on nonmalignant cells. To this aim, our study explains the anti-TNBC effect of strictinin, an ellagitanin previously isolated from docking analysis Ligand and protein structure 3D constructions of the ligands, DB03208 and strictinin, were built in Schrodingers Maestro. For each ligand, the tautomeric claims were generated at pH = 7 using Maestros Epik [18,19]. The lowest tautomeric state was selected and then minimized to the most energetically beneficial structure. Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1) has an extracellular website, a transmembrane website, and an intracellular website. We modeled a truncated version of ROR1 (tROR1) that is identical with the intracellular, C-terminal region of ROR1 but does not contain the transmembrane nor the extracellular domains. The sequence for tROR1 (“type”:”entrez-protein”,”attrs”:”text”:”AAC50714.1″,”term_id”:”1589740″,”term_text”:”AAC50714.1″AAC50714.1) was retrieved from your NCBI protein database. The 3D structure of tROR1 (Fig 1) was then expected using I-TASSER [20C22]. The model for tROR1 was then preprocessed, optimized, and minimized using Maestros Protein Preparation Wizard [23]. We then mapped for active sites using Maestros SiteMap [24,25]. Open in a separate windows Fig 1 Strictinin interacts with ROR1 in TNBC.a) Homology Model of tROR1. Whereas N-terminus is definitely indicated with a reddish colored ball, C-terminus is certainly indicated with a blue ball. b, c) Strictinin docked to tROR1 and ligand connections diagram. Whereas N-terminus is certainly indicated with a reddish colored ball, C-terminus is certainly indicated with a blue ball. d, e) Immunoblot and immunofluorescence looking into basal ROR1 appearance in two TNBC lines and nonmalignant breast epithelial range, MCF-10A. f) MTT evaluating aftereffect of ROR1 siRNA knockdown on strictinin cytotoxicity in TNBC. g) Immunoblot displaying aftereffect of siROR1 knockdown and strictinin on ROR1 appearance in MDA-MB-231. h) Co-immunoprecipitation of ROR1 receptor displaying inhibition of Wnt-5a binding to ROR1 after strictinin treatment. (* = p.worth 0.05, n = 3). Ligand docking The energetic site (S1A Fig) we decided to go with was the main one closest towards the residues indicated within a prior docking of DB03208 to tROR1 by Nath et Al [7]. A receptor grid was produced around the energetic site of tROR1. DB03208 and Strictinin had been independently docked to tROR1 using Glide docking accompanied by Induced Suit Docking [26C31]. To check on the validity of our docking, we likened our ligand relationship diagram compared to that of Nath et Al [7]. Our DB03208 ligand connections showed many equivalent residue connections to theirs (S1B and S1C Fig). The binding site of strictinin is quite near to the binding site of DB03208 (S1D Fig), recommending both of these ligands may have equivalent drug actions against tROR1. Immunoblotting Pursuing various remedies, the cells had been cleaned once in 1X PBS and lysed with RIPA buffer (150 mM NaCl, 1.0% Triton-X-100, 0.5% Sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) with 1 mM PMSF. Proteins concentrations had been dependant on Bradford Assay using PhiKan 083 Pierce 660 nm Proteins Assay reagent (thermofisher, Waltham, MA, USA). Pursuing SDS-PAGE, Proteins had been used in nitrocellulose membranes, that have been obstructed for 1-hour in 5% dairy with soft agitation. Membranes had been incubated right away at 4C with different antibodies then cleaned in TBS-T (20mM Tris, 150mM NaCl, 0.1% Tween 20), and incubated for 1-hour at area temperatures with horseradish-peroxidase-linked.Using LiCl, a well-established GSK3? inhibitor, in conjunction with strictinin, we noticed phenotypic rescue in comparison with strictinin treatment by itself. of ROR1 to assess CK1 binding after strictinin treatment.(TIF) pone.0217789.s002.tif (439K) GUID:?2585BEC2-C3FC-415E-AE03-6ED5927B0965 S3 Fig: Strictinin will not affect nonmalignant MCF-10A cell motility. Wound curing assay looking into strictinin influence on MCF-10A migration (* = p.worth 0.05, n = 3).(TIF) pone.0217789.s003.tif (125K) GUID:?6745E744-B667-4141-9E7B-5D1201B2B120 Data Availability StatementAll relevant data are inside the manuscirpt and its own Helping informaiton files. Abstract Triple Harmful Breast Cancers (TNBC), one of the most intense subtype of breasts cancer, is certainly seen as a the lack of hormone receptors generally targeted by hormone therapies like Tamoxifen. Because therapy achievement and survival prices for TNBC lag significantly behind other breasts cancer subtypes, there is certainly significant fascination with developing novel anti-TNBC agencies that may target TNBC particularly, with minimal results on nonmalignant tissues. To the aim, our research details the anti-TNBC aftereffect of strictinin, an ellagitanin previously isolated from docking evaluation Ligand and proteins structure 3D buildings from the ligands, DB03208 and strictinin, had been built-in Schrodingers Maestro. For every ligand, the tautomeric expresses had been produced at pH = 7 using Maestros Epik [18,19]. The cheapest tautomeric condition was selected and minimized towards the most energetically advantageous framework. Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1) comes with an extracellular area, a transmembrane area, and an intracellular area. We modeled a truncated edition of ROR1 (tROR1) that’s identical using the intracellular, C-terminal area of ROR1 but will not support the transmembrane nor the extracellular domains. The series for tROR1 (“type”:”entrez-protein”,”attrs”:”text”:”AAC50714.1″,”term_id”:”1589740″,”term_text”:”AAC50714.1″AAC50714.1) was retrieved through the NCBI protein data source. The 3D framework of tROR1 (Fig 1) was after that forecasted using I-TASSER [20C22]. The model for tROR1 was after that preprocessed, optimized, and reduced using Maestros Proteins Planning Wizard [23]. We after that mapped for energetic sites using Maestros SiteMap [24,25]. Open up in another home window Fig 1 Strictinin interacts with ROR1 in TNBC.a) Homology Style of tROR1. Whereas N-terminus is certainly indicated with a reddish colored ball, C-terminus is certainly indicated with a blue ball. b, c) Strictinin PhiKan 083 docked to tROR1 and ligand connections diagram. Whereas N-terminus is certainly indicated with a reddish colored ball, C-terminus is certainly indicated with a blue ball. d, e) Immunoblot and immunofluorescence looking into basal ROR1 appearance in two TNBC lines and nonmalignant breast epithelial range, MCF-10A. f) MTT evaluating aftereffect of ROR1 siRNA knockdown on strictinin cytotoxicity in TNBC. g) Immunoblot displaying aftereffect of siROR1 knockdown and strictinin on ROR1 appearance in MDA-MB-231. h) Co-immunoprecipitation of ROR1 receptor displaying inhibition of Wnt-5a binding to ROR1 after strictinin treatment. (* = p.worth 0.05, n = 3). Ligand docking The energetic site (S1A Fig) we decided to go with was the main one closest towards the residues indicated within a prior docking of DB03208 to tROR1 by Nath et Al [7]. A receptor grid was produced around the energetic site of tROR1. DB03208 and Strictinin had been independently docked to tROR1 using Glide docking accompanied by Induced Suit Docking [26C31]. To check on the validity of our docking, we likened our ligand relationship diagram compared to that of Nath et Al [7]. Our DB03208 ligand relationships showed many identical residue relationships to theirs (S1B and S1C Fig). The binding site of strictinin is quite near to the binding site of DB03208 (S1D Fig), recommending both of these ligands may have identical drug actions against tROR1. Immunoblotting Pursuing various remedies, the cells had been cleaned once in 1X PBS and lysed with RIPA buffer (150 mM NaCl, 1.0% Triton-X-100, 0.5% Sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) with 1 mM PMSF. Proteins concentrations had been dependant on Bradford Assay using Pierce 660 nm Proteins Assay reagent (thermofisher, Waltham, MA, USA). Pursuing SDS-PAGE, Proteins had been used in nitrocellulose membranes, that have been clogged for 1-hour in 5% dairy with mild agitation. Membranes had been incubated over night at 4C with different antibodies then cleaned in TBS-T (20mM Tris, 150mM NaCl, 0.1% Tween 20), and incubated for 1-hour at space temp with horseradish-peroxidase-linked anti-Mouse or anti-Rabbit IgGs (Cell Signaling, Danvers, MA, USA). Proteins bands had been recognized by chemiluminescence. Antibodies: ROR1 (Cell sign, #D6T8C), GAPDH (Cell sign, #D16H11), CK1 (Cell sign, #12448S), p-AKT-ser473 (Cell sign, #9271S), AKT (Cell sign, #C67E7), p-GSK3-ser9 (Cell sign, #D17D2), GSK3 (Cell sign, #D7SD3), XIAP (Cell sign, #D2Z8UL), caspase-9 (Cell sign, #9502S), Poor (Cell sign, #D24A9), p-Bad-ser136 (Cell sign, #4366T), -catenin (Invitrogen, MA1-301), p–catenin-ser33/37, Wint-5a (Novus-Bio NBP2-24752SS) Co-immunoprecipitation Co-immunoprecipitation of ROR1 and its own interacting protein was performed using the Pierce Basic IP Package (thermofisher, # 26146) relating to manufacturer process. Briefly, following remedies, cells had been lysed inside a moderate-strength IP-Lysis buffer without SDS (thermofisher, #87787) and.g) Immunoblot teaching aftereffect of siROR1 knockdown and strictinin about ROR1 expression in MDA-MB-231. 0.05, n = 3).(TIF) pone.0217789.s003.tif (125K) GUID:?6745E744-B667-4141-9E7B-5D1201B2B120 Data Availability StatementAll relevant data are inside the manuscirpt and its own Helping informaiton files. Abstract Triple Adverse Breast Tumor (TNBC), probably the most intense subtype of breasts cancer, can be seen as a the lack of hormone receptors generally targeted by hormone therapies like Tamoxifen. Because PhiKan 083 therapy achievement and survival prices for TNBC lag significantly behind other breasts cancer subtypes, there is certainly significant fascination with developing novel anti-TNBC real estate agents that may target TNBC particularly, with minimal results on nonmalignant cells. To the aim, our research identifies the anti-TNBC aftereffect of strictinin, an ellagitanin previously isolated from docking evaluation Ligand and proteins structure 3D constructions from the ligands, DB03208 and strictinin, had been built-in Schrodingers Maestro. For every ligand, the tautomeric areas had been produced at pH = 7 using Maestros Epik [18,19]. The cheapest tautomeric condition was selected and minimized towards the most energetically beneficial framework. Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1) comes with an extracellular site, a transmembrane site, and an intracellular site. We modeled a truncated edition of ROR1 (tROR1) that’s identical using the intracellular, C-terminal area of ROR1 but will not support the transmembrane nor the extracellular domains. The series for tROR1 (“type”:”entrez-protein”,”attrs”:”text”:”AAC50714.1″,”term_id”:”1589740″,”term_text”:”AAC50714.1″AAC50714.1) was retrieved through the NCBI protein data source. The 3D framework of tROR1 (Fig 1) was after that expected using I-TASSER [20C22]. The model for tROR1 was after that preprocessed, optimized, and reduced using Maestros Proteins Planning Wizard [23]. We after that mapped for energetic sites using Maestros SiteMap [24,25]. Open up in another windowpane Fig 1 Strictinin interacts with ROR1 in TNBC.a) Homology Style of tROR1. Whereas N-terminus can be indicated with a reddish colored ball, C-terminus can be indicated with a blue ball. b, c) Strictinin docked to tROR1 and ligand relationships diagram. Whereas N-terminus can be indicated with a reddish colored ball, C-terminus can be indicated with a blue ball. d, e) Immunoblot and immunofluorescence looking into basal ROR1 manifestation in two TNBC lines and nonmalignant breast epithelial range, MCF-10A. f) MTT evaluating aftereffect of ROR1 siRNA knockdown on strictinin cytotoxicity in TNBC. g) Immunoblot displaying aftereffect of siROR1 knockdown and strictinin on ROR1 manifestation in MDA-MB-231. h) Co-immunoprecipitation of ROR1 receptor displaying inhibition of Wnt-5a binding to ROR1 after strictinin treatment. (* = p.worth 0.05, n = 3). Ligand docking The energetic site (S1A Fig) we select was the main one closest towards the residues indicated inside a earlier docking of DB03208 to tROR1 by Nath et Al [7]. A receptor grid was produced around the energetic site of tROR1. DB03208 and Strictinin had been separately docked to tROR1 using Glide docking accompanied by Induced Match Docking [26C31]. To check on the validity of our docking, we likened our ligand discussion diagram compared to that of Nath et Al [7]. Our DB03208 ligand relationships showed many identical residue connections to theirs (S1B and S1C Fig). The binding site of strictinin is quite near to the binding site of DB03208 (S1D Fig), recommending both of these ligands may have very similar drug actions against tROR1. Immunoblotting Pursuing various remedies, the cells had been cleaned once in 1X PBS and lysed with RIPA buffer (150 mM NaCl, 1.0% Triton-X-100, 0.5% Sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) with 1 mM PMSF. Proteins concentrations had been dependant on Bradford Assay using Pierce 660 nm Proteins Assay reagent (thermofisher, Waltham, MA, USA). Pursuing SDS-PAGE, Proteins had been used in nitrocellulose membranes, that have been obstructed for 1-hour in 5% dairy with soft agitation. Membranes had been incubated right away at 4C with several antibodies then cleaned in TBS-T (20mM Tris, 150mM NaCl, 0.1% Tween 20), and incubated for 1-hour at area heat range with horseradish-peroxidase-linked anti-Mouse or anti-Rabbit IgGs (Cell Signaling, Danvers, MA, USA). Proteins bands had been discovered by chemiluminescence. Antibodies: ROR1 (Cell indication, #D6T8C), GAPDH (Cell indication, #D16H11), CK1 (Cell indication, #12448S), p-AKT-ser473 (Cell indication, #9271S), AKT (Cell indication, #C67E7), p-GSK3-ser9 (Cell indication, #D17D2), GSK3 (Cell indication, #D7SD3), XIAP (Cell indication, #D2Z8UL), caspase-9 (Cell indication, #9502S), Poor (Cell indication, #D24A9), p-Bad-ser136 (Cell indication, #4366T), -catenin (Invitrogen, MA1-301), p–catenin-ser33/37, Wint-5a (Novus-Bio NBP2-24752SS) Co-immunoprecipitation Co-immunoprecipitation.Whereas N-terminus is indicated with a crimson ball, C-terminus is indicated with a blue ball. strictinin influence on MCF-10A migration (* = p.worth 0.05, n = 3).(TIF) pone.0217789.s003.tif (125K) GUID:?6745E744-B667-4141-9E7B-5D1201B2B120 Data Availability StatementAll relevant data are inside the manuscirpt and its own Helping informaiton files. Abstract Triple Detrimental Breast Cancer tumor (TNBC), one of the most intense subtype of breasts cancer, is normally seen as a the lack of hormone receptors generally targeted by hormone therapies like Tamoxifen. Because therapy achievement and survival prices for TNBC lag considerably behind other breasts cancer subtypes, there is certainly significant curiosity about developing novel anti-TNBC realtors that may target TNBC particularly, with minimal results on nonmalignant tissues. To the aim, our research represents the anti-TNBC aftereffect of strictinin, an ellagitanin previously isolated from docking evaluation Ligand and proteins structure 3D buildings from the ligands, DB03208 and strictinin, had been built-in Schrodingers Maestro. For every ligand, the tautomeric state governments had been produced at pH = 7 using Maestros Epik [18,19]. The cheapest tautomeric condition was selected and minimized towards the most energetically advantageous framework. Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1) comes with an extracellular domains, a transmembrane domains, and an intracellular domains. We modeled a truncated edition of ROR1 (tROR1) that’s identical using the intracellular, C-terminal area of ROR1 but will not support the transmembrane nor the extracellular domains. The series for tROR1 (“type”:”entrez-protein”,”attrs”:”text”:”AAC50714.1″,”term_id”:”1589740″,”term_text”:”AAC50714.1″AAC50714.1) was retrieved in the NCBI protein data source. The 3D framework of tROR1 (Fig 1) was after that forecasted using I-TASSER [20C22]. The model for tROR1 was after that preprocessed, optimized, and reduced using Maestros Proteins Planning Wizard [23]. We after that mapped for energetic sites using Maestros SiteMap [24,25]. Open up in another windows Fig 1 Strictinin interacts with ROR1 in TNBC.a) Homology Model of tROR1. Whereas N-terminus is usually indicated by a reddish ball, C-terminus is usually indicated by a blue ball. b, c) Strictinin docked to tROR1 and ligand interactions diagram. Whereas N-terminus is usually indicated by a reddish ball, C-terminus is usually indicated by a blue ball. d, e) Immunoblot and immunofluorescence investigating basal ROR1 expression in two TNBC lines and non-malignant breast epithelial collection, MCF-10A. f) MTT assessing effect of ROR1 siRNA knockdown on strictinin cytotoxicity in TNBC. g) Immunoblot showing effect of siROR1 knockdown and strictinin on ROR1 expression in MDA-MB-231. h) Co-immunoprecipitation of ROR1 receptor showing inhibition of Wnt-5a binding to ROR1 after strictinin treatment. (* = p.value 0.05, n = 3). Ligand docking The active site (S1A Fig) we selected was the one closest to the residues indicated in a previous docking of DB03208 to tROR1 by Nath et Al [7]. A receptor grid was generated around the active site of tROR1. DB03208 and Strictinin were individually docked to tROR1 using Glide docking followed by Induced Fit Docking [26C31]. To check the validity of our docking, we compared our ligand conversation diagram to that of Nath et Al [7]. Our DB03208 ligand interactions showed many comparable residue interactions to theirs (S1B and S1C Fig). The binding site of strictinin is very close to the binding site of DB03208 (S1D Fig), suggesting these two ligands might have comparable drug action against tROR1. Immunoblotting Following various treatments, the cells were washed once in 1X PBS and lysed with RIPA buffer (150 mM NaCl, 1.0% Triton-X-100, 0.5% Sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) with 1 mM PMSF. Protein concentrations were determined by Bradford Assay using Pierce 660 nm Protein Assay reagent (thermofisher, Waltham,.