These data indicate that phosphorylation of GSK3 in BEC stimulated with PGN induced a significant reduction of GSK3 activity

These data indicate that phosphorylation of GSK3 in BEC stimulated with PGN induced a significant reduction of GSK3 activity. Open in a separate window Fig 3 Inhibition of glycogen synthase phosphorylation at Ser641 in BEC treated with PGN, LiCl or SB, and stabilization of -catenin levels by GSK3 or GSK3 gene silencing.A) BEC were either stimulated with 10 g/mL of PGN for 30 min, treated with 10 mM NaCl Lanolin for 60 min, 10 mM LiCl or 10 M SB and then stimulated with 10 g/mL of PGN for 30 min. blots were stripped and reprobed with antibodies that recognize -actin (A) or the nonphosphorylated form of Akt Lanolin (B-D). Blots are representative of three self-employed experiments. Graphs on the right show the band intensity acquired by densitometric analysis. Results are indicated as the mean S.E.M. (n = 3). *p 0.05, compared with the unstimulated control.(PDF) pone.0132867.s001.pdf (317K) GUID:?44A43E76-CF5E-4760-B62A-285A2F0171EE S2 Fig: Temporal course of GSK3 and GSK3 phosphorylation induced by PGN in BEC. A and B) BEC were remaining unstimulated (0) or stimulated with 10 g/mL of PGN for 15, 30, 60 or 120 min. Protein extracts were analyzed by western blot and probed with monoclonal antibodies against the phosphorylated forms of GSK3 (pGSK3 Ser21) or GSK3 (pGSK3 Ser9). To verify equivalent protein loading, blots were stripped and reprobed with an antibody that recognizes the nonphosphorylated form of GSK3. Blots are representative of three self-employed experiments. Graphs on the right indicate the band intensity acquired by densitometric analysis. Results are indicated as the mean S.E.M. (n = 3). *p 0.05; **p 0.01, compared with the unstimulated control.(PDF) pone.0132867.s002.pdf (351K) GUID:?043C0CC6-AEA9-42FF-83AE-6301224C355D S3 Fig: Phosphorylation of Akt at Ser473, GSK3 at Ser21 and GSK3 at Ser9 in BEC treated with inhibitors. BEC were left untreated, treated with 10 M of LY294002 (LY) for 30 min, treated with 1 M of Akt inhibitor IV (Akt-i IV) for 30 min or treated with 10M of SB216763 (SB) for 30 min. Untreated cells were incubated with 10 M of DMSO. Then, total protein from untreated and treated cell was acquired. A) Phosphorylation of Akt at Ser473; B) Phosphorylation of GSK3 at Ser21; C) Phosphorylation of GSK3 at Ser9. Detection of Cactin and GAPDH were used as control Lanolin of protein loading. Data offered are representative of two self-employed experiments.(PDF) pone.0132867.s003.pdf (104K) GUID:?DCF1D350-46E0-41F1-AD17-3EA44A078367 S4 Fig: GSK3 inhibition induces phosphorylation of NF-B. A) BEC were remaining untreated and unstimulated (-), treated for 60 min with 10 mM of NaCl, stimulated with 10 g/mL of PGN for 30 min, treated for 60 min with 10 mM of LiCl and then stimulated with 10 g/mL of PGN for 30 min or treated for 60 min with 10 mM of LiCl. B) BEC were transfected with control siRNA (siRNA Rabbit polyclonal to AKAP13 control), transfected with siRNA control and then stimulated with 10 g/mL of PGN for 30 min, transfected with siRNA focusing on GSK3 (siRNA GSK3), transfected with siRNA GSK3 and then stimulated with 10 g/mL of PGN for 30 min, transfected with siRNA focusing on GSK3 (siRNA GSK3) or transfected with siRNA GSK3 and then stimulated with 10 g/mL of PGN for 30 min. Protein extracts were analyzed by western blot and probed with monoclonal antibodies against the phosphorylated forms of p65 (NF-B p65 Ser536). To check for equivalent amount of proteins, blots were stripped and reprobed with antibodies that identify the nonphosphorylated forms of p65 (A) or -actin (B). Blots are representative of three self-employed experiments. Graphs show the band intensity acquired by densitometric analysis. Results are indicated as the mean S.E.M. (n = 3). *p 0.05; **p 0.01, compared with the unstimulated control.(PDF) pone.0132867.s004.pdf (212K) GUID:?A8EDA4FE-5438-4E30-9671-F04470357CD7 S5 Fig: GSK3 and GSK3 gene silencing differentially regulates IL-12p40 expression in BEC stimulated with PGN. BEC were remaining untransfected and unstimulated (-), stimulated with 10 g/mL of PGN for 9 h, transfected with control siRNA (siRNA control), transfected with control siRNA and then stimulated with 10 g/mL of PGN for 9 h, transfected with siRNA concentrating on GSK3 (siRNA GSK32 or siRNA GSK33) or transfected with siRNA concentrating on GSK3 (siRNA GSK31 or siRNA GSK32) and activated with 10 g/mL of PGN for 9 h. A) Cell-free supernatants had been examined by ELISA for creation of IL-12p40 and B) Proteins extracts had been analyzed by traditional western blot and probed using a monoclonal antibody against the phosphorylated types of GSK3 and GSK3. To verify that identical amount.