Mkp 3

The following agents U 0126, SB 203580, Bay 11-7082, KT 5720 (Calbiochem Corp, USA) and rolipram, foskolin (Sigma-Aldrich, USA) were dissolved in DMSO. Final concentration of DMSO was less than 0.1%. 8-Bromo-cAMP (8-Bromoadenosine 3′,5′-cyclic monophosphate sodium salt) was purchased from Sigma-Aldrich. MKP-1, total-IkBα, total-ERK2 antibodies obtained from Santa Cruz Biotechnology. MKP-3, MKP-5, phospho-IkBα (Ser32/36), phospho-p38 (Thr180/Tyr182), total-p38, phospho-ERK1/2 (Thr202/Tyr204), phospho-CREB (Ser133) and PKA-cα were purchased from Cell Signaling Technology. β-actin was purchased from Sigma-Aldrich. Dulbecco’s modified Eagle’s medium (DMEM), penicillin and streptomycin were from Invitrogen Life Technologies. All other reagents were purchased from Sigma-Aldrich, unless indicated otherwise.

30 μg of different protein fractions from rat tissues was subjected to SDS-polyacrylamide gel electrophoresis. Proteins were then transferred to Immobilon polyvinylidene difluoride transfer membranes (Millipore, Billerica, MA, USA), blocked for 1 h at room temperature with 5% non-fat milk solution in 0.1% Tween-20-Tris-buffered saline (TBS), and incubated as described previously15 (link). Detection was performed using the ECL chemiluminescence kit for HRP (Amersham GE Healthcare Europe GmbH, Barcelona, Spain). To confirm the uniformity of protein loading, blots were incubated with β-tubulin or β-actin antibodies (Sigma-Aldrich, St. Louis, MO, USA) as a control. Primary antibodies for phospho-and total AKT, phospho- and total AS160, phospho-ERK, GLUT4, phospho-PKA and phospho-TSC-2 were supplied by Cell Signaling (Danvers, MA, USA), those for 14-3-3, ChREBP, phospho-and total FoxO1, IRS-1, IRS-2, MKP-3 and phospho-SGK-1 were obtained from Santa Cruz Biotechnologies (Dallas, TX, USA), and antibody against phospho-mTOR was from Millipore (Billerica, MA, USA).

Human recombinant GPx3 (hGPx3) was purchased from Enzo Life Sciences (MI, USA). Antibodies specific for GPx3, MKP3, Erk, NF-κB, cyclinB1, lamin B1, and actin were purchased from Santa Cruz Biotechnology (CA, USA) and Cell Signaling Technology (MA, USA). PDTC (pyrrolidine dithiocarbamate) was purchased from Sigma (CA, USA).