Socs3

Proteins were fractionated on a SDS-PAGE gel and blotted for 2 h at 90 V. Western Blot analyses was performed by antibody incubation in PBS containing 0.05% Tween and 5% (w/v) milk powder according to standard protocols.
The following primary antibodies were used in this study: β-actin (Sigma, 1:1000 dilution), SOCS-3 (Santa Cruz, 1:500), pY-1146-IRβ (Sigma, 1:500), IRβ (Santa Cruz, 1:500), pY-632 and pS-307 IRS-1 (SAB Signaling, 1:500), IRS-1 (Cell Signaling, 1:500), pT-183-JNK (Abcam, 1:500), JNK (Abcam, 1:500), Y-705 and S-627 STAT-3 (Cell Signaling, 1:500), STAT-3 (Cell Signaling, 1:500), pS-50-PTP-1B (Abcam, 1:500), PTP-1B (Abcam, 1:500), NF-κB (Abcam, 1:500). Goat anti-rabbit IgG-HRP and rabbit anti-Mouse IgG-HRP (Sigma) were used as secondary antibodies, and ECL Prime (Amersham) reagent was used for developing. Bands were quantified by scanning densitometry with a G-Box densitometer with exposure in the linear range using Gene Tools software (Synergy, Cambridge, UK). The relative levels of phosphorylated and total proteins were normalized to the corresponding amount of total protein mass and β-actin, respectively, in the same sample.

Cells were lysed directly in lysis buffer to collect whole cell extracts. Protein samples were separated on polyacrylamide gels, transferred onto nitrocellulose membrane by iblot (Invitrogen) and detected using horseradish peroxidase-conjugated secondary antibodies and chemiluminesce (SantaCruz) exposure of BioMax film (Kodak). The following antibodies were used: antibodies to SOCS3, actin, STAT5 and p-STAT5 (Tyr 694/Tyr 699) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). JAK2 and p-JAK2 (Tyr1007/1008) were obtained from Cell Signaling Technology (Beverly, MA, USA).

Liver proteins were quantified by western blot analysis as follows using BioRad, (Hercules, CA) Criterion and ChemiDoc systems following manufacturers’ instructions. PGC1α antibody was purchased from EMD Millipore, Billerica, MA, Cat# ST1202. All other antibodies purchased from Santa Cruz Biotechnology, Santa Cruz, CA were as follows: FOXO1Ser256 Cat# sc-101681; G6Pase-α Cat# sc-27198; PEPCK Cat# sc-32879; PPARα Cat# sc-9000; SREBP-1 Cat# sc-13551; mTORC Cat# sc-8319; PPARγ Cat# sc-7273; PGC1β Cat# sc-67286, NFκB p65 Cat# sc-8008; IKKαβ Cat# sc-7607; SOCS3 Cat# sc-9023; JNK Cat# sc-571; Actin Cat# sc-47778. Criterion gradient tris-glycine precast gels, secondary antibodies, PVDF blotting membranes, molecular weight markers, and Enhanced Chemiluminescence (ECL) reagents were also purchased from BioRad. Samples from 8 SHR Vehicle, 8 SHR Bromocriptine treated rats, and 6 Wistar wild type controls were loaded onto the same 26 well Criterion gel along with the molecular weight markers; band intensity was compared only within the samples loaded onto the same gel. A housekeeping protein (Actin) was concurrently quantified on all gels, and the test protein amount was normalized to Actin in the Western blot analysis. Protein bands were quantified with BioRad ImageLab 4.1 software.

Cells were lysed directly in lysis buffer to collect whole cell extracts. Protein samples were separated on polyacrylamide gels, transferred onto nitrocellulose membrane by iblot (Invitrogen), detected using horseradish peroxidase-conjugated secondary antibodies, and exposed to BioMax film (Kodak) following chemiluminescence (Santa Cruz, CA, USA). The following primary antibodies were used: SOCS3, Actin, and B4GALT1 (Santa Cruz, CA, USA).

After harvesting, the cells were lysed on ice using NETN buffer (20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) containing 10 mM NaF and 1 mM PMSF. Proteins were separated by 10% SDS-PAGE, and transferred to PVDF membrane, to be probed with corresponding primary antibodies at 4 ℃, overnight. Next day, the membranes were incubated with corresponding secondary antibodies, conjugated with horseradish peroxidase, for 1 h, and then ECL detection reagent was used to visualize the target proteins. Primary antibodies against AKT (#9272, 1:1000), phospho-AKT (Ser473) (#9271, 1:1000), phospho-FOXO3a (Thr32) (#9464, 1:1000), and ERK (#9102, 1:1000) were all purchased from Cell Signaling Inc. (Boston, MA, USA). IRS1 (#559, 1:1000), SOCS3 (#73045, 1:200), SIRT1 (#74504, 1:200), SNAI1 (#271977, 1:200), and phospho-ERK (E-4, 1:1000) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

For Western blotting, HeLa cells were lysed in RIPA buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% SDS, and 1% Triton 100) at 4° C, separated by SDS-PAGE, and analyzed by immunoblotting with antibodies against Zac1 (MW: 51 kDa; 1:2000 dilution) and p-Stat 3 (Y705) (MW: 88 kDa; 1:1000 dilution) (Abcam, Cambridge, UK); ACTN (MW: 100 kDa; 1:10000 dilution), aggrecan (MW: 200 kDa; 1:1000 dilution), cadherin-11 (MW: 110 kDa; 1:500 dilution), HIF-1α (MW: 132 kDa; 1:1000 dilution), IL-11 (MW: 23 kDa; 1:1000 dilution), IL-6 (MW: 21/27 kDa; 1:1000 dilution), SOCS3 (MW: 30 kDa; 1:1000 dilution), p53 (MW: 53 kDa; 1:2000 dilution), PCNA (MW: 36 kDa; 1:10000 dilution), and β-actin (MW: 43 kDa; 1:10000 dilution) (Santa Cruz, TX, USA); and Akt (MW: 60 kDa; 1:5000 dilution), p-Akt (MW: 60 kDa; 1:1000 dilution), ERK (MW: 42/44 kDa; 1:2000 dilution), p-ERK (MW: 42/44 kDa; 1:2000 dilution), Stat 3 (MW: 79/86 kDa; 1:1000 dilution), ChIP-grade Hemagglutinin (HA), ChIP-grade c-Fos, and ChIP-grade HIF-1α (Cell Signaling, MA, USA).