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.
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.