Rabbit anti gapdh ab

The IL-6, TNF-α, IL-1β, STAT3, and the reference protein [glyceraldehyde-phosphate dehydrogenase (GAPDH)] samples were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto 0.45-µm polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA, USA) for 90 minutes at 350 mA, and then blocked with tris-buffered saline with Tween 20 (TBST) buffer (50 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween-20, pH 7.4) containing 5% non-fat dry milk (w/v) at 4 ℃ overnight. After washing thrice with TBST for 30 minutes, the blots were incubated with rabbit anti-phospho-STAT3 Ab (1:1,000), rabbit anti-TNF-α Ab (1:1,000), rabbit anti-IL-1β Ab (1:1,000), rabbit anti-STAT3 Ab (1:1,000), rabbit anti-IL-6 Ab (1:1,000), or rabbit anti-GAPDH Ab (1:3,000) (Abcam, Cambridge, MA, USA). The blots were washed thrice with TBST for 30 minutes, and then incubated for 1 hour with the HRP-conjugated goat anti-rabbit IgG Ab (Absin) at room temperature. After washing with TBST, the immunoreactive proteins were visualized using a chemical luminescent immunodetection system (Tanon 4500, Tanon Technology Company, Shanghai, China).