Anti stat3 clone d3z2g

Immobilon-P PVDF membranes (Millipore) were probed with the primary antibodies at 4°C overnight and then washed and incubated with HRP-conjugated goat anti-mouse IgG (H+L) or anti-rabbit IgG (H+L) secondary antibody (Promega). The following primary antibodies were used: anti-STAT3 (clone D3Z2G, Cell Signaling Technology), anti-phospho-STAT3 (Tyr705) (clone D3A7, Cell Signaling Technology), anti-STAT5 (Cell Signaling Technology), anti-phospho-STAT5 (Tyr694) (clone D47E7, Cell Signaling Technology), anti-β-actin (clone C4, Santa Cruz Biotechnology, sc-47778), HRP-conjugated anti-mouse IgG (H+L) (Promega, W4021), and HRP-conjugated anti-rabbit IgG (H+L) (Promega, W4011). All images were acquired with ChemiDoc MP system (Bio-Rad) and Image Lab software (Bio-Rad). Protein levels for each blot were quantified with ImageJ software.

Cellular protein was extracted from cells using RIPA buffer (Thermo) supplemented with proteinase and phosphatase inhibitor (Thermo). Same amount of protein was separated on pre-cast 4-15% gradient gels (BioRad) by SDS-PAGE and transferred to PVDF membranes (BioRad). The membranes were blocked with 5% Milk in TBS-5% Tween buffer and probed with primary antibodies at 4 degree overnight. Then, the membranes were washed and incubated with secondary antibodies conjugated to HRP. The following primary and secondary antibodies were used: anti-IL23R (Novus Biological 1:1000 Dilution), anti-GAPDH (clone 6C5, Santa Cruz, 1:1000 Dilution), anti-phospho-STAT3(Tyr705) (clone D3A7, Cell Signaling Technology, 1:1000 dilution), anti-phospho-STAT3(Ser727) (clone 6E4, Cell Signaling Technology, 1:1000 Dilution), anti-STAT3 (clone D3Z2G, Cell Signaling Technology, 1:1000 Dilution) and anti-CD3ζ (clone 6B10.2, Santa Cruz, 1:1000 Dilution). Blot images were acquired with ChemiDoc MP system (BioRad) and the densitometry was calculated by Image Lab software (Bio-Rad).