Rabbit anti stat3

Harvested cells were lysed in RIPA buffer (150 mM sodium chloride, 1% triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 7.5 and 2 mM EDTA) (GenDEPOT, TX, USA) containing 1% protease inhibitor cocktail (GenDEPOT). 20 μg of each sample is separated by SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare, Chalfont St Giles, UK). Western blotting was performed as previously described by Liu et al.³⁸ with primary antibodies to mouse anti-E-cadherin 1:1000 (Cell Signalling, Danvers, MA, USA), rabbit anti-Vimentin 1:500 (Abcam, Cambridge, UK), rabbit anti-Zeb1 1:500 (Sigma), rabbit anti-IL-6 1:1000 (Abcam), rabbit anti-STAT3 1:500 (phosphor Y705: Abcam), mouse anti-STAT3 1:1000 (Cell signalling), rabbit anti-α-lamin 1:1000 and goat anti-β-actin 1:5000 (Santa Cruz Biotechnology, Santa Cruz, USA). All primary antibodies were diluted in 5% Bovine Serum Albumin (BSA) in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) and incubated overnight at 4 °C. Secondary antibody included goat anti-rabbit IgG-HPR 1:5000 (Santa Cruz Biotechnology), goat anti-mouse IgG-HPR 1:2000 (Santa Cruz Biotechnology) and rabbit anti-goat IgG-HPR (GenDepot, TX, USA). Protein bands were visualised using enhanced chemiluminescence reagents (Western Lighting Plus, PerkinElmer, USA).

The total protein was extracted by using protein lysis buffer from the spinal cord tissues. The protein concentration was measured using BCA Protein Kit (Thermo Fisher). The protein sample (20 µg) was separated on 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The blots were probed with rabbit anti-SOCS3 (Abcam, Cambridge, U.K.; 1:200 dilution), rabbit anti-pSTAT3 (Abcam, 1:1000 dilution), rabbit anti-STAT3 (Abcam, 1:1000 dilution), rabbit anti-pJAK2 (Abcam, 1: 1000 dilution), rabbit anti-JAK2 (Abcam, 1:5000 dilution) for overnight at 4°C. The blots were incubated with horse radish peroxide-conjugated secondary antibody. The imaging was performed with electron chemiluminescence (ECL) emitting solution. Finally, the blots were visualized with an Immobilon Western Chemiluminescent HRP Substrate system (Millipore Corp., Billerica, MA, U.S.A.).