Anti gapdh ab181602

CD4⁺ T cells were lysed in 1 ml of lysis buffer containing protease inhibitor (Thermo Scientific) at 4°C. Lysates were centrifuged for 15 min at 14,000g at 4°C, and protein concentration was determined by Bradford protein assay (Thermo Scientific). Soluble lysates were loaded in each lane and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore). Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween-20 buffer (TBST) for 1 hr, washed with TBST, and incubated with primary antibodies. Antibodies include anti-STAT5 (ab126832), anti-TET2 (ab94580), anti-FOXP3 (ab10901), and anti-GAPDH (ab181602); all antibodies were obtained from Abcam Company. Blots were rinsed with TBST and subsequently incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hr at room temperature. The intensity of the identified bands was quantified using Quantity One software (Bio-Rad).

Anti-YAP (A1002) was purchased from ABclonal (Wuhan, China). Anti-mouse (sc-2005), and anti-rabbit (sc-2004) HRP conjugated secondary antibodies were purchased from Santa-Cruz Biotechnology (Dallas, TX, USA); anti-GAPDH (ab181602) was purchased from Abcam (Shanghai, China). The anti-pMLC (3674 s) was purchased from Cell Signaling Technologies (Denvers, CO, USA); anti-RhoA (ARH04) was purchased from Cytosketon (Denvers, CO, USA); anti-ARHGAP29 (sc-377022) was purchased from Santa-Cruz Biotechnology (Dallas, TX, USA); Alexa Fluor 488- or Alexa Fluor 555-conjugated secondary antibodies were obtained from Life (Carlsbad, CA, USA). Rho activator II were from Cytoskeleton. Myosin II inhibitor (blebbistatin) was from EMD_Millipore. Bromophenol Blue was generously donated by Prof. Yuxin Yin’s lab. Dimethyl sulfoxide (DMSO) was purchased from VWR (branch company in Shanghai, China). DNA transfection reagent was purchased from NEOFECT (Beijing, China).

Cells grown in 24-well plates were lysed on ice using 200 μL RIPA buffer (Beyotime Biotechnology, Shanghai, China) for 30 min. Next, the lysis mixtures were centrifuged and the supernatants were collected. BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA) was used for quantification of proteins. Then, proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and electrophoretically transferred to PVDF membranes. Afterwards, membranes were blocked in 5% bovine serum albumin (BSA) for 2 h at room temperature and then probed with primary antibodies at 4 °C overnight. The primary antibodies used in this study include anti-GAPDH (ab181602, Abcam, Cambridge, UK), anti-RIP2 (70R-10459, fitzgerald), anti-IL1β (abx132185, Abbexa, Cambridge, UK), anti-IL8 (abx100965, Abbexa), anti-IL6 (abx177189, Abbexa), anti-NFκB p65 (10745-1-AP, Proteintech, Wuhan, China), and anti-IκBα (10268-1-AP, Proteintech). The primary antibodies were used at a dilution of 1:1000. Then, the membranes were incubated with secondary antibodies marked by horseradish peroxidas (Sigma-Aldrich, St. Louis, MI, USA) at a 1:10,000 dilution at room temperature for 2 h. Immunoblots were visualized by enhanced chemiluminescence (ECL kit, Santa Cruz Biotechnology, Dallas, TX, USA). The images were analyzed using Image Lab™ Software (Bio-Rad, Hercules, CA, USA).

Total protein was extracted from FC tissue and HMC3 cells using PMSF and RIPA buffer and quantified with the bicinchoninic acid method. The western V3 workflow (Bio-Rad) was used for western blot analysis. The primary antibodies [anti-GAP-43 (ab12274 and ab11136) and anti-GAPDH (ab181602)] and secondary antibody [horseradish peroxidase-conjugated goat anti-rabbit IgG H&L (ab6721)] were purchased from Abcam (Shanghai, China).

The harvested cells were digested by RIPA buffer. Following sonication, the samples were centrifuged for 15 min at 12,000 g under 4 °C. For the determination of total protein density, a DC Protein Assay Kit I (Bio-Rad, Richmond, CA, USA) was applied. Then, after separating the proteins were on 12% SDS-PAGE, transfer the proteins onto the hybond nitrocellulose membrane (Amersham, NJ, USA). In all, 5% nonfat milk was utilized for the sealing of membranes in the Tris-buffered saline (pH 7.5). The membrane went through overnight hybridization with the primary antibodies, and then second antibodies. The protein bands were revealed by an ECL kit (Millipore, Billerica, MA, USA). The expression levels of proteins expression levels were evaluated by the Image J (National Institutes of Health, USA). The primary antibodies were: anti-ENO1 (ab155102) anti-E-cadherin (ab1416), anti-N-cadherin (ab18203), anti-PARP (ab74290), anti-cleaved-caspase 3 (ab2302), caspase-3 (ab13847), anti-cleaved-caspase 6 (ab2326), anti-caspase 6 (ab185645), anti-cleaved-caspase 9 (ab2324), anti-caspase 9 (ab52298), and anti-GAPDH (ab181602) all from Abcam (Cambridge, UK).