Mouse anti human gapdh monoclonal antibody

Total protein was isolated from tissue samples or cells with RIPA lysis buffer (Beyotime Biotechnology, Jiangsu, P.R. China) containing 1% protease inhibitors (Pierce, Rockford, IL, USA). The concentration of total protein was examined by Bradford assay (Bio-Rad Laboratories). Equal amounts of protein samples (about 30 μg) were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore). After blocking with 5% nonfat milk in Tris-buffered saline with Tween (TBST; Sigma-Aldrich, St. Louis, MO, USA), the membranes were incubated with primary antibodies overnight at 4°C. The primary antibodies used in this study include rabbit anti-human polyclonal SRPK1 (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti-human monoclonal GAPDH antibody (1:1,000 dilution; Santa Cruz Biotechnology). The membranes were then washed with TBST and incubated with corresponding HRP-conjugated secondary antibodies (1:1,000 dilution; Santa Cruz Biotechnology) at room temperature for 2 h. Band signals were visualized using an enhanced chemiluminescence kit (Pierce, Minneapolis, MN, USA) and analyzed with Quantity One software version 4.6.2 (Bio-Rad Laboratories). GAPDH was used as an internal control.

At 72 h post-transfection, total protein was isolated from transfected cells using RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Briefly, equal quantities of protein (20 µg) were separated by 10% SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). After blocking with 5% non-fat dried milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature, the membranes were probed with primary antibodies at 4°C overnight. The primary antibodies used in the present study included mouse anti-human monoclonal ADAM9 antibody (cat. no. sc-377233; dilution, 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and mouse anti-human monoclonal GAPDH antibody (cat. sc-166574; dilution, 1:1,000; Santa Cruz Biotechnology, Inc.). Thereafter, the membranes were washed with TBST for three times, incubated with the corresponding horseradish peroxidase-conjugated goat anti-mouse secondary ImmunoglobulinG (cat. ab150113; dilution, 1:1,000; Abcam, Cambridge, UK) at room temperature for 1 h, and visualized by chemiluminescence using the ECL detection system (Pierce; Thermo Fisher Scientific, Inc.). The protein expression levels were normalized to GAPDH. Protein expression was analyzed using BandScan 5.0 software (Glyko, Inc., Novato, CA, USA). All experiments were repeated ≥3 times.