Mouse anti gapdh antibody

Cultured cells were homogenized and lysed in M-PER TM mammalian protein extraction reagent (UC282138; Thermo Fisher Scientific, Waltham, MA, United States) containing protease and phosphatase inhibitors (Roche Applied Science, Indianapolis, IN, United States). Protein concentrations were quantified using the Pierce TM BCA Protein Assay (UD281372; Thermo Fisher Scientific). Protein samples were loaded onto a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was then electrotransferred to a PVDF membrane (IPVH00010; Amersham Biosciences, Piscataway, NJ, United States). The following antibodies purchased from ABclonal Technology (Woburn, MA, United States) were used in this study: rabbit anti-ERVWE1 antibody (dilution 1:1000, A16522), rabbit anti-CPEB1 antibody (dilution 1:1000, A5913), rabbit anti-NDUFV2 antibody (dilution 1:1500, A7442), mouse anti-GAPDH antibody (dilution 1:50000, AC002), goat anti-mouse IgG-horseradish peroxidase (HRP) (dilution 1:5000, AS003), and goat anti-rabbit IgG-HRP (dilution 1:5000, AS014). The bands were visualized using the Millipore Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500; Millipore, Burlington, MA, United States). Images were captured using the Tanon 5200 chemiluminescence imaging system (Tanon, Shanghai, China). Target protein expression levels were normalized to GAPDH.

The procedures of total proteins extraction and Western blot were performed according to our previous description.17 (link) Simply, the cells were disrupted by RIPA lysis and the total proteins were collected by centrifugation. The denatured proteins (30 μg per lane) were separated by SDS-PAGE and subsequently transmitted into 0.22μm PVDF membrane. After blocked with 5% nonfat milk at room temperature for 1 hr, the membrane was incubated with rabbit anti-RKIP antibody (BBI, Shanghai, China) and mouse anti-GAPDH antibody (Abclonal, Wuhan, China), respectively, overnight at 4°C. Next, after incubated with anti-rabbit or anti-mouse IgG HRP-conjugated secondary antibodies, the protein amounts were visualized by chemiluminescent HRP substrate (EpiZyme, Shanghai, China). The equipment and the software of visualization are FluorChem FC3 (Protein Simple, CA, USA) and AlphaView SA (Protein Simple, CA, USA), respectively.