Anti glyceraldehyde 3 phosphate dehydrogenase gapdh antibody

Cells were promptly homogenized in a homogenization buffer and centrifuged at 10,000×g for 30 min to collect the supernatant. Protein concentrations were determined with a BioRad protein assay (BioRad, Hercules, CA, USA). Total protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were washed, blocked, and incubated with anti-Nrdp1 antibody (1:1,000; Santa Cruz Biotechnology, CA, USA) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:1,000; Sigma). Membranes were washed and incubated with HRP-conjugated secondary antibodies (1:500; Santa Cruz Biotechnology). Proteins were detected using an enhanced chemiluminescence reagent (NEN, Boston, MA). The relative intensity of each band was determined using Image Lab 3.0 (BioRad Laboratories, Inc., Hercules, CA, USA).

Protein from H20 and H446 SCLC cell lines was extracted by radioimmunoprecipitation and loading buffer and then boiled for 10 min until the samples were denatured. A 10% SDS-polyacrylamide gel was applied to separate the protein. Then the protein was transferred onto a nitrocellulose membrane (Life technologies, Carlsbad, CA, USA), and 5% milk in Tris-buffered saline containing 0.1% (v/v) Tween-20 (TBST) was used to block the membrane for 1 h at 37°C. Anti-hTERT antibody, anti-EZH2 antibody (Sigma, St. Louis, USA) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) a ntibody (Sigma, St. Louis, USA), diluted by TBST at a concentration of 1:1000, were incubated with the membrane overnight at 4°C. The horse radish peroxidase secondary antibody (Sigma, St. Louis, USA), diluted by TBST at a concentration of 1:10 000, was incubated with the membrane after it was washed three times with TBST. An exposer (Thermo Fisher Labsystems, Waltham, MA, USA) was then used to display the protein bands. GAPDH served as the endogenous control.

Tissue samples (~1 g) of the distal colon and the spinal cord were extracted in three volumes of extract buffer, centrifuged at 13,000× g for 10 minutes at 4°C; the supernatant was saved at −70°C until usage. Western blotting protocol was developed for the Bio-Rad protein gel and transfer apparatus. The membranes were blocked and incubated with the primary antibodies at 4°C, overnight, and subsequently with the secondary antibodies for 2 hours. The gel was transferred to a polyvinylidene difluoriizde membrane and blotted with primary (overnight) and then with secondary (1 hour) antibodies. The primary antibodies included a rabbit polyclonal anti-TRPA1 antibody (1:200; Novus Biologicals) and a mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:500; Sigma). The secondary antibodies included anti-mouse Ig for GAPDH (1:1,000) and anti-rabbit Ig for TRPA1 (1:3,000). The integrated density of Western blotting bands was quantitatively analyzed by the Gel Image analysis software. TRPA1 protein expression was normalized to GAPDH.