Anti gapdh monoclonal antibody

Cell lysates were prepared, electrotransferred, immunoblotted with antibodies, and then visualized via incubation with colorigenic substrates (nitroblue tetrazolium, NBT, and 5-bromo-4-chloro-3-indolyl phosphate, BCIP) (Sigma Chemical Co., St. Louis, MO, USA). Expression of GAPDH was used as a control to ensure equal protein loading [19 (link)].
Immunodetection was performed by probing with appropriate dilutions of specific antibodies at room temperature for 2 h. Anti-p53, anti-p27/Kip1, anti-p21/Cip1, anti-GAPDH monoclonal antibodies (Santa Cruz, Inc. CA, USA), anti-E cadherin [SP64] (ab227639) (Abcam Inc. Shanghai, China), and anti-cyclin D1 monoclonal antibodies (Transduction Laboratories, Lexington, KY) were used. Membranes were incubated with secondary alkaline phosphatase-coupled anti-mouse and anti-rabbit antibodies (Jackson, Westgrove, PA, USA) at room temperature for 1 h at dilutions of 1:5,000 and 1:1,000, respectively. Immunoreactive proteins were visualized with a chemiluminescent detection system (PerkinElmer Life Science, Inc., Boston, MA, USA) and BioMax LightFilm (Eastman Kodak Co., New Haven, CT, USA) according to the manufacturer’s instructions. Results were analyzed by densitometry analysis.

African green monkey kidney fibroblast-like COS-7 cells were purchased from the Cell Center of the Chinese Academy of Medical Sciences (China). Human neuroblastoma SK-N-SH and SK/NEST cells that stably expressed NEST were from our previous study [44 (link)]. The NTE-GFP and ΔR-NTE-GFP constructs were generous gifts from Dr. Paul Glynn [6 (link)]. Plasmid pEGFP-N3 was purchased from Clontech (Mountain View, CA, USA). Transfection reagent Lipofectamine 2000 and HCS LipidTOX™ Deep Red neutral lipid stain were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Cell culture reagents and OA were from Sigma-Aldrich (St. Louis, MO, USA). The E1003 triglyceride assay kit was purchased from Applygen Technologies (Beijing, China). The Q5 Site-Directed Mutagenesis Kit was purchased from New England Biolabs (Ipswich, MA, USA). Mouse anti-GFP, anti-GAPDH monoclonal antibodies, and goat anti-mouse IgG HRP were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Enhanced chemiluminescence (ECL) reagents were obtained from Beyotime Biotechnology (Shanghai, China).

Proteins were resolved on SDS–PAGE and electroblotted onto PVDF membranes. Membranes with transferred proteins were incubated with the primary monoclonal antibody and successively with the specific peroxidase conjugated secondary antibody. Monoclonal antibodies against Ub and p27 were obtained from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). SQSTM1/p62 (sequestosome 1, herein p62) mouse monoclonal antibody was from Sigma‐Aldrich S.r.L. (Milano, Italy) and the anti‐LC3B antibody was purchased from Cell Signaling Technology, Inc. The immunoblot detection was performed with ECL Western blotting detection reagents using a ChemiDoc MP system. Each gel was loaded with molecular weight markers in the range of 12 to 225 kDa (GE Healthcare). Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was utilized as a control for equal protein loading: membranes were stripped and re‐probed with anti‐GAPDH monoclonal antibody (Santa Cruz Biotechnology, Heidelberg, Germany). Stripping buffer contained 200 mM glycine, 0.1% SDS, and 1% Tween 20. Immunoblot images were quantified using ImageJ 1.52p software (NIH, USA).