Mouse anti human gapdh

Western blot was used to detect the expression of E6 protein and/or G6PD protein in tumor tissues or HeLa cells with different treatments as described previously (20 (link)). Briefly, cells or samples were lysed on ice for 30 min in CytoBuster Protein Extraction Buffer (Novagen, USA) and then all samples were centrifuged at 12,000 rpm at 4°C. Quantitative 50-g proteins of each sample was extracted and isolated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then, the protein was transferred to a nitrocellulose (NC) membrane and sealed with Tris-Buffered Saline Tween-20 (TBST) containing 5% non-fat milk. The membrane was subsequently incubated with mouse anti-human HPV E6 antibody (1:800, Merck Millipore, MAB874, USA), goat anti-human G6PD proteins (1:500, Santa Cruz, sc-46971) or mouse anti-human GAPDH (1:500, Santa Cruz, sc-81545) antibodies at 4°C overnight. After washing in TBST, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2,000) at 25°C for 4 h. The protein was visualized and the quantity of targeting protein was determined using electrochemiluminescence (ECL) technique (BestBio, USA). All results were photographed using the Bio-Rad Gel Imagining system (ChemiDoc™XRS+) and using Quantity One software v4.6.6 (all Bio-Rad Laboratories, Inc., Hercules, CA, USA) to calculate the gray density.

Proteins were prepared using Radioimmunoprecipitation assay buffer (RIPA) as previously reported[24 (link)]. Lysate protein concentrations were measured by BCA Assay (Pierce, Rockford, IL, USA). For Western blotting, protein extracts were separated by 10% SDS-PAGE and transferred to the nitrocellulose membranes. The membranes were incubated with primary antibodies overnight at 4°C after blocking. The primary antibodies used were mouse anti-human SGPL1 (Santa Cruz, Santa Cruz, CA, USA), goat anti-human IL-8 (Abcam, CA, USA) and mouse anti-human GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and anti-mouse-HRP-conjugated secondary antibody was used as secondary antibody (Invitrogen, CA, USA). The signals were detected using an Enhanced Chemiluminescence Plus kit (Amersham, NJ, USA) and visualized after exposure to a Kodak film. Relative densities of SGPL1 and IL-8 were normalized to GAPDH of the same blot. The band intensity were analyzed by Image J v1.50 (NIH, USA).

Total lysates from RPMI and U266 cell lines were lysed in a lysis-buffer containing protease inhibitor cocktail (Sigma-Aldrich, Milan, Italy). Proteins were separated on 8% SDS polyacrylamide gel, transferred onto Hybond-C extra membranes (GE Healthcare, Milan, Italy) and blotted with the specific Abs. 5% low-fat dry milk, 5% bovine serum albumin (BSA) in phosphate-buffered saline 0.1% Tween 20 O/N were used to block non-specific binding sites. Membranes were incubated with the mouse anti-human TRPML2 (Santa Cruz Biotechnology, Dallas, TX, USA) or mouse anti-human GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA) primary Abs for 1h at room temperature followed by HRP-conjugated anti-mouse Ab for 1h at room temperature. The detection was performed using the LiteAblot PLUS (EuroClone, Milan, Italy) kits, and densitometric analysis was carried out by a Chemidoc using the Quantity One software (Bio-Rad, Hercules, CA, USA). For quantification, GAPDH was used as loading control. One representative out of three independent experiments is shown.