Mouse anti human hif 1α antibody

Whole cell lysates were prepared using lysis buffer containing mammalian protein extraction reagent (Thermo Fisher Scientific Inc, Rockford, IL, USA) and a protease inhibitor cocktail (Calbiochem, La Jolla, CA, USA). Samples were centrifuged at 10 000 × g to pellet the cell debris. The protein concentrations were quantified using Bio‐Rad protein assay reagent (Bio‐Rad Lab., Hercules, CA, USA). Equivalent amounts of lysate protein (20 μg/lane) were electrophoresed on a 7.5% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and electro‐transferred onto membranes using the Trans‐Blot Turbo^™ Transfer System (Bio‐Rad, Laboratories, Inc). Non‐specific‐binding sites were blocked with Blocking One solution (Nacalai tesque, Kyoto, Japan) for 20 minutes. The membranes were probed with purified mouse anti‐human HIF‐1α antibody (1:1000; BD Transduction Laboratories, Tokyo, Japan) and mouse monoclonal β‐actin antibody (1:5000; Sigma‐Aldrich) as the primary antibody, and anti‐mouse IgG peroxidase‐labeled secondary antibody (1:5000; GE Healthcare Life Science) as the secondary antibody. Immune complexes were visualized using enhanced chemiluminescence plus Western blotting detection reagents (GE, Little chalfont, UK). Experiments were performed in triplicate, and representative blots are shown.

Primary antibodies: We have used undiluted hybridoma medium of mouse monoclonal antibody M87, specific for nucleoprotein of LCMV strain MX [15 (link), 77 (link)], undiluted hybridoma medium of mouse monoclonal antibody M75 (specific for the PG domain of the CA IX protein) [77 (link)] and VII/20 antibody (internalizing monoclonal antibody, specific for CA domain of the CA IX protein) [51 (link)]. Purified mouse anti-human HIF-1α antibody diluted 1:500 (BD Transduction, San Jose, CA, USA); rabbit anti HIF-2α antibody diluted 1:500 (Novus Biologicals, Littleton, CO, USA); mouse anti-enolase antibody in concentration 2 μg/ml (Abcam, Cambridge, UK); rabbit anti-lactate dehydrogenase-A (LDH-A) antibody diluted 1:1000 (Abcam), rabbit anti-β-catenin antibody diluted 1:500 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-E-cadherin antibody diluted 1:500 (Santa Cruz Biotechnology) and mouse anti β-actin diluted 1:5000 (Cell Signalling Technology, Danvers, MA, USA) were also used.
Secondary antibodies: polyclonal goat anti-mouse immunoglobulins conjugated with horseradish peroxidase, diluted 1:5000 (Dako, Glostrup, Denmark) and polyclonal swine anti-rabbit immunoglobulins conjugated with horseradish peroxidase, diluted 1:5000 (Dako). Donkey anti-mouse IgG (H + L) secondary antibody conjugated with Alexa Fluor 488 (Termo Fisher Scientific, Paisley, Scotland).