Rabbit anti hif 2α nb100 122

Cells were seeded at 1 × 10⁵ cells in 35‐mm dishes, pre‐incubated for 12 h in normoxia, and further cultured in hypoxia (1% O₂) for the indicated time. Cell lysates were collected after addition of 200 μL of 2× sample buffer (125 mM Tris‐HCl [pH 6.8], 2% SDS, 20% glycerol, 10% β‐mercaptoethanol, and 0.01% bromophenol blue). The samples were electrophoresed on a 10% SDS‐polyacrylamide gel and separated proteins were transferred to a PVDF membrane filter (Merck, Darmstadt, Germany). Actin, HIF‐1α, and HIF‐2α on the filter were probed with the following antibodies: mouse anti‐actin (A4700; Sigma‐Aldrich), rabbit anti‐HIF‐1α (NB100‐134) and rabbit anti‐HIF‐2α (NB100‐122) (Novus Biologicals, Littleton, CO, USA), anti‐mouse IgG conjugated with HRP (#7076) and anti‐rabbit IgG conjugated with HRP (#7074) (Cell Signaling Technology, Danvers, MA, USA). Binding was detected with Chemi‐Lumi One Ultra, a chemiluminescence detection reagent (Nacalai Tesque).