Western blot analyses were carried out as described previously [23] (link). In brief, lysates from NIH 3T3-scr, NIH 3T3-MECR-KD, HepG2-scr, HepG2-MnSOD-KD, Mpv17+/+ and Mpv17-/- cells were collected and 100 µg of total protein was loaded onto a 7.5% or 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel. After electrophoresis and electroblotting onto a nitrocellulose membrane, proteins were detected with primary antibodies against human HIF-1α (1:2000; BD Bioscience), mouse HIF-1α (1:1000; Novus Biologicals), MnSOD (SOD2) (1:1000; Calbiochem), MECR (1:1000; Proteintech), and against α-tubulin (1:10.000; Sigma). The secondary antibody was either an anti-mouse, an anti-rabbit or an anti-sheep immunoglobulin G conjugated to horseradish peroxidase (1:5000; Bio-Rad Laboratories). The ECL system (Amersham) was used for detection.
For half-life studies, NIH 3T3-scr, NIH 3T3-MECR-KD, HepG2-scr, HepG2-MnSOD-KD, Mpv17+/+ and Mpv17-/- cells were cultured under normoxic or hypoxic conditions. After 20 h, cycloheximide (10 µg/ml; Sigma) was added to the cell culture medium, cells were scraped in lysis buffer (50 mM Tris/HCL, pH 7.5, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 2 mM EGTA, 1 mM PMSF and complete protease inhibitor cocktail tablet (Roche)) at indicated time points and protein levels were measured by immunoblot analysis.
For half-life studies, NIH 3T3-scr, NIH 3T3-MECR-KD, HepG2-scr, HepG2-MnSOD-KD, Mpv17+/+ and Mpv17-/- cells were cultured under normoxic or hypoxic conditions. After 20 h, cycloheximide (10 µg/ml; Sigma) was added to the cell culture medium, cells were scraped in lysis buffer (50 mM Tris/HCL, pH 7.5, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 2 mM EGTA, 1 mM PMSF and complete protease inhibitor cocktail tablet (Roche)) at indicated time points and protein levels were measured by immunoblot analysis.