Immunoblots and immunoprecipitation

Cells were lysed in lysis buffer (25 mM Sodium Phosphate (pH 7.2), 150 mM NaCl, 10% Glycerol, 1 mM EDTA, 1% TritonX-100) and Complete protease inhibitor mixture (Sigma, 4693116001). Samples were electrophoresed on 4-12% gradient SDS-PAGE gels after reducing with dithiothreitol-based reducing agents (Thermo Scientific Inc., NP0009). Western blotting was performed with an iBlot Gel Transfer device and nitrocellulose membrane (Thermo Scientific Inc., IB301002). Chicken polyclonal anti-GFP antibody (Aves, GFP-1020) and mouse monoclonal anti-HA (MBL, M180-3) were used for probing. Bands were detected and quantified using an Odyssey Infrared Imaging System (LI-COR). In some experiments, band intensities of PC1 fragments were normalized to PC1 full-length band intensity.

Mitochondrial isolation was performed (n = 7 independent experiments) using Qproteome Mitochondrial Isolation Kit (QIAGEN, 37612) according to manufacturer’s manual. Sub-fractional enrichment was confirmed by probing with anti-Timm23 (BD bioscience, 611222) and anti-Heterochromatin protein-1β (Millipore, MAB3448) antibody.

For immunoprecipitation, cell lysates from MDCK and MEF were incubated with mouse monoclonal anti-HA antibody (MBL, M180-3) and Protein G agarose (Sigma, 11719416001). MDCK cells with tetracycline-inducible stable PC1 expression (with GFP tag on its N-terminus and HA tag on its C-terminus) and control were induced with 10 μg/ml tetracycline for 48 h prior to studies. Cells were exposed to: 1) hypoxia (0.01%O₂, 5%CO₂) for 2 hours (n = 3 experiments); 2) treatment of 1 μM CCCP (Carbonyl cyanide 3-chorophenylhydrazone; Sigma, Aldrich, St. Louis MO) for 20 min. followed by 16 hour incubation in standard medium (n = 3 experiments); 3) starved for 48 h in serum-free DMEM (n = 1); 4) 24 h treatment with “inhibitors mix” (n = 2 experiments): protease inhibitor cocktail (1:200 P1860; Sigma), calpain inhibitor (26 μM ALLN, SantaCruz, sc-221236), matrix metalloproteinase inhibitor (6 pg/μl Batimastat; ApexBio, A2577); 5) 3 h treatment with proteasome inhibitor (20 μM MG132, C2211, Sigma; n = 2 experiments); 6) 24 h γ-secretase inhibitor (50 μM DAPT, D5942, Sigma; n = 3 experiments). Band intensity was measured using Fiji⁷¹, and normalized to PC1-FL band intensity. Rabbit polyclonal anti HIF-1 alpha Antibody (Novus Biologicals, NB100-479) was used to confirm Hif1α induction in the total lysate.