Anti bnip3

Western blotting was performed as previously described.12, 15 Dilutions and manufacturers of the antibodies were as follows: anti‐α‐fodrin (1:1000; ENZO, New York, NY, USA), anti‐Bnip3 (1:1000; Cell Signalling Technology, Danvers, MA, USA), anti‐β‐actin (1:5000; Sigma‐Aldrich, Saint Louis, MO, USA), anti‐mouse IgG (H + L) HRP (1:2500; Promega, Madison, WI, USA), and anti‐Rabbit IgG (H + L) HRP (1:2500; Promega). The signals were detected and quantified using ECL Prime Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, England) and quantified using ImageQuant™ LAS 4000 biomolecular imager (GE Healthcare).

Protein extraction and immunoblot analysis were performed using a modified Laemmli sample buffer (125 mM Tris-HCl, pH 6.8 buffer containing 2% SDS and 20% glycerol) in the presence of protease and phosphatase inhibitors (Roche, Vilvoorde, Belgium). Lysates were separated by SDS-PAGE under reducing conditions, transferred to a nitrocellulose or PVDF membrane, and analyzed by immunoblotting. Primary antibodies used were anti-LC3 and anti-BNIP3 from Cell Signaling (Leiden, The Netherlands), anti-VE-cadherin from R&D Systems (Abingdon, UK), anti-phospho-FAK from BD-Transduction Laboratories (Ermebodegem, Belgium), anti-ZO-1 from Invitrogen, anti-Vinculin and anti-Integrin from Millipore (Merck, Overijse, Belgium), anti-Actin, anti-Vinculin and anti-Vimentin from Sigma (Diegem, Belgium). Equal loading was verified by actin immunostaining. Appropriate secondary antibodies were from Thermo Scientific (Erembodegem, Belgium). The LICOR Odyssey System (Westburg, Leusden, The Netherlands) was used for western blot detection according to the manufacturer's instructions. Quantifications were performed using the Odyssey System software. Representative blots of at least three independent experiments are shown.

Fifty mg skeletal muscle was divided and added into RIPA buffer, phosphatase inhibitors, and protease. Proteins were extracted and quantified to 5 μg/ml with BCA protein quantitation Kit, then were separated by 7.5% and 12.5% SDS-PAGE, electrophoretically transferred to a polyvinylidene difluoride membrane and blocked by BSA. After incubation overnight at 4°C with anti-LC3A/B, anti-Bnip3 (1:1,000, Cell Signaling Technology, Danvers, MA) and anti-PINK1 (1:1,000; Abcam, Cambridge, UK), membranes were washed by TBST and incubated with anti-mouse or anti-rabbit Ig. Immunoreactive bands were visualized by enhanced chemiluminescence solution (Thermo Scientific, Waltham, MA). Band quantitative analysis was performed using ImageJ software ver. 1.53t.