Lysosome Inhibition and Protein Interaction

The lysosome inhibitors NH₄Cl and leupeptin (Fisher Scientific) were used together at 20 mM and 200 µM respectively for 4 h. The lysosomal inhibitor concanamycin A (Sigma–Aldrich) was used at 1 µM for 30 min. Primary antibodies used for immunoprecipitation and Western blotting were as follows: anti-NRBF2 (Cell Signaling Technology), anti-Vps34 for immunoprecipitation [24 (link)], anti-Vps34 for Western blotting [25 (link)], anti-Vps15 [18 (link)], anti-Beclin-1 (BD Biosciences), anti- Atg14L (MBL International), anti-UVRAG (Cell Signaling Technology), anti-LC3 (Cell Signaling Technology), anti-p62 (MBL International), anti-V5 for immunoprecipitation (Thermo Scientific); anti-V5 for Western blotting (Life Technologies), anti-FLAG (Sigma–Aldrich); anti-actin (Sigma–Aldrich), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase; MBL International) and anti-(rabbit IgG) (Jackson ImmunoResearch). Anti-HA (haemagglutinin) and anti-Myc antibodies were produced in-house.