Immunoblotting

We trypsinized and collected cells and lysed in RIPA buffer (25 mM Tris, pH 7.6, 150 mM NaCl, 1% NP-40, 1% Na-deoxycholate, and 0.1% SDS) with protease and phosphatase inhibitors (Thermo Fisher Scientific). We removed insoluble material by centrifugation, and for GFP immunoblots we resuspended the pellets in equivalent volumes of SDS sample buffer and passed through a needle 10 times to solubilize. We then resolved proteins using either 4–12% (for GFP) or 12% (for LC3) polyacrylamide NuPage Bis–Tris gels. We transferred the proteins to nitrocellulose and incubated for 1 h in blocking buffer (5% BSA, 0.05% Tween20, 0.01% Triton X-100, and TBS) at room temperature. We then probed using antibodies for GFP (Invitrogen Molecular Probes A6455, 1:7500), TUBULIN (Cell Signaling 2144, 1:5000), or LC3B (Sigma-Aldrich L7543, 1:1000) at 4°C overnight, washed, and incubated with a secondary antibody (LiCor 926-32211, 1:10,000, 1 h, RT). We used a LiCor Odyssey CLx Imager to scan the blot and quantified band intensities using the LiCor Image Studio software. For GFP immunoblots we divided the GFP signal by the tubulin signal in soluble samples. For LC3 we divided LC3B-II (processed, lipidated LC3B) band intensities by the sum of the intensities for unprocessed LC3B-I and processed LC3B-II.