Uvrag

The lentivirus knockdown plasmids contain shRNA targeting mouse beclin 1 at nucleotides 405–423 [5 (link)] or a scrambled control shRNA in the pSIH-H1 vector from SBI System Biosciences. This plasmid also contains a copepod GFP (copGFP) to monitor infection efficiency. All lentiviruses were generated by the Stanford Neuroscience Gene Vector and Virus Core. Plasmids for VPS34, UVRAG, ATG7, and Atg14 shRNA were obtained from Santa Cruz Biotechnology Inc. Plasmids were provided as pools of three 19–25 bp target shRNA. The Rab5-GFP and Rab7-GFP plasmids were generously provided by Dr. Craig Garner.

Cells were lysed in buffer (10 mM HEPES pH 7.4, 50 mM NaPyrophosphate, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 100 µM Na₃VO₄, 0.1% Triton X-100) and Western blotting was conducted using the following primary antibodies: LC3B (Novus Biologicals, NB100-2220), β-actin (ACTB, Sigma-Aldrich, A5316), ATG5 (Santa Cruz, sc-33210), FIP200 (Cell Signaling Technology, D10D11), BECN1 (Santa Cruz, sc-48341), PIK3C3 (Santa Cruz, sc365404), WIPI2 (Cell Signaling Technology, #8567), ATG13 (Cell Signaling Technology, D4P1K), ATG9A (Cell Signaling Technology, D4O9D), ULK1 (Santa Cruz, sc-390904), ULK2 (GeneTex, GTX111476), UVRAG (Santa Cruz, sc-293268), ATG14 (Cell Signaling Technology, #5504), ATG16L1 (Santa Cruz, sc-393274), cleaved-caspase 3 (Cell Signaling Technology, #9661), viral capsid protein 1 (VP1, Dako, M706401-1), FLAG (Sigma, F1804), PI4KIIIβ (Sigma 06578), and PIKfyve (Santa Cruz, sc-100408). Blots were cropped to improve clarity and conciseness. Uncropped blots are provided in Supplemental Figure 2.