Anti usp5

Antibodies used for this study were anti-LC3 (Cell Signaling, 4108), anti-LC3 (Novus, NB100-2220), anti-Ref(2)P (Abcam, ab178440), anti-GABARAP (Abcam, ab109364), anti-pULK1-Ser555 (Cell signaling, 5869), anti-ULK1 (Cell signaling, 8054), anti-USP5 (Proteintech, 10473-1-AP), anti-p62 (MBL, PM045), anti-Flag M2 (Sigma, F1804), anti-MYC (Santa Cruz, SC-40), anti-HA (Sigma, H9658), anti-ACTB/β-actin (Novus, NB600-501), anti-tubulin (Sigma, T0198), anti-GFP (Abcam, ab290), anti-ubiquitin (FK2; Sigma, ST1200), and anti-ubiquitin (P4D1; Cell signaling, 3936). To generate antiserum against Drosophila Atg1, the C-terminal segment of Atg1 (amino acid residues 562-855) was cloned to pET32a vector to generate 6xHis-tagged fusion protein. The fusion protein was purified using a Ni Sephsrose 6 FF column (GE Healthcare). The purified protein was used to immunize rabbits by LTK BioLaboratories and the resulting antiserum was purified by NAb Protein A Plus Spin Kit (Thermo).

The growth medium of adherent cells in a 10 cm cell culture dish was removed by aspiration and washed once with PBS. The cells were then lysed with 1 mL IP buffer, mixed with 2 μg anti‐HuR (Proteintech, 11910‐1‐AP) and incubated with rotation overnight at 4°C. Protein A/G Agarose was fully resuspended in 20 μL buffer A and shaken slowly. IP complex was washed in IP buffer five times and 20 μL of 1 × SDS‐PAGE loading buffer was added and mixed. The pellet was vortexed and then centrifuged to concentrate the sample at the bottom of the tube. Western blot analysis was performed using antiubiquitin (Proteintech, 10201‐2‐AP) and anti‐USP5 (Proteintech, 10473‐1‐AP).