Wblur0500

Cells were lysed in buffer containing 50 mM Tris–HCl pH 7.5, 0.5% Triton X‐100, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF and protease inhibitor cocktail for 45 min at 4°C with rotation. Lysates were then centrifuged at 21,000 g for 15 min and the supernatant collected for inputs and subsequent immunoprecipitation. GFP‐tagged proteins were pulled down with GFP‐trap agarose beads (ChromoTek, gta‐10) for 2 h. Beads were then washed three times with the lysis buffer. Samples were run on SDS–PAGE gel and transferred onto nitrocellulose membrane. Membranes were blocked with 4% (w/v) milk in PBS with 0.1% Tween 20 (PBST). Primary antibodies were incubated overnight at 4°C, washed three times with PBST and incubated with the secondary for 45 min at room temperature. Following three washes with PBST, the membrane was developed by exposure to ECL substrate (Millipore, WBLUR0500) and imaged on the ImageQuant LAS4000 mini (GE Healthcare).

Cells were washed with cold PBS and lysed in RIPA buffer (Thermo Fisher Scientific). Protein concentrations were quantified using the BCA protein assay (Thermo Fisher Scientific). Equal amounts of protein were separated on 8‒15% gels using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked for 1 h at room temperature with 5% skim milk diluted in TBS-T. Next, the membrane was probed overnight with primary antibodies against Cyclin E (Santa Cruz #sc-481), CDK2 (Santacruz #sc-748), Cyclin D1 (Santa Cruz #sc-8396), CDK4 (Santa Cruz #sc-56277), GAPDH (Santa Cruz #sc-47724), P16^INK4a (Abcam #ab108349), P53 (Abcam #ab32132), P27 (Cell Signaling #2552), mTOR (Cell Signaling #2971), and p-mTOR (Ser2448, Cell Signaling #2532), ATG4A, ATG3) at 4 °C. The membrane was then washed with TBST and incubated with an HRP-conjugated secondary antibody for 1 h at room temperature. The protein bands were visualized using an HRP substrate (Millipore #WBLUR0500) and imaged using an Amersham Imager 600 (GE Healthcare).