Anti hunk

All cells were lysed in buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 1 mM EDTA, 1% Triton X-100 with HALT protease and phosphatase inhibitor cocktail (Thermo Scientific, Waltham, MA, USA). Primary antibodies used for Western blotting include: anti-Flag-M2 (Sigma-Aldrich, St. Louis, MO, USA, #F1804), anti-HUNK (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA, PA5-28765), anti-HA (Cell Signaling, Danvers, MA, USA, #3724), anti-GFP (Santa Cruz, CA, USA, #sc-8334), anti-LC3B (Cell Signaling, #2775), anti-Rubicon (Cell Signaling, #D9F7), anti-phosphoserine (Abcam, Cambridge, UK #9332), anti-phosphoserine/threonine (Abcam, #17464), anti-HA (Cell Signaling, #C29F4), anti-GST (Santa Cruz, sc-53909), anti-Beclin-1 (Santa Cruz, sc-48381), anti-UVRAG (ThermoFisher, #23215), anti-Atg14L (Cell Signaling, #5504), anti-PI3K class III (aka Vps34, Cell Signaling, #4263), and anti-β-tubulin (Santa Cruz, sc-55529). Imaging was performed on the FluorChem-R imaging system and quantitated using Alpha View SA software (Protein Simple, San Jose, CA, USA, version 3.4.0).

All cells were lysed in buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 1 mM EDTA, 1% Triton X-100 with HALT protease and phosphatase inhibitor cocktail (Thermo Scientific). Primary antibodies used for western blotting include: anti-phospho-MBP (EMD Millipore, 05-429), anti-MBP (LifeSpan BioSciences, LS-C312288/59980), anti-flag-M2 (Sigma-Aldrich, #F1804), anti-HUNK (Invitrogen PA5-28765), anti-p27 (Santa Cruz, sc-528), anti-phospho-PKC-substrates (Cell Signaling, #2261), anti-phospho-PKC (Santa Cruz, sc-271920), anti-PKC (Santa Cruz, sc-17769), and anti-β-tubulin (Santa Cruz, sc-55529). Imaging was performed on the Protein Simple FluorChem-R imaging system.

Human breast cancer cell lines MDA-MB-468, BT474, SKBR-3 and normal breast epithelial cell line Hs578Bs were purchased from the Cell Center of Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Human breast cancer cell lines MCF-7, MDA-MB-231 and human embryonic kidney cell line HEK293T were kept in our laboratory. EBSS (Hyclone) starvation was carried out to switch the culture medium from the complete medium to EBSS medium. Commercially available antibodies and dilutions used are as follows: anti-ITM2A (Proteintech, cat: 18306–1-AP, 1:1000 dilution), anti-GAPDH (Proteintech, cat: 60004–1-Ig, 1:1000 dilution), anti-LC3 (Cell Signaling Technology, cat: 12741, 1:1000 dilution), anti-P62/SQSTM1 (Boster, cat: BM4385, 1:1000 dilution), anti-Rabbit Flag (EMD Millipore, cat: PM020A, 1:1000 dilution), anti-pAMPK-T172 (Cell Signaling Technology, cat: 2535, 1:1000 dilution), anti-AMPK (Boster, cat: A30453, 1:500 dilution), anti-p4EBP1-T37/46 (Cell Signaling Technology, cat: 2855, 1:1000 dilution), anti-4EBP1 (Proteintech, cat: 60246–1-Ig, 1:1000 dilution). anti-phospho-MBP (EMD Millipore, cat: 05–429, 1:1000 dilution), anti-MBP (Proteintech, cat: 10458–1-AP, 1:1000 dilution), anti-HUNK (Invitrogen, cat: PA5–28765, 1:1000 dilution).