Anti phosphoserine threonine

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).

Western blot analysis (WB) was carried out essentially as previously described in (63 (link)). The detection and quantification were performed with Odyssey Clx System (LI-COR Biosciences) through the Image Studio Software, or by traditional ECL detection. The following antibodies were used: anti-ZEB1-1642 (from Dr. Douglas Darling Lab), anti-ZEB1 (Santa Cruz,# sc-25388), anti-E-cadherin (BD Biosciences,# 610182), anti-vimentin (Cell Signaling, #5741), anti-SNAIL (Cell Signaling, #3879), anti β-catenin (Cell Signaling, #8480), anti ZO-1 (Cell Signaling, #8193), anti-cytokeratin 18 (Cell Signaling, #4548), anti-phospho serine/threonine (Abcam, #ab9337), anti-phospho-substrates antibodies kit Cell Signaling, #9920) anti-GFP (Abcam, #ab290), anti-PKCα (Santa Cruz, # sc-208), anti-PKCδ (Cell Signaling, #2058) anti-PKCε (Santa Cruz,# sc-1681), anti- Phospho-PKCα/β II (Cell Signaling, #9375) anti-α-tubulin (Sigma-Aldrich, #T5168); anti-β-actin (Sigma-Aldrich, #A2228); anti-PCNA (Santa Cruz, #sc56). As secondary antibodies we used anti-rabbit-Alexa-Fluor 594 (Molecular Probes), anti-rabbit HRP (Cell Signaling, #7074), anti-mouse HRP (Cell Signaling, #7076), goat anti-mouse IRDye 680RD and goat anti-rabbit IRDye 800CW (LI-COR Biosciences).