Pik3c3

Stock solutions of Bafilomycin A1 (LC Laboratories, B-1080), MG132 (Calbiochem, 474790), Cycloheximide (Sigma, 01810) and Rapamycin (Enzo, BML-A275-0025) were prepared in DMSO (Roth, A994.2). Stock solution of Canavanine (Santa Cruz Biotech, sc-202983A) and Puromycin (Sigma, P8833) was prepared in distilled H2O.
Antibody sources were as follows: for Actin (Sigma, A5060), BAG1 (Abcam, ab7976), BAG3 (Proteintech Group, 10599-1-AP), BECN1 (Cell Signaling, 3495), CTSD (Abcam, ab75852), DLP1 (BD Transduction Laboratories, 611113), LAMP2 (DSHB Biology, ABL-93), LC3B (Nanotools, 0260-100), LC3B (Sigma, L7543), OPA1 (BD Transduction Laboratories, 612607), Phospho mTOR (Abcam, ab109268), Puromycin (Millipore, MABE343), mTOR (Calbiochem, OP97), p62 (Progen, GP62-C), PIK3C3 (Cell Signaling, 4263), Poly-Ubiquitin (Dako, Z0458), RAB18 (Sigma, SAB4200173), Tubulin (Millipore, MAB1637), Tubulin (Sigma, T9026), TFEB (Proteintech Group, 13372-1-AP), Vimentin (SCBT, sc-373717), WIPI1 (Sigma, HPA007493).

Whole-cell protein extractions was acquired using RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific, Cat# 89901), supplemented with protease inhibitor (Thermo Fisher Scientific, Cat#78430). The concentration of protein was quantified using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Cat# J63283.QA) before mixed with protein loading buffer (3×). Then the protein samples were separated by 10% SDS-PAGE, and the detailed procedures conducted as previously described[20 (link)].
Antibodies including α-Tubulin, GAPDH, Cyclin D1, P21, PCNA, E2F1, CDK2, CDK4, CDK6, RB1, JAG1, JAG2, Caspase 8, BAX, XIAP, BCL-2L12, ATR, ATM, Rad50, Mre11, PIK3C3, LC3A/B, and ULK1 were purchased from Cell Signaling Technology (Danvers, MA, United States) and Santa Cruz Biotechnology (Dallas, TX, United States).

Flag-tagged mouse WT BECN1 and hemagglutinin-tagged mouse ZFP91 were cloned into the pcDNA3 vector. His-tagged K48, K63, and K63R mutant ubiquitin were cloned into the pcDNA3 vector. Antibodies for GAPDH, lamin B, p-S6K, p-S6, p-AKT (S473), c-Myc, BECN1, PIK3R4, PIK3C3, and K63 ubiquitin were purchased from Cell Signaling Technology, Inc. HRP-conjugated anti-hemagglutinin antibody (3F10) was from Roche, Inc. Anti-Flag (M2) antibody and anti–β-actin antibody were from Sigma-Aldrich, Inc. Ni–nitrilotriacetic acid agarose was from QIAGEN, Inc. Anti-ZFP91 antibody was from Bethyl Laboratories, Inc. Anti-LC3B antibody was purchased from Abcam, Inc. Anti-p62 antibody was from Proteintech Group, Inc. Antibodies for ubiquitin (P4D1) and NIK (A-12) were from Santa Cruz Biotechnology, Inc. The fluorochrome-conjugated antibodies for CD4 (GK1.5), CD8 (53-6.7), CD44 (IM7), CD62L (MEL-14), Foxp3 (FJK-16s), IFN-γ (XMG1.2), IL-17 (eBio17B7), CD25 (PC61.5), CTLA-4 (UC10-4B9), and PD-1 (J43) were purchased from Thermo Fisher Scientific, Inc. c-Myc (D84C12) rabbit mAb (PE conjugate) and phospho-S6 ribosomal protein (Ser235/236; D57.2.2E) XP Rabbit mAb (allophycocyanin conjugate) were purchased from Cell Signaling Technology, Inc. Rapamycin (R0395), chloroquine diphosphate salt (C6628), leptomycin B solution (L2913), and DCA (D54702) were purchased from Sigma-Aldrich, Inc.