Vps34

Lentiviral constructs expressing shRNAs for caspases 1, 3, 6, 7, 9, CAD, EndoG, calpastatin, calpain S1, PAD2, PAD4, Atg5, Vps34 and Beclin1 were purchased from Sigma-Aldrich. cDNAs of caspase 3, CAD, EndoG, calpain 1 and calpain 2 were purchased from OriGene (Rockville, MD, USA), calpastatin cDNA was from Shi-Yong Sun (Emory University School of Medicine), being subcloned into pCDH-EF2-MCS-T2A-Puro, a lentiviral vector for cDNA expression (System Biosciences, Mountain View, CA, USA). Mutation of calpastatin at cleavage sites of caspases was accomplished using a QuickChange site-directed mutagenesis kit (Stratagene, Santa Clara, CA, USA). All the lentiviral vectors were transfected to 293TN cells (System Biosciences) with Lipofectamine 2000 transfection reagent (Invitrogen, Seoul, Korea). Particles were collected 2 days after the transfection of lentiviral plasmids, and infected into the cells. Lentivirus-infected cells were puromycin-selected for 1 week.

Protein was extracted from cells and tissues with RIPA lysis buffer (50 mM Tris-HCl(pH 7.4), 150 mM NaCl, 1% NP-40, 0.1% SDS). Protein concentrations were measured by BCA method (Beyotime, China). Antibodies against FOXK2 (#12008, 1:1,000 dilution), LC3A/B (#4108, 1:1,000 dilution), ULK1 (#8054, 1:1,000 dilution), FOXO3a (#2497, 1:1,000 dilution) were from Cell Signaling Technology. Antibodies against p62/SQSTM1 (P0067, 1:2,000 dilution) and VPS34 (V9764, 1:1,000 dilution) were from Sigma. Tubulin antibody (SG4110-16, 1:1000 dilution) was from Shanghai Genomics Technology.