Atg16

SH-SY5Y (CRL-2266) and SK-N-AS (CRL-2137) human neuroblastoma cell lines were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). Cells cultured in DMEM (PAN-Biotech GmbH, Aidenbach, Germany) medium supplemented with 10% fetal bovine serum (PAN-Biotech GmbH) and 1% penicillin/streptomycin (GIBCO, Invitrogen) in a humidified atmosphere containing 5% CO₂ at 37 °C incubators (Hera Cell 150i, Thermo Lab systems, Beverly, MA, USA). Forced GH was expressing SH-SY5Y cells generated according to our previous work (Coker-Gurkan et al., 2018 (link)). Mycoplasma testing has been done for the cell lines used by PlasmoTest Reagent Kit (InvivoGen, Toulouse, France). Rotenone (Sigma, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO; 10 mM). The following antibodies; BIP, CHOP, PERK, p-PERK (Thr 980), Ire1α, p-Ire1α (Ser724), ATF6, XBP-1, eIF2α, SAPK/JNK, Beclin-1, Atg12, Atg 5, Atg 16, LC3, p62, PARP, caspase-7, Bim, Bax, Puma, Bcl-2, and polyclonal antirabbit/mouse antibodies were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). Each primary antibody was diluted to 1:1000 and HRP-conjugated secondary antibodies were diluted to 1:3000 with in superblock T20 reagent from Thermo Scientific (Beverly, MA, USA).

Cells and tissues were lysed in Lysis buffer (ThermoFisher: 87787) containing protease and phosphatase inhibitors (ThermoFisher: 1861281). Protein concentration was determined with a Bradford reagent (Bio-Rad). Fifty µg proteins from each sample were run on SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked in 5% milk in PBS-T (0.1%Tween 20) for 30 mins followed by overnight incubation in cold in antibodies against Actin (Cell Signaling: 4970), p62 (Cell Signaling: 5114) or LC3 (Cell Signaling: 3868), Atg16 (Cell Signaling: 8089), p65 and phospho-p65 (Cell Signaling: 4764 and 3033, respectively). Antibodies were diluted 1:1000 in 5% BSA in PBS-T as recommended by the manufacturer. Blots were incubated with secondary antibodies (Cell Signaling: 7074) at room temperature for 1 hour (dilution of 1:2000) and developed using enhanced Chemiluminiscence signal (ThermoFisher: 32106).