Mouse anti gadph

For Western blot analysis cells were lysed in RIPA buffer and different amounts of total proteins were separated by electrophoresis through SDS-PAGE gels and blotted into nitrocellulose membranes. Mouse anti-RTN-1C (Abcam, ab8961) was diluted 1:1000 rabbit anti-LC3 (Cell Signaling, 3868 S) was diluted 1:1000, rabbit anti-syntaxin 17 (Abcam, ab116113) was diluted 1:200, rabbit anti-HA (Sigma, H6908) was diluted 1:2000. Rabbit anti-actin (Sigma, A2066) diluted 1:2000, mouse anti-GADPH diluted 1:1000 (Santa Cruz, sc-47724) and mouse anti-tubulin 1:2000 (Sigma, T-4026) were used as loading controls. Secondary antibodies (Biorad, 1721011, 1706515) were diluted 1:5000.

AA was isolated and identified as previously described (Figure 1A) [25 (link)]. AA was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution, which was further diluted in media to the desired working concentration. DMSO was used at ≤0.1% concentration, which was non-toxic to all cells tested.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Invitrogen (Carlsbad, CA, USA). Hoechst 33342 and compound C (CC, Dorsomorphin) were obtained from Sigma Chemical (St. Louis, MO, USA). Annexin-V-FITC and propidium iodide (PI) were purchased from Immuno Tools (Friesoythe, Germany). MG132 was obtained from Merck Millipore (Billerica, MA, USA). Primary and secondary antibodies included: rabbit anti-Bcl-2, rabbit anti-poly-ADP-ribose polymerase (PARP), rabbit anti-caspase 3, rabbit anti-AMPK, rabbit anti-mTOR, rabbit anti-Bax, mouse anti-tubulin, anti-rabbit and anti-mouse IgG HRP-linked (Cell Signaling Technology, Beverly, MA, USA). Mouse anti-GADPH was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

EV and cell extracts were lysed with radio immunoprecipitation assay (RIPA) buffer supplemented with Complete Protease Inhibitor Mixture tablets (Roche Diagnostics). Protein concentration was determined by the Pierce™ BCA Protein Assay Kit (Thermo Scientific). 10 μg proteins were separated by SDS/PAGE and transferred to the PVDF membrane (Bio-Rad). Membranes were incubated with primary antibodies against the following proteins overnight at 4°C: mouse anti-Tsg101 (sc-365062, Santa Cruz), mouse anti-calnexin (sc-23954, Santa Cruz), goat-anti-Hsp70 (EXOAB-Hsp70A-1, SBI), rabbit anti-CD9 (#13174, CST), rabbit anti-flotillin-1 (#18634, CST), and mouse anti-GADPH (sc-365062, Santa Cruz). The membrane was then washed and incubated with an anti-mouse/rabbit/goat peroxidase-conjugated secondary antibody. Immunoreactive bands were visualized by the enhanced chemiluminescence method (Pierce, Thermo Scientific) with a western blotting detection system (FluorChem E, ProteinSimple USA) and were quantified by densitometry with ImageJ software.