Anti tom40

Extracts for immunoblotting were acquired from N2a cells homogenized in RIPA buffer (radioimmunoprecipitation assay buffer, Thermo Fisher Scientific, Catalog Numbers 89900). Briefly, proteins were separated by electrophoresis and subsequently transferred to the PVDF membrane by electroblotting using standard protocols. The primary antibodies used were anti-LC3II (Cell Signaling Technology, catalog #2775s), anti-COX IV (Proteintech, catalog no. 11242–1-AP), anti-TOM40 (Proteintech, catalog no. 18409–1-AP), anti-MnSOD (Proteintech, catalog no. 24127–1-AP), anti-CLS (Proteintech, catalog no.14845–1-AP), anti-PLS3 (Proteintech, catalog no.28028–1-AP), anti-NDPK-D (Bioss, bs-11902R), anti-PINK1 (Novus Biologicals, catalog no. BC100–494), anti-Parkin (Abcam, catalog no. ab77924), anti-Drp1 (Cell Signaling Technology, catalog #8570), anti-Lamp2 (Cell Signaling Technology, catalog #49067), PGC1a monoclonal antibody (Proteintech, catalog no. 66369–1-Ig), recombinant anti-mtTFA antibody (Abcam, ab252432), GAPDH monoclonal antibody (Proteintech, catalog no. 60004–1-Ig), and anti-Tubulin (Proteintech, catalog no. 11224–1-AP).

Anti-β-Catenin (catalog no. 610153) and anti-CD133 antibody (catalog no. 566598) was purchased from BD Bioscience. Anti-PGK1 (catalog no. 68540 S), anti-EEA1 (catalog no. 3288 S), anti-β-actin (catalog no. 4970), anti-H3 (catalog no. 4499) and anti-H3K4me3 (catalog no. 9751 S) antibodies were from Cell Signaling Technology. Anti-ZIC2 (catalog no. ARP35821_P050) antibody was purchased from Aviva Systems Biology. Anti-TOM40 (catalog no. 18409-1-AP), anti-TOM70 (catalog no. 14528-1-AP), anti-c-MYC (catalog no. 10828-1-AP) and anti-AXIN2 (catalog no. 20540-1-AP) antibodies were from Proteintech Group, Inc. Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594 antibody (catalog no. A-11005), Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (catalog no. A-11008), Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647 (catalog no. A-21244) were purchased from Invitrogen. HRP-conjugated Affinipure Goat Anti-Mouse IgG(H + L) antibody (catalog no. SA00001-2) and HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) antibody (catalog no. SA00001-2) were purchased from Proteintech Group, Inc. Polymer HRP and AP detection kits were from Beyotime Biotechnology. Biotin labeled RNA mix (catalog no. 11685597910) was from Roche.

RPMI-8226 and OPM2 cells were stably transduced with lentiviral LentiCRISPR v.2 expressing VAS9 gene and gRNAs targeting GCN2 or control lentivirus for 24 h at 37 °C in antibiotic-free IMDM with polybrene. Lentivirus media was removed and replaced with fresh IMDM media including antibiotic (1% penicillin-streptomycin) and incubated for 48 h. Stably transduced cells were then selected using 2 μM puromycin for 3 days and GCN2 knockouts validated by western blotting using anti-GCN2 (Cell Signalling Technologies, cat.3302, 1:1000 dilution), anti-TOM40 (Proteintech, cat.18409, 1:1000 dilution) and anti-β-Tubulin (Cell Signalling Technologies, cat.2146, 1:1000 dilution). HRP-conjugated anti-rabbit was used as a secondary antibody (Cytiva, cat.NA934, 1:1000 dilution). GCN2 knockout and control vector HMCLs were cultured in IMDM media without L-glutamine and after 5 days, viability was analyzed using trypan blue (0.4% Trypan Blue Solution, Gibco, Grand Island, NY, USA). Two independent constructs were used for CRISPR knockout experiments and viability measurements were obtained in duplicate.