Mouse anti c myc

Immunofluorescence analysis was carried out on cover slips. Cells were fixed with 4% paraformaldehyde for 20 minutes, then a permeabilisation step using 0.2% Triton X-100 in PBS was performed for 20 minutes, followed by a blocking step using 2% bovine serum albumin in PBS for 20 minutes, then stained for 60 minutes with the indicated antibodies. Primary antibodies used were: mouse anti c-MYC (Sigma, M-4439), rabbit anti-catalase, rabbit anti-Mic4 and rabbit anti-GAP45 (gifts from Dominique Soldati), mouse anti-HSP60 (gifts from Boris Striepen), rabbit anti-Tom40 (gifts from Giel Van Dooren), rat anti-DrpB, rabbit anti-Rop13 (gift from Peter Bradley). Subsequently, secondary stain was performed for 60 minutes. Secondary antibodies used were goat anti-mouse, goat anti-rabbit or goat anti-rat AlexaFluor 350, AlexaFluor 488, AlexaFluor 594 or AlexaFluor 633-conjugated antibodies (Life Technologies). These reactions were carried out at room temperature. Co-localisation analysis was performed using FIJI plugin “JACoP” to determine Manders’ coefficient, counting at least 20 images per condition [46 (link)].

Cells (HeLa and primary GCs) on coverslips were washed with PBS followed by incubation in 4% PFA at room temperature for 20–30 min. Coverslips were washed in PBS three times and permeabilised with 0.1% Trition X-100 prior to incubation in blocking solution containing 5% bovine serum albumin (Sigma-Aldrich) and 5% goat serum (Vectorlabs) for 1 h at room temperature. After blocking, coverslips were incubated with primary antibodies for either 1 h at room temperature or overnight at 4 °C. The coverslips were then washed three times in PBS and incubated with secondary antibodies for 2 h (Alexa Fluor, 1:500, Thermofisher). After incubation, coverslips were washed again three times in PBS and mounted in mounting medium with DAPI (Vector Laboratories). Primary antibodies were: mouse anti-c-Myc (1:100, Sigma-Aldrich), rabbit anti-cleaved caspase-3 (Asp175) (1:1400, Cell Signaling), rabbit anti-GABA(A) receptor alpha 6 subunit (1:100, Merck Millipore) and rabbit anti-COX IV (1:1000, Abcam).

Blasticidin hydrochloride (10 mg/ml stock solution), hygromycin B (50 mg/ml solution), Geneticin sulfate (75% [wt/vol] purity), SuperSignal West Pico Plus chemiluminescent substrate, 6×His tag monoclonal antibody horseradish peroxidase (His.H8), and mouse antihemagglutinin (anti-HA) monoclonal antibody were from Invitrogen (Thermo Fisher Scientific). l-Histidine, anti-α-tubulin monoclonal antibody, mouse anti-c-Myc, ammonia assay kit, acid-washed glass beads (425 to 600 μm), protease inhibitor cocktail (catalog no. P2815), resazurin, EDAC [1-ethyl-3-(dimethylaminopropyl)carbodiimide] HCl, and rabbit anti-FLAG were from Sigma Chemical Co., and chicken anti-GFP was from Abcam. Guinea pig anti-vacuolar proton pyrophosphatase (VP1) was produced in-house (30 (link)), rabbit anti-pyruvate phosphate dikinase (PPDK) was a gift from Frédéric Bringaud (University of Bordeaux, France), mouse anti-histidine ammonia lyase (HAL) was kindly provided by Ariel Silber (University of São Paulo, Brazil). Aminomethylenediphosphonate (AMDP) was a gift from Eric Oldfield (University of Illinois at Urbana-Champaign), polyP₁₀₀ (i.e., 100 P_i units) was kindly provided by Shiba Toshikazu (RegeneTiss Co., Japan) or was purchased from Kerafast. Polymethacrylate beads (ReliZyme EA113; particle size, S, EC-HA) carrying an ethylenediamino group were purchased from Resindion srl (Milan, Italy).