Tim23

NRVM were washed twice with PBS and lysed for 5 min with RIPA buffer containing complete EDTA-free protease inhibitor cocktail tablets (Roche) and PhosSTOP phosphatase inhibitor cocktail (Roche) when required. Pellets were clarified by centrifugation at 10000 rpm for 10 min at 4°C. Protein concentration was determined using the BCA assay (Thermo Scientific, MA, USA) or Bradford assay (Sigma). 30 µg of protein were separated by SDS polyacrylamide gel electrophoresis and subsequently transferred to nitrocellulose membranes (Millipore). Primary antibodies used for detection were: PDE2A (Abcam ab125677), DRP1 (Cell Signalling, 8570), phosphoDRP1 (ser637; Cell Signalling 6319), phosphoDRP1 (ser616; Cell Signalling 3455), GAPDH (Santa Cruz, sc-20357), TOM20 FL-145 (Santa Cruz, sc-11415), Total OXPHOS rodent (Abcam, ab-110413), Mitofilin (Abcam, ab110329)., Tim23 (Santa Cruz, sc-514463), Cytochrome C (Abcam, ab1100325), alpha tubulin (Abcam, ab18251), GFP (Santa Cruz, sc-9996).

The mitochondrial membrane protein Tim23 (Santa Cruz Biotechnology) was examined by immunostaining of histological sections from mouse lung and tracheal tissues and in vitro colonies.

Commercially available antibodies to HRP-conjugated p53, cyclin D1, CDK2, TIM23, LC3, p62, phospho-p53 (Ser392 and Ser46), p19-ARF, cMyc, Caspase 8, DRAM1, and LAMP2 were purchased from Santa Cruz Biotechnology, CA. Phospho-MDM2 (Ser166 and Ser186), total MDM2, cleaved PARP, total PARP, pro-caspase 9, Caspase 3, phospho-Drp1 (Ser616), Beclin1, phospho-Ulk1 (Ser757), phospho-AMPKα (Thr172), phospho-Beclin1 (Ser15), and phospho-mTOR (Ser2481) were procured from Cell Signaling Technology, MA. HRP-conjugated antibody to actin, Mdivi1, Z-VAD-fmk, chloroquine, and FH535 were purchased from Sigma-Aldrich, MO. LAPTM4B was purchased from Novus Biologicals, CO. Commercially available AZD5363, rapamycin, and mTOR inhibitor (AZD8055) were procured from Cayman Chemicals, MI.

Cells were plated on glass slides in a 6-well plate at a density of 1 × 106 cells per well. Subsequently, cells were fixed in ice-cold 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100, and blocked with 2% gelatine in PBS at room temperature [38 (link)]. The cells were then incubated with the primary antibodies: LAMP1 (1:1000; Abcam; #ab24170), Tim23 (1:1000, Santa Cruz Biotechnology, #sc-13298), cyt-c (1:1000; Abcam; #ab90529), Parkin (1:1000; Abcam; #ab77924), p-CaMKII (1:1000, Cell Signaling Technology, #12716) overnight at 4 °C. After being washed with PBS, the cells were incubated with secondary antibody and DAPI (1:1000 dilution in PBS) for 1 h at room temperature. Images were obtained using a fluorescence microscope [39 (link)].

Antibodies were obtained as follows: α‐tubulin (Cat No. AF5012; Beyotime), TIM23 (Cat No. sc‐514463; Santa Cruz Biotechnology), GFP‐Trap^® Agarose (Cat No. gta; ChromoTek), anti‐FLAG^® M2 affinity gel (Cat No. A2220; Sigma–Aldrich), m⁶A (Cat. No. 56593; CST), TOM20 (Cat No. 42406; CST) and COXIV (Cat No. 11967; CST).
All of these antibodies were purchased from Proteintech: glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (Cat No. 60004‐1‐Ig), FLAG (Cat No. 20543‐1‐AP), FSCN1 (Cat No. 14384‐1‐AP), GFP (Cat No. 50430‐2‐AP), IGF2BP1 (Cat No. 22803‐1‐AP), IGF2BP2 (Cat No. 11601‐1‐AP), IGF2BP3 (Cat No. 14642‐1‐AP), Ki67 (Cat No. 27309‐1‐AP), MYC (Cat No. 10828‐1‐AP), MARCKSL1 (Cat No. 10004‐2‐Ig), LC3 (Cat No. 14600‐1‐AP), Parkin (Cat No. 14060‐1‐AP), TK1 (Cat No. 15691‐1‐AP), ubiquitin (Cat No. 10201‐2‐AP), VDAC1 (Cat No. 10866‐1‐AP).
Recombinant proteins were purchased from Novus: ubiquitin‐activating Enzyme/UBE1 (Cat No. E‐305), UbcH7/UBE2L3 (Cat No. E2‐640), ubiquitin (Cat No. U‐100H), Parkin (Novus, E3‐160). Recombinant IGF2BP3 protein was purchased from Abnova (Cat No. H00010643‐P01).
All chemicals were purchased from MedChemExpress: actinomycin D (Cat No. HY‐17559), CCCP (Cat No. HY‐100941), MG132 (Cat No. HY‐13259), CHX (Cat No. HY‐12320), SAHA (Cat No. HY‐10221).