Ubiquitin

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).

Total cell lysates were prepared with a detergent buffer, as described [55 (link)]. Protein concentrations were measured with the BCA Protein Assay according to the manufacturer's manual (Beyotime Institute of Biotechnology, Shanghai, China). Equal amounts (80 μg) of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA). Membranes were incubated overnight at 4°C with a 1:1000 dilution of anti-GAPDH (Sigma) and antibodies for HIF-2α (Abcam), VHL protein (Cell Signaling Technology, Beverly, MA), and ubiquitin (Novus, Littleton, CO). After additional incubation with a 1:1000 dilution of an anti-immunoglobin horseradish peroxidase-linked antibody for 1 h, the immune complexes were detected by enhanced chemiluminescence (Cell Signaling Technology). For densitometric analyses, protein bands on the blots were measured by the use of Eagle Eye II software.

The whole cell lysates, cytosol fraction lysates, or mitochondrial fraction lysates were prepared from TH1 cells and renal cortex tissue. Protein concentration was determined by employing the BCA method, and 30 μg of protein per sample was loaded for western blotting. Electrophoresis in 8–12% sodium dodecyl sulfate-polyacrylamide gel was followed by a transfer to polyvinylidene difluoride membranes. The blots were washed with TBST (10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20) and blocked with 5% skim milk for 1 h at room temperature. Subsequently, we incubated the blot with the appropriate primary antibodies: against PINK1 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-517353), SIAH3 (Novus, Littleton, CT, USA, NBP2-83524), LC3B (Novus, NB100-2220), P62/SQSTM1 (Novus, NBP1-48320), p-DRP1 (Ser637) (Cell Signaling Technology, #4867S), DRP1 (Santa Cruz Biotechnology, sc-271583), MFN1 (Santa Cruz Biotechnology, sc-166644), OPA1 (Novus, NBP1-71656), ubiquitin (Novus, NB300-130), VDAC1 (Novus, NB100-695), α-tubulin (Santa Cruz Biotechnology, sc-8035), and β-actin (Santa Cruz Biotechnology, sc-47778). Then, the membranes were washed again and incubated for detection with either goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies (Cell signaling, Danvers, MA, USA). The bands were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK).