Flag tag (flag)

Purified baculoviruses and infected cell lysates were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Primary antibodies (1:4000 dilution), including against 6×His (Beyotime, Shanghai, China), V5 (Thermo Fisher, Waltham, MA, USA), and Flag (Beyotime, Shanghai, China) tags, were used to detect fusion proteins by western blot analysis. The secondary antibodies were goat anti-pig and goat anti-mouse IgG conjugated to HRP (1:3000 dilution, Thermo Fisher, Waltham, MA, USA). Protein bands were visualized using the ECL chemiluminescence system and Hyper-Max films, as recommended by the manufacturer.

Mouse Cylc1 cDNA was chemically synthesized by GenScript Biotech Corporation (Suzhou, China) and inserted into FLAG- or Myc-tagged pCMV vectors (Beyotime, Shanghai, China). Full-length cDNA encoding CCIN, ACTRT1, ACTL7A, SPACA1, FAM209, and SPATA46 was amplified by PCR and cloned into FLAG- or Myc-tagged pCMV vectors as described in our previous studies (Zhang et al., 2022a (link); Zhang et al., 2022b (link)). Primers for CAPZA3: 5′-GGTACCATGTCACTCAGCGTCTTGAGTAGG-3′ and 5′-TCTAGATATTATCCAGTTGCACAACACAC TTC-3′.
HEK293T line was obtained from ATCC (American Type Culture Collection) and authenticated using STR profiling test by Shanghai Biowing Applied Biotechnology Co, Ltd. HEK293T cell lines were tested negative for mycoplasma contamination. HEK293T cells were cultured at 37°C in a 5% CO₂ incubator with Dulbecco’s modified Eagle’s medium (Gibco, NY, USA) with 10% fetal bovine serum (HyClone, UT, USA) and 1% penicillin‒streptomycin (Gibco). Transient transfection of HEK293T cells was performed using Lipofectamine 3000 transfection reagent (Invitrogen, Shanghai, China) following the manufacturer’s protocol. Cells were then harvested 48 hr after transfection.

Proteins were extracted from tissues or cells in protein lysis buffer (Keygen Biotech, Nanjing, China). The proteins were separated by 8%, 10% or 12% SDS–PAGE and then transferred to PVDF membranes (Merck Millipore). The membranes were blocked in 5% skim milk for 2 h and then membranes were incubated overnight at 4 °C with antibodies against Prok2 (Abcam, ab76747, rabbit polyclonal, 1:1000), NeuN (Abcam, ab279296, mouse monoclonal, 1:1000), Acsl4 (Santa Cruz, sc-365230, mouse monoclonal, 1:400), Gpx4 (Santa Cruz, sc-166570, mouse monoclonal, 1:400), Tomm20 (Abcam, ab56783, mouse monoclonal, 1:1000), Tfam (Abcam, ab131607, rabbit polyclonal, 1:1000), MT-ND1(Abcam, ab181848, rabbit monoclonal, 1:1000), GAPDH (YI FEI XUE BIOTECH, YFMA0037, mouse monoclonal, 1:1000), Flag (Beyotime, China, AF519, mouse monoclonal, 1:1000), HA (Beyotime, China, AH158, mouse monoclonal, 1:1000), Fbxo10 (Novus, NBP1-91889, rabbit polyclonal, 1:1000) followed by incubation with horseradish peroxidase-conjugated secondary antibody (Beyotime, China, A0208, A0216, 1:5000) for 2 h. After washing with PBST, protein bands were visualized using SuperSignal^® Maximum Sensitivity Substrate (Thermo Fisher Scientific). Samples derive from the same experiment and blots are processed in parallel. Uncropped and unprocessed scans of blots are included in a Source Data file.