Anti abcd3

Reagents and antibodies used were as follows: anti-Calnexin (#ab22595, Abcam, Cambridge, MA, USA), anti-LC3 (#L8918, Sigma-Aldrich, St. Louis, MO, USA), anti-NBR1 (#16004-1-AP, Proteintech, Rosemont, IL, USA), anti-PEX3 (#247042, Abcam), anti-PEX14 (#sc-23197, Santa Cruz Biotechnology, Dallas, TX, USA), anti-PEX16 (#14816-1-AP, Proteintech, Rosemont, IL, USA), anti-PEX19 (#PA5-22129, Invitrogen, Carlsbad, CA, USA), anti-ABCD3 (#ab3421, Abcam, Cambridge, MA, USA and #SAB4200181, Sigma-Aldrich, St. Louis, MO, USA), anti-SQSTM1/p62 (#H00008878-M01, Abnova, Taipei, Taiwan), anti-VDAC (#55259-1-AP, Proteintech, Rosemont, IL, USA), anti-β-actin (#sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA), and chloroquine (#C6628, Sigma-Aldrich, St. Louis, MO, USA).

Western blot analysis was performed as previously described with a minor change (Wu et al., 2021 (link)). In short, 48 h after inhibition using Ints7 MO oligos, BM-MSC protein lysates were separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to 0.22-μm polyvinylidene difluoride membranes (Millipore, Billerica, MA, United States) and incubated with specific anti-INTS7 (Proteintech, Chicago, IL, United States) or anti-ABCD3 (Abcam, Cambridge, MA, United States) antibodies. A beta-tubulin antibody (Beyotime, Nantong, China) was used as an internal control. After incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Thermo Scientific, Waltham, MA, United States), signals were detected by enhanced chemiluminescence substrate (Thermo Scientific). The resulting bands were analyzed using Image-Pro Plus Software.

Briefly, 5 μg of anti-INTS7 antibody (Proteintech) was incubated with total BM-MSC lysates for 2 h, followed by the addition of 20 μl of washed protein A/G beads (Santa Cruz Biotechnology) and incubated for another 16 h at 4°C. Beads were then pelleted and washed three times in 20× bed volume of lysis buffer. The bound protein was eluted by heating the beads at 95°C for 5 min with 2× SDS buffer. Finally, anti-INTS7 (Proteintech), anti-ABCD3 (Abcam), or anti-HDLBP (Proteintech) antibodies were used to detect the presence of each protein in the immunoprecipitation (IP) product. Inputs represented approximately 1/10 of the extract volume used for the IP experiment.