Pex19

400 nM PEX19^Bpa was incubated without and with 100 nM PEX26 at room temperature for 1 min. The reaction was further incubated with 400 nM PEX3ΔN-WT and its variants at room temperature for 5 min. The reaction was UV-irradiated as described in the PEX26^Bpa crosslinking assay. The crosslinked samples were loaded onto 8% Tris-glycine gels and analyzed by western blotting using Strep (1:3000 dilution, Genscript), PEX19 (1:3000 dilution, Novus Biologicals), Flag (1:3000 dilution, Genscript), and IRDye800 secondary antibodies (1:20,000 dilution, LiCor).

HeLa cells on the culture dish were semi-permeabilized, as described above. To remove cytosolic proteins, the cells were extensively washed with SP buffer. The semi-permeabilized HeLa cells were incubated with 200 nM PEX19-F125^Bpa protein at room temperature for 1 h. The cells were irradiated at ∼4 cm from a UVP BLAK-RAY B-100AP lamp (Analytik Jena) for 10 min on ice. After UV crosslinking, the cells were scraped and lysed by the incubation in the lysis buffer (50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM MgCl₂, 0.5% SDS, 0.15 U/μl benzonase) supplemented with 1× Halt protease inhibitor cocktail for 30 min on ice. After adding 1.5% SDS and 50 mM DTT, the crosslinked cell lysate was boiled at 95°C for 10 min, loaded onto 10% Tris-Glycine gel, and analyzed by western blotting using PEX19 (1:3000 dilution, Novus Biologicals) and PEX3 (1:3000 dilution, Novus Biologicals) primary antibodies and IRDye800 secondary antibody (1:20,000 dilution, LiCor).