Hpa012145

Cells were washed with phosphate-buffered saline (PBS) and fixed by incubation with 2% paraformaldehyde for 15 min. Next, membranes were permeabilized by incubation with PBS solution containing 0.1% (v/v) Triton X-100 (Bio-Rad, 1610407) for 5 min, and the coverslips were blocked using 10 g/L bovine serum albumin dissolved in PBS for 1 h. Primary and secondary antibodies were diluted in 10 g/L bovine serum albumin in PBS and used for incubation for 1 h at room temperature. We used primary antibodies against GFP (Santa Cruz, sc-8334; 1:1000) or ACBD5 (Sigma, HPA012145; 1:500) and secondary antibodies anti-rabbit IgG Alexa 594 (Invitrogen, A-11012; 1:500) or sequentially biotinylated anti-rabbit IgG (DAKO, E0432; 1:200) and a streptavidin–fluorescein isothiocyanate (FITC) complex (DAKO, 11-4317-87; 1:200). The glass coverslips were fixed on objective slides with the mounting medium ProLong^TM Gold anti-fade reagent with DAPI (Invitrogen, P36935).

All plasmids were cloned and amplified using DH5α Competent Cells (Invitrogen, Cat. No. 18265–017). The plasmids encoding EGFP-SKL, myc-ACBD5(human), FLAG-ACBD5(human)-FFAT, ACBD5(human)-ACB and FLAG-ACBD5(human)-WT were generated as described in a previous publication (16). The plasmids mCherry2-C1, mPlum-N1, mPlum-Mito-3 and mCherry-Mito-7 were gifts from Michael Davidson (Addgene plasmid #54563, #54629, #55988, #55102) [20 ]. The primary and secondary antibodies used in this study include mouse monoclonal anti-GFP (MAB3580, Chemicon), mouse monoclonal anti-myc (#2276, Cell signaling), rat monoclonal anti-RFP (5F8, Chromotek), rabbit polyclonal Pex14 (kind gift from D. Crane, Brisbane, Australia), rabbit polyclonal ACBD5(human) (HPA012145, Sigma), rabbit polyclonal VAPB (HPA013144, Sigma), Alexa Fluor 488 anti-mouse IgG, Alexa Fluor 568 anti-rat IgG and Alexa Fluor 647 anti-rabbit (A32723, A11036, A32733, Invitrogen).