Detection of PSKH2 in membrane-enriched fractions is dependent on N-myristoylation.
(A) Co-precipitation of mitochondrial protein TIMM23 with PSKH2. (B) Mitochondrial fractionation of HEK-293T cells overexpressing EGFP of CT-Myc PSKH2. (C) Immunoprecipitation of PSKH2 from HEK-293T cells metabolically labelled with alkynyl myristic acid. Myristoylated protein was labelled with biotin azide using a copper-catalyzed click reaction and detected after western blotting using neutravidin-HRP. (D) Detection of myristoylated PSKH2 in immunoprecipitations from HEK-293T cells treated in the absence of presence of IMP-1088. (E) Mitochondrial fractionation of HEK-293T cells overexpressing EGFP, WT PSKH2 (CT-Myc) or PSKH2 mutated at putative sites of acylation. (F) Mitochondrial fractionation of PSKH2-expressing HEK-293T treated in the presence or absence of IMP-1088. For all fractionations; T = total cell lysate, C = cytoplasmic fraction, M = mitochondrial-enriched fraction.