Fig. 2

KAI1 localizes at the lipid rafts of PCs, which depends on palmitoylation. a Kai1 localization confirmed in MS1 and 10T1/2 cells using immunofluorescence. Scale bar, 50 μm. b Lipid rafts isolated from MS1 and 10T1/2 cells transfected with Kai1-GFP fusion vector using OptiPrep medium (frozen and crosslinks method). Flotillin-1 (planar type) and caveolin-1 (caveolae type) was detected in the lipid-raft membrane fraction. Fraction 1: no cellular protein. Fraction 2; Lipid raft proteins. Fraction 3; Non-raft membrane proteins. Fraction 4 and 5; Cytosolic proteins. Input samples are shown below. c Lipid raft marker (Cholera toxin B, red) co-localization with Kai1 (green) on MS1 and 10T1/2 cells transfected with Kai1-GFP fusion vector, observed using structured illumination microscopy. Arrowheads, co-localization of Kai1 protein and cholera toxin B. Scale bar, 2 μm. d Kai1 palmitoylation was analyzed by Acyl-Biotin Exchange (ABE) assay in ECs (MS1) and PCs (10T1/2). e zDHHC3 and zDHHC4 knockdown in 10T1/2, and their effects on Kai1 palmitoylation levels. f Changes in Kai1 localization after inhibition of palmitoylation using 2-bromopalmitate (2-bp) in 10T1/2. Scale bar, 50 μm. g Left, changes in Kai1 localization in the membrane and cytosol of 10T1/2 cells following the inhibition of palmitoylation using 2-bp. Right, quantification of the obtained results (**p < 0.05, ***p < 0.001, Means ± SEM, 3 technical replicates)