Sequence confirmation and surface expression of different CD36 mutants. (A) Sequence of WT (bold) compared with CD36^G12V, CD36^G16V, and CD36^G12V/G16V. The change in codon from glycine to valine is shown in a black solid box with the nucleotides highlighted in red color. (B) Two-color TIRFM images of HaloTag-fused WT (CD36^WT) and mutated CD36 (CD36^G12V, CD36^G16V, and CD36^G12V/G16V) expressed in TIME cells, labeled using JF549-conjugated Halo ligand to assess total expression (first column) and an anti-CD36 antibody that targets the CD36 ectodomain to assess surface expression (second column). Scale bar, 5 μm. (C) To assess the fraction of CD36^WT and CD36^G12V expressed on the cell surface, the fraction of Halo-labeled puncta (labeling CD36 regardless of its surface expression) colocalized with antibody-labeled puncta (labeling only CD36 on the cell surface) was calculated. The plot shows these fractions after compensating for the slightly different overall expression levels of WT and mutant CD36, achieved by nullTR equalization. NullTR equalization = multiplying the data and nullTR values of one condition (CD36^WT in this case) with a factor to make its nullTR have the same median as the nullTR of the condition it is compared with (CD36^G12V in this case). The idea is that this compensates for different overall expression levels, as nullTR reflects the number of objects used in colocalization analysis (Vega-Lugo et al., 2022), thus allowing the direct comparison of the colocalization fractions between the two conditions. Plot details are as in Fig. S2. N = 32 cells for each of CD36^WT and CD36^G12V, from four experimental repeats.