G12V mutation increases CD36 localization at caveolin-1 structures and abolishes CD36 signaling in response to TSP-1. (A) Representative two-color fixed-cell images of CD36 (magenta) and caveolin-1 (Cav-1) (cyan) on the surface of a TIME cell imaged via TIRFM, for CD36^WT (top) and CD36^G12V (bottom). Solid lines show the segmented cell edge, dashed lines specify the boundary between periphery and center regions. Scale bar, 5 μm. (B) −log₁₀(P value) of test for significance of CD36 (WT or mutant) colocalization with Cav-1 puncta in the whole, center and periphery regions. Blue color intensity of each entry reflects level of significance, as shown in color bar below. Values in black and highlighted by a solid red outline are significant (P value ≤ 0.05; −log₁₀(P value) ≥1.301), values in gray are not significant. (C) Representative three-color fixed-cell images of CD36 (magenta), pSFK(Y419) (cyan), and Fyn (yellow) on the surface of a TIME cell imaged via TIRFM for CD36^WT (top) and CD36^G12V (bottom). Solid and dashed lines and scale bars are as in A. (D and E) -log₁₀(P value) of test for significance of CD36-Fyn colocalization (D) and of CD36-pSFK(Y419) colocalization (E), for WT and mutant CD36 in the whole, center, and periphery regions, in the absence or presence of TSP-1. Entry details are as in B. See Table 4 for number of cells, number of experimental repeats, and number of objects per channel per ROI used for analysis.