Fig. 1
FRAP studies characterizing the lateral diffusion of various TGF-β-superfamily receptors. COS7 cells were transfected with an expression vector encoding myc- or HA-tagged receptor (myc-ACVR2A, myc-ALK6, myc-ALK3, HA-ALK4, or HA-ALK2), or empty vector (control). After 24 h, live cells were labeled by monovalent fluorescent Fab’ fragments as detailed under “Methods.” a Point-confocal measurements of the cell surface levels of the tagged receptors. Measurements were conducted as described under “Methods,” using the FRAP setup under identical non-bleaching conditions. Results are mean ± SEM of 30 independent measurements (each on a different cell) under each condition. “Control” designates cells transfected with empty vector (i.e., not expressing tagged receptors) incubated with αmyc (myc-Control) or αHA Fab’ (HA-Control) followed by secondary fluorescent Fab’ to yield background fluorescence levels. No significant differences were found between the expression levels of the various receptors, excluding the control samples (one-way ANOVA and Bonferroni post hoc test; P > 0.99). b A representative FRAP curve of the lateral diffusion of myc-ACVR2A. FRAP studies were conducted at 15 °C to minimize internalization. Solid lines are the best-fit of a nonlinear regression analysis to the lateral diffusion equation [56]. c A representative FRAP curve of HA-ALK4, which shows a lower mobile fraction (Rf). d, e Average Rf and D values derived from multiple FRAP measurements. Bars are mean ± SEM; the number of measurements (each conducted on a different cell) is depicted on each bar. Some of these numbers are lower in panel D because FRAP curves yielding less than 20% recovery could be accurately analyzed only for Rf