Fig. 8
Stabilin-2 promotes BAI3 GPCR activity and facilitates the recruitment of ELMO2 at the membrane. a Stabilin-2 activates BAI3. Increasing amounts of Stabilin-2 lead to an increase in β-arrestin2 coupling to BAI3. b Immunofluorescence experiments assessing ELMO2 (purple) recruitment at the membrane of C2C12 cells in the presence of either Flag-BAI3WT, BAI3ΔELMO2 or V5-Stabilin-2 (green). c–e Quantification of experiment shown in b. f Membrane fractionation experiment followed by western blot analyses of HEK293t cells demonstrate that Stabilin-2 cooperates with BAI3 to promote the recruitment of ELMO2 at the membrane. BAI3ΔELMO2 fails to recruit ELMO2 at the membrane in the presence of Stabilin-2. g The extracellular domain and the Elmo binding site of BAI3 are both required to promote myoblast fusion. C2C12 overexpressing either Flag-BAI3, V5-Stabilin-2 or both together exhibit an increase in fusion. Cells expressing Flag-BAI3∆N mutant together with V5-Stabilin-2 display a decrease in fusion. Similarly, the expression of BAI3∆ELMO2 together with V5-Stabilin-2 blocked myoblast fusion. h Quantification of the experiment shown in g. Myofibers were stained for Myosin Heavy Chain (MyHC, MF20 antibody (green)) and nuclei were revealed by Hoechst (purple). Error bars indicate standard deviation. Scale bar = 100 μm. One-way ANOVA followed by a Bonferroni test was used to calculate the P-values; ****P < 0.0001
