Fig. 7
Loss of CD98hc impairs GEF-H1and Src family kinase activation, and integrin dynamics. a Loss of CD98hc impairs LARG and GEF-H1 activation by mechanical force application on integrins. One experiment representative of n = 2. b Loss of CD98hc impairs Src activity but does not affect ERK1/2 phosphorylation by mechanical force application on integrins. Src activity was monitored by tracking substrate FAK Y576 phosphorylation and SFK phosphorylation at Y416. c Loss of CD98hc induces constitutive recruitment of GEF-H1 to the membrane. s supernatant, p membrane pellet. One experiment representative of n = 3. d Control or CD98hc-null dermal fibroblasts were stimulated with FN-coated magnetic beads. Cells were lysed and lysates were fractionated into cytosolic (s) and membrane (p) fractions by centrifugation. Active RhoGEFs was pulled-down from each fraction with GST-RhoA17A beads. One experiment representative of n = 2. e, f Diffusion rate (t-half) of integrin α5 and β3 as calculated from the curve fit generated from the FRAP measurements performed on n = 27, 41, and 67; and 73, 36, and 57 adhesions, respectively, for control, CD98hc-null and C330S cells on integrin α5 and β3. Error bars are 95% CI calculated from that fit. g, h Integrin α5 and β3 mobile fraction as calculated from the curve fit generated from the FRAP measurements. Error bars are 95 CI calculated from that fit. i Trafficking of integrin β1 in control or CD98hc-null cells. Integrin β1 was labeled with Alexa 488 coupled antibody then integrin trafficking was chased for indicated time. Extracellular staining was quenched and only intracellular labeled integrin is observed. Scale bar is 50 µm
