Fig. 1
From: Sharpin suppresses β1-integrin activation by complexing with the β1 tail and kindlin-1
Sharpin suppresses integrin α5β1 activation but not integrin αIIbβ3 activation. a Sharpin (SH) was co-expressed in CHO cells together with DsRed-fused talin head (TH) and EGFP-fused kindlin-1 (K1) by transient transfection. Activation of endogenous integrin α5β1 in transfected cells were measured by the GST-Fn-III binding assay. b CHO cells that stably express αIIbβ3 (CHO-αIIbβ3) were used to express the indicated regulators. Their effects on integrin αIIbβ3 activation were evaluated by the PAC-1 antibody binding assay. c CHO cells were transfected with two different siRNA (siRNA1 and siRNA2) specifically targeting endogenous sharpin in CHO cells or non-targeting siRNA (NCS) as a control. 24 h after transfection, expression of sharpin protein in CHO cells was evaluated by immunoblotting. d, e NSC or two siRNA targeting hamster sharpin were co-transfected either in CHO cells (d) or CHO-αIIbβ3 cells (e) together with DsRed-fused talin head (TH) or TH plus EGFP-kindlin-1 (K1); their effects on integrin α5β1 activation in CHO cells or integrin αIIbβ3 activation in CHO-αIIbβ3 cells were evaluated by the GST-Fn-III binding assay and the PAC-1 antibody binding assay, respectively. f Sharpin (SH) was co-expressed in 3 T3 cells together with DsRed-fused talin head (TH) and EGFP-fused kindlin-1 (K1) by transient transfection. Activation of endogenous integrin α5β1 in transfected 3 T3 cells was measured by the 9EG7 antibody binding assay. g 3 T3 cells were transfected with three different siRNA (siRNAa, siRNAb and siRNAc) targeting endogenous sharpin in 3 T3 cells or non-targeting siRNA (NCS) as a control. 24 h after transfection, expression of endogenous sharpin protein in 3 T3 cells was evaluated by immunoblotting. h NSC or two different siRNA (siRNAb and siRNAc) were co-transfected in 3 T3 cells together with DsRed-fused talin head (TH) or TH plus EGFP-kindlin-1 (K1), and their effects on integrin α5β1 activation in 3 T3 cells were evaluated by the 9EG7 antibody binding assay. The results represent the mean ± SD of at least 3 experiments. (MFI: median of fluorescence intensity; ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001)