Fig. 3: Kindlin cooperates with intact active talin via paxillin to activate integrin.

a Kindlin-2 synergizes with intact active talin (tlnM3 + K2) to trigger PAC-1 binding to integrin-αIIbβ3. This effect is drastically lower in cells expressing similar level of talin head and kindlin-2 (tlnH+K2). ****p = 0.0001 with 95% confidence interval 13.02–22.32 (t-test), N = 6 biologically independent samples. Values are shown as mean ± S.E.M. b Representative histogram of (a) where tlnM3 + K2 (dark blue) enhances PAC-1 binding much more drastically than tlnH+K2 (light blue). c Talin WT does not cooperate with kindlin-2 in PAC-1-binding tests while tlnM3 does. ****p < 0.0001 with confidence interval 19.58–24.34 (t-test), N = 3 biologically independent samples; ns, p = 0.0815 with confidence interval −0.0459 to 0.5080 (t-test), N = 3 biologically independent samples. Values are shown as mean ± S.E.M. d Kindlin cooperation with full-length active talin (tlnM3 + K2WT) was significantly impaired when the paxillin-binding defective kindlin-2 mutant G42K/L46E (tlnM3 + K2GLKE) was expressed. **p = 0.0014 with 95% confidence interval −9.039 to −3.018 (t-test), N = 6 biologically independent samples. Values are shown as mean ± S.E.M. e Paxillin knockdown substantially reduced the synergy of tlnM3/kindlin-2 to activate integrin. Note that the effect of tlnM3 alone on PAC-1 binding was also significantly reduced (~15%), albeit less than that by tlnM3/kindlin-2 co-expression (~25%) and the latter had higher effect probably due to the overexpression of both tlnM3 and kindlin-2 vs tlnM3 alone in the former. **p = 0.0058 with 95% confidence interval −2.228 to −0.7115 (t-test); ***p = 0.0001 with 95% confidence interval −10.86 to −7.379 (t-test), N = 3 biologically independent samples. Values are shown as mean ± S.E.M. f Western blot showing that paxillin was efficiently knocked down. One out of two independent experiments is shown here. g Paxillin synergizes with tlnM3 to induce potent integrin activation. The synergy (tlnM3+paxiWT) was impaired by kindlin-2-binding defective paxillin mutant F577E (tlnM3 + paxiF577E). ****p = 0.0001 with 95% confidence interval −9.442 to −7.087 (t-test), N = 3 biologically independent samples. Values are shown as mean ± S.E.M. h Kindlin-2 knockdown substantially reduces the synergy of tlnM3/paxillin to activate integrin. ****p < 0.0001 with 95% confidence interval −21.65 to −19.14 (t- test), N = 3 biologically independent samples. Values are shown as mean ± S.E.M. i Western blot showing that paxillin was efficiently knocked down. One out of two independent experiments is shown here. Uncropped images (f, i) and raw data (a, c–e, g, h) are provided as a Data Source file.