Figure 6.

(A) Cartoon schematics of two cell populations to assess EphB4 forward signaling. We used BT16 cells that do not express endogenous ephrinB2 for the assay, and kindlin2 is a major kindlin isoform (Fig S4 and https://depmap.org). Stable BT16 cells expressing exogenous flag-ephrinB2 and HEK293 cells expressing HA-EphB4 were generated by lentivirus-mediated gene integration, respectively. The two cell populations were harvested and combined for 2 h. Cell lysates were produced and immunoprecipitated with anti-HA antibody. Bound immunocomplex was separated on SDS?PAGE, and tyrosine phosphorylation of EphB4 was assessed by Western blotting with anti-phosphotyrosine?specific antibody. Cell lysate immunoprecipitated by normal IgG was used as a negative control. (B) Kindlin2 binding to the ephrinB2 cytoplasmic tail in BT16 cells regulates in trans EphB4 activation in HEK293 cells. BT16 stable cells expressing flag-ephrinB2 WT or 4A are incubated with HEK293 stable cells expressing HA-EphB4. Cells were lysed, and EphB4/ephrinB2 complex in cell lysates was immunoprecipitated by anti-HA antibody. And bound EphB4 and EphrinB2 were recognized with anti-HA and anti-flag antibodies in Western blot, respectively. The status of tyrosine phosphorylation of EphB4 was analyzed by anti-phosphotyrosine?specific antibody. (C) Ratio of tyrosine-phosphorylated EphB4 to total EphB4 was determined by densitometry in Western blot. The data are shown as the mean ± SEM (n = 3). A one-sample t test was used. **P < 0.01. (D) Cartoon showing disrupting kindlin2 binding to the ephrinB2 cytoplasmic tail in BT16 cells decreases EphB4 activation in HEK293 cells. (E) Silencing kindlin2 expression in BT16 cells reduces EphB4 activation in HEK293 cells. (F) Endogenous kindlin2 expression in BT16 cells expressing flag-ephrinB2 was silenced by shRNA (F) and co-cultured with HEK293 cells expressing HA-EphB4. Cell lysates were produced and immunoprecipitated by anti-HA antibody to isolate EphB4/ephrinB2 complex. Captured proteins were fractionated on SDS?PAGE and analyzed by Western blot with anti-phosphotyrosine, anti-HA, and anti-flag antibodies, respectively. (G) Tyrosine-phosphorylated EphB4/total EphB4 ratio was assessed by densitometry in Western blot. The data are evaluated as the mean ± SEM (n = 3). A one-sample t test was used. **P < 0.01. (H) Cartoon illustrates that silencing kindlin2 expression in BT16 cells diminishes EphB4 activation in HEK293 cells. (I) Silencing integrin expression in HEK293 cells decreases kindlin2-dependent EphB4 activation. (J) BT16 stable cells were co-cultured with HEK293 stable cells that were treated with integrin ?1 shRNA to knock down endogenous integrin ?1 expression (J). After cells were lysed, EphB4/ephrinB2 complex was immunoprecipitated by anti-HA antibody and the status of EphB4 phosphorylation was assessed by anti-phosphotyrosine antibody. (K) Tyrosine phosphorylation status of EphB4 was measured by scanning band intensity in Western blot. Bar graph shows the mean ± SEM (n = 3). A one-sample t test was used. **P < 0.01. (L) Cartoon displays that repressing endogenous integrin ?1 expression in HEK293 cells decreases kindlin2-mediated EphB4 activation. (M) Kindlin2 regulates EphB2/ephrinB1 forward signaling. BT16 stable cells expressing flag-ephrinB1 were incubated with HEK293 stable cells expressing HA-EphB2. Cell lysates were produced, and EphB2/ephrinB1 complex was immunoprecipitated by anti-HA antibody. And immunoprecipitated proteins were analyzed for the activation status of EphB2 by Western blotting with anti-phosphotyrosine antibody. (N) Quantified band intensities are displayed as phosphoEphB4/total EphB4 ratio. The data are presented as the mean ± SEM (n = 3). Statistical analysis used a one-sample t test. ***P < 0.001. (O) Cartoon illustrating that disrupting kindlin2 binding to ephrinB1 diminishes EphB2 activation in HEK293 cells.

Source data are available for this figure.