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. 2023 Jul 25;14:4101. doi: 10.1038/s41467-023-39586-z

Fig. 4. Septin 11 mediates endothelial activation by the 27HC-ERα tandem by disrupting the interaction between MKK7 and its inhibitory protein GADD45β and thereby activating Jnk.

Fig. 4

a HAEC were treated with 27HC and immunoblotting was performed to detect Jnk Thr183/Tyr185 phosphorylation and total Jnk. b Following transfection of HAEC with control shRNA or shRNA targeting Jnk1 (upper panel, findings for 2 samples/group are shown), the effects of vehicle versus 27HC on NF-κB activity were determined (lower panel, n = 8). Following transduction of HAEC with control shRNA or shRNA targeting ERα (c) or septin 11 (d), HAEC were treated with 27HC, and immunoblotting was performed to detect activating phosphorylation of MLK3, MKK7 and Jnk, and total MLK3, MKK7 and Jnk. HAEC were treated with 27HC for 0 to 10 min, MKK7 was IP’d and immunoblotting was performed for MKK7 and GADD45β (e), or septin 11 was IP’d and immunoblotting was done for MKK7 and septin 11 (f). g In pull-down experiments a complex between HA-tagged bound MKK7 and GADD45β was formed. Wild-type septin 11 (WT) or septin 11 deletion mutants lacking AA184-192 (septin 11-∆G4) or AA38-304 (septin 11-∆GTP) were added, and protein associations were evaluated by immunoblotting. h, i Requirement for septin 11 interaction with MKK7 in 27HC action was evaluated in HAEC in which endogenous septin 11 was knocked down by shRNA, and reconstitution was performed with either wild-type septin 11, septin 11-∆G4, or septin 11-∆GTP. (h) Cells were treated with vehicle or 27HC, and immunoblotting was performed to detect activating phosphorylation of MKK7 and Jnk, and total MKK7 and Jnk. (i) In the same study groups as in (h), NF-κB activity was evaluated in cells treated with vehicle or 27HC. n = 8. In (b) and (i), NF-κB activity is expressed relative to values with vehicle treatment. Data are mean ± SEM. In (b) and (i), P values by ANOVA with Tukey’s post-hoc testing are shown. The findings in (a) and (c)–(h) were confirmed in two independent experiments. Source data are provided as a Source Data file.