FIGURE 5.

MRTF-A interacts with RelA and RelB. To explore if MRTF-A binds RelA and RelB, we performed co-immunoprecipitations followed by dot blotting (A). Lysates from cells where MRTF-A was overexpressed were incubated with control resin and with MRTF-A-antibody-conjugated resin. Flow through (FC: flow through control; FM: flow through MRTF-A) from both columns contained RelA, RelB, and MRTF-A. RelA, RelB and MRTF-A were moreover detectable in the eluate from the MRTF-A column (EM: eluate MRTF-A) but in the in the eluate from the control column (EC: eluate control). Western blotting (B) of the eluates showed the RelA band at the expected molecular weight. These findings support a model where MRTF binding to Rel proteins suppresses inflammation (C). (C) Also depicts the hypothesis that RelA suppression, by glucocorticoid receptor stimulation or silencing, should mitigate inflammatory suppression by MRTF-A. (D,E) Test this hypothesis using dexamethasone (glucocorticoid receptor agonist, 3 μM) or short hairpin silencing (200 MOI of shRELA). For the experiments in (D,E), the fold repression is given. Independent testing showed that the fold repression of CCL2 and IL1B by MRTF-A was reduced by dexamethasone. In contrast, fold suppression of CXCL8 and IL6 was unaffected. For RelA silencing, CXCL8 and IL6 suppression by MRTF-A were significantly reduced, but the overall effect, where MRTF-A suppression of inflammation was reduced by ≈25%, was also significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.