NF-κB pathway activation in Arp2/3-depleted or -inhibited cells requires MEKK3 and CCM2. (A) Blot showing phospho–p38 MAPK (Thr180/Tyr182; p-p38), total p38, and GAPDH levels in NS and 2×KD cells. (B) Blot showing p-p38, total p38, and GAPDH levels in NS cells treated with CK689 or CK666 for 6 h (right). (C) Blot showing p-p65, p65, p-HSP27, and GAPDH levels of IA32 cells with 100 µM CK666 only and simultaneous 10 µM SB203580 + 100 µM CK666 treatments at the indicated time points. Decreased p-HSP27 levels indicate effective inhibition of p-38 activation by SB203580. (D) Blot showing p-p65, p65, and GAPDH levels of control and MEKK3^−/− MEFs upon CK666 treatment for the indicated times. (E) Blot showing p-p65, p65, and GAPDH levels of control siRNA and two CCM2 siRNA–treated IA32 cells with CK689 or CK666 treatment for 6 h. (F, left) Time-lapse images showing representative cells expressing YPET-CCM2 treated in 0.25 M sorbitol (top)- or 100 µM CK666 (bottom)-containing media for the indicated times. (right) Quantification of nuclear CCM2 from time-lapse videos. Error bars show 95% CI. (G) Blot showing p34Arc immunoprecipitation (IP) experiments of HEK293 FT cells expressing YPET-CCM2 treated with DMSO or CK666 for 6 h before cell lysis. YPET-CCM2 was detected with an anti-GFP antibody. Fold changes in band intensity averaged across the three experiments (normalized to loading control and experimental control) are shown below each band. A.U., arbitrary unit; IB, immunoblot.