MASTL regulates SRF activity independent of its kinase activity. (A) Quantification of SRF, VCL, and FOS mRNA levels relative to TATA-box binding protein (TBP) in siMASTL (72 h silencing) MCF10A cells (n = 2 biologically independent experiments, mean ± SD). (B) Western blot analysis of vinculin, paxillin, and MASTL protein levels in siControl and siMASTL MCF10A cells after 48 h of silencing. GAPDH was used as a loading control. (C) Quantification of vinculin levels in B after MASTL silencing. Protein levels are normalized to GAPDH (n = 3 individual experiments). (D) Relative SRF luciferase activity in siControl, siMASTL and siSRF (SRF-silenced) MCF10A cells following 48 h of silencing under serum-starved conditions and after stimulation with 20% serum (n = 3 individual experiments). (E) Relative SRF luciferase activity. MCF7 cells expressed EGFP control, EGFP-MASTL WT, and EGFP-MASTL G44S together with the SRF reporter 3D.Aluc and RLTK Renilla luciferase transfection control under serum-starved conditions and after stimulation with 20% serum (n = 4 individual experiments). Unpaired t test and mean ± SD is used in all graphs. See also Table S1.