(A) 293FT cells were co-transfected with siSLMAP and the indicated FLAG-MST2 plasmids. The total cell lysates were blotted with the indicated antibodies. Anti-GAPDH blot was used as the loading control. (B) Immunoblots with the indicated antibodies of lysates of 293FT and MCF10A cells with SLMAP deleted. KO, knockout; HM, hydrophobic motif. (C) Quantification of the ratios of pMST/MST, pMOB1/MOB1, pLATS/LATS, and pYAP/YAP signals in (B). The total and phosphorylated protein levels were individually normalized to GAPDH levels. Normalized values were used to calculate the ratios. Data are plotted as mean ± SEM of three biological replicates (*p<0.05; **p<0.01; ***p<0.001). (D) Immunofluorescence staining of YAP localization in control and SLMAP KO MCF10A cells. Cells were fixed, permeabilized, and stained with anti-YAP (red) and DAPI (blue). Scale bars, 10 μm. (E) Quantification of immunofluorescence signal intensities in (D). Approximately 50 cells were counted from 7 random fields. N < C (blue), N = C (grey), and N > C (red) categories indicate YAP localization in cytoplasm, both cytoplasm and nucleus, and nucleus, respectively. Data are plotted as mean ± SEM of three biological replicates. (F) Relative expression of YAP target genes CTGF and CYR61 in control and SLMAP KO MCF10A cells. Data are plotted as mean ± SEM of three biological replicates (****p<0.0001).