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. 2022 Nov 9;298(12):102685. doi: 10.1016/j.jbc.2022.102685

Figure 5.

Figure 5

B1 signaling activity is reduced in cells lacking ANO6.A, confirmation of ANO6 KO in HEK293T cell line: on left, Western blot of wildtype HEK293T lysates versus lysates from ANO6KO cell line immunoblotted for ANO6 and β-tubulin. B, flow histogram of mock-transfected (gray) wildtype HEK293T cells (+annexin V) overlaid with mock-transfected ANO6KO cells (blue; +annexin V) demonstrates that ANO6KO cells exhibit lower baseline PS exposure than do wildtype HEK293T cells. Lower probe-positive cells were observed in the ANO6KO cells. C, quantification of PS exposure in ANO6KO mutant cell line compared with WT HEK293T cells: Mean fluorescence intensity (MFI) shown with ANO6KO condition normalized to mock-transfected condition. PS exposure was reduced by 26% in the ANO6KO cells (mean ± SEM is shown, unpaired t test, p = 0.0004, n = 5). D, B1FL activation of SRF-luciferase in ANO6KO cells was reduced by 67% relative to matched wildtype HEK293T cells (normalized to cell line, mean ± SEM is shown, ordinary one-way ANOVA with Tukey’s multiple comparisons test, p < 0.0001, n = 6 for HEK293T condition, n = 5 for ANO6KO condition, ROUT method used at 10% to remove one outlier from both conditions). ANO6, anoctamin 6; B1FL, full-length B1; HEK293T, human embryonic kidney 293T cell line; PS, phosphatidylserine.