Fig. 2: PLCε/β3 chimeras confirm the EF hands are essential for RhoA-dependent activation.
From: RhoA allosterically activates phospholipase Cε via its EF hands
a Schematic of the PLCε/β3 EF hand chimeras. b Basal and RhoAG14V-stimulated activities of PLCε, PLCβ3, and PLCε/β3 chimeras. (Left) Only PLCε variants that retain the EF1/2 hands are responsive to RhoA-dependent activation. At least three independent experiments from independent transfections were performed for each variant, and data are shown as the average of triplicate measurements ± SD. Data were analyzed using an unpaired, one-tailed t-test with Welch’s correction comparing the basal and RhoA-stimulated activities of each variant. ****p < 0.0001, **p < 0.0067, *p < 0.0167. (Right) Changes in maximal activity of each variant were analyzed using a one-way ANOVA and Kruskal–Wallis test comparing each variant to PLCε, followed by a Dunn’s multiple comparisons test. ***p < 0.0485, *p < 0.0198. Representative Western blots are shown below, with empty pCMV vector (EV) and β-actin used as loading controls. PLCε variants express a C-terminal FLAG tag and are detected with an anti-FLAG antibody, and RhoA contains an N-terminal HA tag for detection using an anti-HA antibody.
