Fig. 1: The PLCε EF1/2 hands and Y-box are required for maximum RhoA-dependent activity.
From: RhoA allosterically activates phospholipase Cε via its EF hands
a Domain architecture of R. norvegicus PLCε, with domain boundaries shown above. Variants used in this study are shown below, open boxes indicate internal deletions. b (Left) Basal and RhoAG14V-stimulated activities of PLCε and variants retaining EF1/2 and/or the Y-box. At least three independent experiments from independent transfections were carried out for each variant, and data are shown as the average of triplicate measurements ± SD. Data was 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, for PH-C **p < 0.0053, EF-C **p < 0.0034, PLCεΔY **p < 0.0075, EF-CΔY *p < 0.012. (Right) The change in maximal activity ± SD was calculated by subtracting RhoAG14V-stimulated activity from the basal activity of each variant. Data were analyzed using a one-way ANOVA and Kruskal–Wallis test comparing each variant to PLCε, followed by a Dunn’s multiple comparisons test. For EF3-C **p < 0.0053, PLCεΔY **p < 0.0030, and EF-C-ΔY *p > 0.0159. Representative Western blots are shown below, with empty pCMV vector (EV) and β-actin used as loading controls. Differences in expression were not found to be statistically significant but may still contribute to variation in activities. PLCε variants express a C-terminal FLAG tag and are detected with an anti-FLAG antibody, while RhoA contains an N-terminal HA tag and is detected with an anti-HA antibody.
