Fig. 3: Identification of RalGAPα1 as a regulator of SERCA2a.
From: The RalGAPα1–RalA signal module protects cardiac function through regulating calcium homeostasis
A Identification of proteins associated with RalGAPα1. GFP-RalGAPα1 was expressed in HEK293 cells together with HA-RalGAPβ, and empty vectors were expressed in cells as a control. Immunoprecipitation was carried out using the GFP-binder, and proteins in the immunoprecipitates were separated via SDS-PAGE and identified via mass-spectrometry. B GFP-RalGAPα1/HA-RalGAPβ complex was co-expressed with Flag-SERCA2a or free Flag in HEK293 cells. After immunoprecipitation with the Flag antibody, GFP-RalGAPα1/HA-RalGAPβ complex was detected in the immunoprecipitates via western blot. C Endogenous RalGAPα1 was immunoprecipitated from heart lysates, and endogenous SERCA2a was detected in the immunoprecipitates via western blot. D Endogenous SERCA2a was immunoprecipitated from heart lysates, and endogenous RalGAPα1 was detected in the immunoprecipitates via western blot. E Ca2+ transients in HEK293 cells expressing mCherry-SERCA2a together with HA- RalGAPα1 or HA-RalGAPα1N1949K proteins. Ca2+ transients were recorded using confocal microscopy in cells that were stimulated with ATP. n = 59 (HA-vector), 75 (HA-RalGAPα1), and 73 (HA-RalGAPα1N1949K). FDHM: p = 0.0003 (HA-vector vs HA-RalGAPα1), and p < 0.0001 (HA-RalGAPα1 vs HA-RalGAPα1N1949K). Tau: p = 0.031 (HA-vector vs HA-RalGAPα1), and p < 0.0001 (HA-RalGAPα1 vs HA-RalGAPα1N1949K). Amplitude: p = 0.0094 (HA-vector vs HA-RalGAPα1), and p < 0.0001 (HA-RalGAPα1 vs HA-RalGAPα1N1949K). F Ca2+ transients in RalGAPα1-depleted neonatal rat cardiomyocytes upon field stimulation. Amplitude, FDHM and Tau of Ca2+ transients were quantified from 58 (siNC) and 72 (siRalGAPα1) cells. p = 0.0008 (FDHM), 0.0054 (Tau) and 0.0023 (Amplitude). G, H Ca2+ transients in primary cardiomyocytes isolated from the male RalGAPα1f/f and RalGAPα1-cKO mice (2-month-old) upon field stimulation. Amplitude, FDHM and Tau of Ca2+ transients were measured in 66 RalGAPα1f/f cells (3 mice) and 56 RalGAPα1-cKO cells (2 mice), respectively. H representative Ca2+ transient images and curves. p = 1.46e-5 (FDHM), 7.84e-7 (Tau) and 0.00041 (Amplitude). I Expression of Rcan1.4 mRNA in the hearts of male RalGAPα1-cKO and RalGAPα1f/f mice (3-month-old). n = 9 (RalGAPα1f/f) and 7 (RalGAPα1-cKO). p = 0.036. J, K SERCA2a Ca2+-transporting activity J and ATPase activity K in microsomes isolated from hearts of male RalGAPα1-cKO and RalGAPα1f/f mice (2-month-old). SR Ca2+-uptake: n = 5 (RalGAPα1f/f) and 6 (RalGAPα1-cKO). SERCA2a-ATPase: n = 6 (RalGAPα1f/f) and 4 (RalGAPα1-cKO). p = 0.011 (SR Ca2+-uptake) and 0.048 (SERCA2a-ATPase). The data are given as the mean ± SEM. Statistical analyses were carried out using one-way ANOVA for 3E and two-sided t-test for 3F–G, I–K One-asterisk indicates p < 0.05, two-asterisk indicates p < 0.01 and three-asterisk indicates p < 0.001. Source data are provided as a Source Data file.
