Fig. 5: Regulation of SERCA2a oligomerization by the RalGAPα1-RalA axis.
From: The RalGAPα1–RalA signal module protects cardiac function through regulating calcium homeostasis
A Oligomerization of SERCA2a in HEK293 cells in which SERCA2a was co-expressed with RalGAPα1 or an empty vector. B Oligomerization of SERCA2a in the heart of RalGAPα1-cKO mice and RalGAPα1f/f littermates (2-month-old, female). C GFP-RalGAPα1/HA-RalGAPβ complex was co-expressed with Flag-SERCA2a or free Flag in HEK293 cells that were infected with lentivirus-shNC or lentivirus-shRalA. After immunoprecipitation with the Flag antibody, GFP-RalGAPα1 was detected in the immunoprecipitates via western blot. D HA-RalGAPα1 or HA-vector was co-expressed with Flag-SERCA2a in HEK293 cells that were infected with lentivirus-shNC or lentivirus-shRalA. Oligomerization of Flag-SERCA2a was analyzed via western blot. E Oligomerization of SERCA2a in HEK293 cells in which SERCA2a was co-expressed with HA-RalA, HA-RalAG23V, HA-RalAS28N or an empty vector. F Flag-SERCA2a and mCherry-SERCA2a were co-expressed with HA-RalA, HA-RalAG23V, HA-RalAS28N or HA-vector in HEK293 cells. After immunoprecipitation with the Flag antibody, mCherry-SERCA2a was detected in the immunoprecipitates via western blot. G, H SERCA2a Ca2+-transporting activity G and ATPase activity H in microsomes isolated from HEK293 cells in which SERCA2a was co-expressed with RalGAPα1 or an empty vector. SERCA2a Ca2+-transporting activity: n = 7 (GFP) and 6 (GFP-RalGAPα1). SERCA2a ATPase activity: n = 3 (GFP) and 4 (GFP-RalGAPα1). p = 0.027 (SR Ca2+-uptake) and 0.013 (SERCA2a-ATPase). I, J SERCA2a Ca2+-transporting activity I and ATPase activity J in microsomes isolated from HEK293 cells in which SERCA2a was co-expressed with HA-RalA, HA-RalAG23V, HA-RalAS28N or an empty vector. SR Ca2+-uptake: n = 9 (vector, HA-RalA and HA-RalAG23V) and 8 (HA-RalAS28N). SERCA2a-ATPase: n = 8 (vector and HA-RalAS28N) and 9 (HA-RalA and HA-RalAG23V). SR Ca2+-uptake: p = 0.358 (HA-RalA vs HA-RalAG23V) and 0.0075 (HA-RalAG23V vs HA-RalAS28N). SERCA2a-ATPase: p = 0.504 (HA-RalA vs HA-RalAG23V) and 0.814 (HA-RalAG23V vs HA-RalAS28N). K GST-pulldown of Flag-SERCA2a from HEK293 cells. Recombinant GST-RalAS28N protein was used as a prey to pull down Flag-SERCA2a from HEK293 cells. L SERCA2a Ca2+-transporting activity in microsomes isolated from HEK293 cells expressing Flag-SERCA2a. Recombinant GST or GST-RalAS28N proteins were added to microsomes before the assay. n = 7. p = 0.030. The data are given as the mean ± SEM. Statistical analyses were carried out using two-sided t-test for 5G, H and one-way ANOVA for 5I, J. One-asterisk indicates p < 0.05. Source data are provided as a Source Data file.
