Fig. 4: iRhoms bind to IP3Rs.
From: iRhom pseudoproteases regulate ER stress-induced cell death through IP3 receptors and BCL-2
a Levels of iRhom2 and IP3R1 were analysed by immunoblotting in whole cell lysate (WCL) and immunoprecipitation (IP: HA or IP: FLAG) from HEK293T cells transiently transfected for 36 h with FLAG-IP3R1 and iRhom2-HA. b Levels of iRhom2 and endogenous IP3R1, STIM1 and SERCA2 were determined by immunoblotting in whole cell lysate (WCL) and after immunoprecipitation (IP: HA) from iRhom1/2 DKO MEFs stably expressing iRhom2-HA. c Proximity ligation assay (PLA) in iRhom1/2 DKO HEK293T cells reconstituted with HA-iRhom2 under a tetracycline-inducible promoter were stained using antibodies to detect endogenous IP3R1 and N-terminally HA-tagged iRhom2 after 24 h of 250 ng/ml of doxycycline to induce iRhom2. Dots per cell were quantified using ImageJ and shown as mean ± SEM, uninduced (n = 4 images/166 cells), induced (n = 7 images/259 cells) from two biologically independent experiments. Two-tailed unpaired Student’s t-test ****p < 0.0001. Bottom panels are magnified sections of above corresponding panels. Scale bars = 10 μm. d Levels of WT and deletion mutants of iRhom2 and endogenous IP3R1 were determined by immunoblotting in whole cell lysate (WCL) and immunoprecipitation (IP: HA) from iRhom1/2 DKO MEFs stably expressing iRhom2-WT-HA or iRhom2-ΔIRHD-HA or iRhom2-ΔN-HA. Schematics show iRhom2 mutants with domains deleted (numbering refers to TMDs and IRHD refers to iRhom homology domain). e Levels of WT and deletion mutant of iRhom2 and IP3R1 were determined by immunoblotting in whole cell lysate (WCL) and immunoprecipitation (IP: HA) from HEK293T cells transiently transfected with iRhom2_WT-HA or iRhom2_TMD1_IRHD-HA with FLAG-IP3R1 for 36 h. Schematics show iRhom2 mutant with domains deleted. * denotes unspecific band. f Levels of iRhom2 and IP3R1 were analysed by immunoblotting in whole cell lysate (WCL) and after immunoprecipitation (IP: HA) from HEK293T cells transiently transfected with GFP-IP3R1 and iRhom2-HA for 36 h, with tunicamycin (2 µg/ml) added for last 18 h. Bar chart shows quantification of co-IP GFP-IP3R1 levels relative to total GFP-IP3R1 and shown as mean ± SEM (n = 3, biologically independent experiments). Two-tailed paired Student’s t test, ** denotes p < 0.01. Immunoblotting data are representative of 2–3 independent experiments. Source data are provided as a Source data file.
