Fig. 5: BCL-2 forms a complex with iRhom2 and IP3Rs to regulate ER stress-induced cell death.
From: iRhom pseudoproteases regulate ER stress-induced cell death through IP3 receptors and BCL-2
a Cell lysates from two independent pairs of WT and iRhom1/2 DKO MEFs were analysed by immunoblotting with indicated antibodies (left panel). Levels of mRNA transcripts for all IP3Rs in one pair of WT and iRhom1/2 DKO MEFs were determined by quantitative RT-PCR (right panel). Bar chart shows fold change relative to WT cells for each gene as mean ± SEM (n = 3, biologically independent experiments). Two-way ANOVA (Sidak’s), * denotes p < 0.05, ns denotes not significant. b Cell lysates of WT and iRhom1/2 DKO MEFs transiently transfected for 36 h with GFP-IP3R1 (2 µg) and GFP-IP3R3 (2 µg), with tunicamycin (0.5 µg/ml) added for the last 18 h were analysed by immunoblotting with indicated antibodies. c Cell lysates from WT and iRhom1/2 DKO MEFs treated with tunicamycin (0.5 µg/ml, 18 h) were analysed by immunoblotting with indicated antibodies (top panel). Bar chart shows quantification of BCL-2 and BCL-XL proteins (bottom left panel) and mRNA transcripts levels determined by quantitative RT-PCR (bottom right panel). Data presented as fold change relative to WT cells as mean ± SEM (n = 3, biologically independent experiments). Two-way ANOVA (Sidak’s), **** denotes p < 0.0001, *** denotes p < 0.001, ** denotes p < 0.01. d Cell lysates from WT and iRhom1/2 DKO MEFs transfected with control or Bcl-2 RNAi for 72 h, with tunicamycin (0.5 µg/ml) added in the last 18 h were analysed by immunoblotting with indicated antibodies. Bar chart shows quantification of cleaved caspase-3 levels relative to its precursor after tunicamycin shown as mean ± SEM (n = 3, biologically independent experiments), Two-way ANOVA (Sidak’s), **p < 0.01 and ns = not significant (bottom panel). e Levels of iRhom2 and endogenous IP3R1, IP3R3, BCL-2 and BCL-XL were determined by immunoblotting in whole cell lysate (WCL) and immunoprecipitation (IP: HA) from iRhom1/2 DKO MEFs stably expressing iRhom2-HA. f Levels of iRhom2 and BCL-2 were analysed by immunoblotting in whole cell lysate (WCL) and immunoprecipitation (IP: HA) from HEK293T cells transiently co-transfected for 36 h with FLAG-BCL-2_WT (0.25 µg) in combination with wild-type or indicated N-terminal iRhom2 deletions constructs (0.75 µg). g Levels of iRhom2, BCL-2 (WT, ER-targeted, Mitochondria-targeted) were analysed by immunoblotting in whole cell lysate (WCL) and immunoprecipitation (IP: HA) from HEK293T cells transiently transfected for 36 h with iRhom2-HA (0.75 µg) in combination with FLAG-BCL-2_WT (0.25 µg), FLAG-BCL-2_ER (0.25 µg), and FLAG-BCL-2_Mito (0.25 µg). Immunoblotting data are representative of 2–3 independent experiments. Source data are provided as a Source data file.
