Fig. 7
Active STIM1 restores antigen proteolysis and cross-presentation in 3d/3d DCs. a Detection of STIM1 active (STIM1-D76A) and UNC93B1 interaction using Duolink proximity ligation assay (PLA) with anti-GFP (STIM1-D76A) and anti-UNC93B1-specific antibodies in WT or 3d/3d DCs. Quantification of mean fluorescence using ImageJ software (n = 12 cells; mean ± S.E.M.; ns for non-significant). One experiment out of three is shown. Bars = 10 μm. b Fibroblasts expressing GFP-tagged STIM1-D76A and WT or 3d-mutated UNC93B1-FLAG-tagged plasmids were immunoprecipitated with GFP beads (STIM1-D76A) and immunoblotted with anti-GFP and anti-FLAG antibodies. One experiment representative out of two is shown. c Fibroblasts were transiently co-transduced with STIM1-D76A-GFP and WT or 3d-mutated UNC93B1-Cherry-tagged plasmids. Representative TIRF and EPI images of STIM1 active and UNC93B1 (left panel) and quantification of percentage of GFP fluorescence intensity per cell area using ImageJ software (n = 15 cells). One experiment out of two is shown. Bars = 10 μm. d DCs from WT and 3d/3d mice, transfected with STIM1-D76A-GFP or control GFP plasmids, were challenged with OVAb for 6 h before co-culture with CFSE-labeled OT-I T cells. T cell proliferation was monitored by flow cytometry 3 days later (n = 3; mean ± S.E.M.; *P < 0.05, ***P < 0.001 via unpaired t-test). e Phagosomal OVA degradation was measured at different time points in WT and 3d/3d DCs transfected with control GFP or with STIM1-D76A-GFP plasmids (n = 3 experiments; mean ± S.E.M.; *P < 0.05, **P < 0.01 via unpaired t-test). f WT and 3d/3d DCs were treated with ConB (20 nM) 10 min prior addition of OVAb (left panel) or SIINFEKL peptide (right panel) for 6 h. Cells were then extensively washed and co-cultured with B3Z CD8+ T cell hybridoma. T cell activation was monitored by measuring β-galactosidase activity (n = 3 experiments; mean ± S.E.M.; ***P < 0.001, ****P < 0.0001 via unpaired t-test)
