Fig. 4
From: Regulation of the ER stress response by a mitochondrial microprotein
Validation of PIGBOS-CLCC1 interaction via split GFP bimolecular complementation. a (Top) Transfection of COS-7 cells with PIGBOS-3 × GFP11 and CLCC1-GFP(1-10) resulted in a GFP signal, which could only occur if the two proteins are close enough to interact and reconstitute a functional GFP. Scale bar: 2 µm. (Bottom) The region in the white box was enlarged, and a cross-sectional analysis of the normalized fluorescence distribution of the Tom20 (MOM), Sec61b (ER), and GFP signals places the GFP signal between the ER and MOM. Scale bar: 0.5 µm. b U2OS cells were co-transfected with CLCC1-GFP(1-10)-HA and PIGBOS-3 × GFP11-FLAG (or PIGBOS-ΔC-3 × GFP11-FLAG). Forty-eight hours later, cells were fixed and stained with FLAG and HA antibodies overnight before imaging. Scale bar: 10 µm. c Flow cytometry measurement of PIGBOS-CLCC1 interaction in HEK293T cells. HEK293T cells were co-transfected with CLCC1-GFP(1-10)-HA and PIGBOS-3 × GFP11-FLAG (or PIGBOS-ΔC-3 × GFP11-FLAG). GFP signals were assessed by flow cytometry 72 hours after transfection. d Quantification of mean GFP intensity in (c). Error bars, s.d., ***p < 0.001 (two tailed unpaired t-test), n = 3 independent experiments. e Flow cytometry measurement of reconstituted PIGBOS-CLCC1 split GFP intensity in HEK293T cells expressing a known ER-mitochondrial tether, VAPB/PTPIP51. Error bars, s.d., **p < 0.01 (two tailed unpaired t-test), n = 5 independent experiments. Source data for Fig. 4a, d and e are provided as a Source Data file
