Figure 2: The N terminus of iRHOM2 interacts with K16.
From: Rhomboid family member 2 regulates cytoskeletal stress-associated Keratin 16
(a) Confocal microscopy analysis of GFP-tagged iRHOM2 overexpressed in CTRL keratinocytes showing co-localization with endogenous K16. Pseudocoloured ‘product of the differences from the mean’ (PDM) images, in which each pixel is equal to the PDM value at that location, are shown with a PDM scale. Scale bar, 20 μm. (b) Control (CTRL) keratinocyte lysates were immunoprecipitated with anti-K16 antibody and immunoblotted with anti-iRHOM2 antibody. (c) Left panel: cell extracts from CTRL keratinocytes transfected with a vector expressing NT-iRHOM2-GFP were immunoprecipitated with anti-K16 antibody and analysed by western blotting with anti-GFP and anti-K16 antibodies. Right panel: cell extracts from CTRL keratinocytes transfected with a vector expressing HA-tagged iRHOM2 lacking its N terminus (iRHOM2-ΔNT-HA) were immunoprecipitated with anti-K16 antibody and analysed by western blotting with anti-HA and anti-K16 antibodies. (d) CTRL and TOC keratinocyte lysates were immunoprecipitated with anti-K16 antibody and analysed by western blotting with anti-iRHOM1 and anti-iRHOM2 antibody. Graph depicting fold change of iRHOM2 pull-down by K16 in TOC cells compared with CTRL (n=3); error bars denote s.d., *P<0.05 (Student’s t-test). (e) PLA between iRHOM2 and K16 analysed by confocal microscopy in CTRL and TOC keratinocytes. Graph represents quantification of red fluorescent PLA signals relative to 4,6-diamidino-2-phenylindole (DAPI). Mean of 3 experiments, each experiment comprising 12 separate images. Error bars denote s.d. *P<0.05 (Student’s t-test). Scale bar, 20 μm. (f) PLA between K6 and K16 analysed by confocal microscopy. The graph shows quantification of red fluorescent PLA signals relative to DAPI. Mean of 3 experiments, each experiment comprising 12 separate images. Error bars denote s.d.*P<0.05 (Student’s t-test). Scale bar, 20 μm.
