Fig 1: Confirmation of membralin-interacting protein components by FRET and co-IP. a FRET images of cells co-transfected with vectors expressing membralin-mCherry and either SYVN1-EGFP, AMFR-EGFP, EMC3-EGFP or Ubxd8-EGFP. Scale bar, 10 µm. b Quantification of FRET efficiency between membralin and SYVN1, AMFR, EMC3, Ubxd8, Derlin1, EMC1, Ubac2, FAM8A1, respectively. mCherry-7AA-EGFP was used as a positive control (n = 10 cells). Data represent mean ± s.e.m. from three independent experiments. c Co-IP analysis of endogenous membralin interactions with endogenous SYVN1, or AMFR in N2a cells under normal and ER stress conditions induced by tunicamycin (5 µg/ml) or thapsigargin (500 nM) for 4 hrs. The fold effect shown was calculated using the ratio from AMFR or SYVN1 (IP/Input) normalized to the membralin IP (IP/ß-actin). Non-treated cell samples were set to 1.0
Fig 2: Proteomic analysis of membralin-interacting proteins. a Venn diagram showing the number of interacting proteins identified from cell lysates prepared in NP40 (red), Digitonin (yellow), and TritonX-100 (blue). b Among 180 proteins identified in the membralin interactome (see Supplementary Fig. 1), a heatmap of 14 known ERAD components is presented. The color intensity represents protein intensity (summed peptide area under the curve) (c) Interaction network of the membralin interactome by IPA. Three main ERAD subnetworks were highlighted in green (SYVN1 subnetwork), blue (AMFR subnetwork) and yellow (EMC subnetwork). Nicastrin was highlighted in orange color. Solid and dashed lines represent direct interactions and indirect interactions, respectively
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