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. 2020 May 29;6(22):eaba5412. doi: 10.1126/sciadv.aba5412

Fig. 2. SND1 is a novel ER-associated protein interacting with SEC61A on ER membrane via N-terminal peptide.

Fig. 2

(A) Immunostaining for cellular colocalizations, followed by confocal microscopic analysis by using antibody against SND1, RRBP1, SEC61A, and HLA-A–FLAG. Scale bar, 10 μm. (B) HeLa cells were transfected with the ER reporter plasmids, GFG, HLA-SP-GFG, UGGT1-SP-GFG, GAPDH-NP-GFG, and SND1-NP-GFG, respectively. Western blot for molecular weight of these GFG-tagged fusion proteins expressed in HeLa cells. (C) Colocalizations of these GFG-tagged fusion proteins with SEC61A were detected by confocal microscopy. UGGT1 was used as a positive control for ER-associating protein, while GAPDH was used as a negative control. Scale bar, 20 μm. Fluorescence intensity profiles of regions indicated by short lines are shown in the bottom. (D) Co-IP by antibody against SEC61A for interaction with SND1-GFP or SND1-NP−/−-GFP in HeLa cells transfected with either SND1-GFP (lane 3) or SND1-NP−/−-GFP vector (lane 4). (E) Co-IP by antibody against FLAG for interaction with SND1-NP in HeLa cells transfected with either GFG (lane 3) or SND1-NP-GFG vector (lane 4). (F) Ectopically increased expression of either SND1-GFP or SND1-NP−/−-GFP in SND1-KO HeLa cells followed by Western blot for SND1 and HLA-A expression. WT, wild type.