Fig 2: Normal SNARE complex partners in BET1 fibroblasts with reduced colocalization at the cis-Golgi in transfected cells Quantification of protein levels of BET1 SNARE complex partners and the novel interaction partner ERGIC-53 in fibroblasts. Values are normalized to GAPDH. No significant differences between the control and patient fibroblasts were detected. Data represented are mean ± SD of four biological replicates. Significant differences were analyzed by one-way ANOVA followed by Tukey's post hoc test.Immunofluorescence images of BET1 knockdown HeLa pC4 cells transfected with BET1 siRNA #1 10 min after induction of ER-to-Golgi transport with solubilizer. Scale bar: 20 µm.HeLa pC4 cells were transfected with control siRNA (ctrl) or siRNA against BET1 (#1 and #2) and protein levels of BET1 and ER-to-Golgi complex SNAREs SEC22b, Syntaxin-5, and GOSR2 were analyzed by immunoblot. *residual SEC22b signal after immunoblot stripping.Quantification of EV2C. Data represented are mean ± SD of three biological replicates. Significant differences to ctrl cells were analyzed by one-way ANOVA followed by Dunnett’s post hoc test. ***P = 0.001.Table showing a spectral count-based summary of three independent AP-MS experiments with significant hits shown in Fig 5A. Hits are sorted by the difference of BET1-WT and BET1-Ile51Ser. Hits which do not fulfill the criteria to have a mock-transfected spectral count of two or less are colored in grey.

Fig 3: The expression of the SARS-CoV-2 E protein is restricted to intracellular compartments of the cell.COS-7 cells were transfected with the vector expressing the SARS-CoV-2 E-HA protein. At 24 h, cells were processed for immunostaining. The cells on coverslips were reacted with a mouse monoclonal antibody against HA-tag and with rabbit antibodies against ERGIC53, Golgin-97, or LAMP-1. The cells were washed and reacted with a secondary goat anti-rabbit antibody conjugated to AlexaFluor488 and with a chicken anti-mouse antibody conjugated to AlexaFluor594 for 1 h. Cells on coverslips were counterstained with DAPI and mounted on glass slides with a glycerol-containing mounting medium. To examine the intracellular localization of the E protein with the RER, cis-medial Golgi, or trans Golgi network (TGN), COS-7 cells were co-transfected with the plasmid expressing the E-HA protein, and vectors expressing ERMoxGFP, mNeonGreen-Giantin, or TGN38-GFP. Cells were prepared as above and immunostained with an anti-HA antibody and mounted as above. Coverslips were viewed with a Leica TCS SP8 Confocal Microscope with a 100X objective and a 2X digital zoom using the Leica Application Suite X (LASX) as previously described. A 405nm filter was used to visualize the DAPI, a 594nm filter to visualize HA staining, and a 488 nm filter to visualize the cellular markers. Panels A. Cells co-transfected with vectors expressing SARS-CoV-2 E-HA and ERMoxGFP and immunostained with an anti-HA antibody. Scale bar= 20µm. Panel B. Cells transfected with a vector expressing SARS-CoV-2 E-HA and immunostained with anti-HA and anti-ERGIC53. Panel C. Cells co-transfected with vectors expressing SARS-CoV-2 E-HA and mNeonGreen-Giantin and immunostained with an anti-HA antibody. Panel D. Cells transfected with a vector expressing SARS-CoV-2 E-HA and immunostained with anti-HA and anti-Golgin97. Panel E. Cells co-transfected with vectors expressing SARS-CoV-2 E-HA and TGN38-GFP and immunostained with an anti-HA antibody. Panel F. Cells transfected with the vector expressing SARS-CoV-2 E-HA and immunostained with an anti-HA and anti-LAMP1.

Fig 4: The expression of the SARS-CoV-2 E protein is restricted to intracellular compartments of the cell. COS-7 cells were transfected with the vector expressing the SARS-CoV-2 E-HA protein. At 24 h, cells were processed for immunostaining. The cells on coverslips were reacted with a mouse monoclonal antibody against HA-tag and with rabbit antibodies against ERGIC53, Golgin-97, or LAMP-1. The cells were washed and reacted with a secondary goat anti-rabbit antibody conjugated to AlexaFluor488 and with a chicken anti-mouse antibody conjugated to AlexaFluor594 for 1 h. Cells on coverslips were counterstained with DAPI and mounted on glass slides with a glycerol-containing mounting medium. To examine the intracellular localization of the E protein with the RER, cis-medial Golgi, or trans Golgi network (TGN), COS-7 cells were co-transfected with the plasmid expressing the E-HA protein, and vectors expressing ERMoxGFP, mNeonGreen-Giantin, or TGN38-GFP. Cells were prepared as above and immunostained with an anti-HA antibody and mounted as above. Coverslips were viewed with a Leica TCS SP8 Confocal Microscope with a 100X objective and a 2X digital zoom using the Leica Application Suite X (LASX) as previously described. A 405 nm filter was used to visualize the DAPI, a 594 nm filter to visualize HA staining, and a 488 nm filter to visualize the cellular markers. A. Cells co-transfected with vectors expressing SARS-CoV-2 E-HA and ERMoxGFP and immunostained with an anti-HA antibody. Scale bar = 20 µm. B. Cells transfected with a vector expressing SARS-CoV-2 E-HA and immunostained with anti-HA and anti-ERGIC53. C. Cells co-transfected with vectors expressing SARS-CoV-2 E-HA and mNeonGreen-Giantin and immunostained with an anti-HA antibody. D. Cells transfected with a vector expressing SARS-CoV-2 E-HA and immunostained with anti-HA and anti-Golgin97. E Cells co-transfected with vectors expressing SARS-CoV-2 E-HA and TGN38-GFP and immunostained with an anti-HA antibody. F Cells transfected with the vector expressing SARS-CoV-2 E-HA and immunostained with an anti-HA and anti-LAMP1