Journal: bioRxiv

Article Title: β-Coronavirus Nsp6 hijacks host ER translocation machineries into viral replication centers

doi: 10.64898/2025.12.16.694738

Figure Lengend Snippet: (A) Representative images of FL83B cells (top) mock transfected, (middle) transfected with TurboID-mNe empty vector or (bottom) TurboID-mNe-Nsp6 (magenta) and treated with biotin for 1 hour prior to immunostaining with streptavidin-405 antibody (green). (B) TurboID biotinylation experimental approach. (C) Log 2 iBAQ values plotted for the top 30 ER-specific biotinylation hits identified by mass-spectrometry. Nsp6 self-biotinylation (blue dot), translocation machineries (magent dots), post-translocon-associated factors (green dots), and all other ER proteins (black dots). Representative 10 x 10 µm images of (D) EMC5-mNe (H) mNe-OPTI, (L) TMCO1-mNe, or (P) DDOST-mNe (green) and mCh-KDEL (magenta) co-transfected in the absence (top) or presence of HALO-Nsp6 (bottom, blue). MCC analysis of EMC5-mNe, (I) mNe-OPTI, (M) TMCO1-mNe, or (Q) DDOST-mNe with mCh-KDEL without (Control) and with (+Nsp6) HALO-Nsp6 including an mCh-KDEL channel rotated control (Control 90° and +Nsp6 90°). MCC analysis of (E) EMC5-mNe, (I) mNe-OPTI, (M) TMCO1-mNe, or (Q) DDOST-mNe with HALO-Nsp6 (+Nsp6) compared to HALO-Nsp6 channel rotated control (+Nsp6 90°). MCC analysis of (G, K, O, S) HALO-Nsp6 with mCh-KDEL (+Nsp6) and mCh-KDEL rotated control (+Nsp6 90°). For MCC graphs, the large blue square, green circle, and magenta triangle represent the mean for independent biological replicates and corresponding small symbols are each individual data point within the respective replicate (3 biological replicates, n = 15 cells per replicate, error bars = ± standard error of the mean). Scale bars = 5 µm.

Article Snippet: For TurboID proximity biotin ligase immunostaining experiments, FL83B cells transfected with TurboID-mNe-Nsp6, TurboID-mNe-empty vector, or mock transfected were treated as described above however, a Streptavidin Alexa Fluor 405 conjugated antibody (Invitrogen S32351) was used as the primary antibody at 1:100 (10 μg/mL) diluted in blocking buffer.

Techniques: Transfection, Plasmid Preparation, Immunostaining, Mass Spectrometry, Translocation Assay, Control