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) (D) Cartoon of domain organization and topology of MHV nonstructural proteins Nsp3-6 derived from literature models and alphafold 3 predictions . (B-C) Representative images of FL83B cells co-transfected with (B) mNe-Nsp3 (green), mCh-Nsp4 (yellow), HALO-Nsp6 (JFX650 conjugated, magenta) and with either (B) BFP-KDEL (blue) or (C) BFP-Sec61β (blue). (D) Images of FL83B cells co-transfected with BFP-KDEL (magenta) and mNe-Nsp3 (top, green), mCh-Nsp4 (middle, green), or HALO-Nsp6 (conjugated to JFX650, bottom, green). (E) Images of FL83B cells co-transfected with BFP-Sec61β (magenta) and mNe-Nsp3 (top, green), mCh-Nsp4 (middle, green), or HALO-Nsp6 (conjugated to JFX650, bottom, green). (F-G) Images of FL83B cells co-transfected with BFP-KDEL (F, blue) or BFP-Sec61β (G, blue) and with mNe-Nsp3 (green) and mCh-Nsp4 (magenta). (H-J) Images of FL83B cells co-transfected with BFP-KDEL (H and J, blue) or BFP-Sec61β (I, blue) and Halo-Nsp6 (magenta) with either (H) mNe-Rtn3L, (I) mNe-SigmaR1, or (J) mCh-Climp63 (green). Inset white boxes shown in left panels are magnified in panels on the right. Scale bars = 5 µm.

Article Snippet: 293T cells (ATCC CRL-3216) were used to produce lentiviral particles for generating acGFP-Sec61β FL83B stable cells.

Techniques: Derivative Assay, Transfection

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: Representative images of FL83B cells co-transfected with (A) mCh-KDEL (magenta), mNe-Sec61β (green), and in the absence (top panels) or presence (bottom panels) of HALO-Nsp6 (JFX650 conjugated, blue). (B-D) A 10 x 10 µm ROIs were used to score protein co-localization (between channels indicated) within ER domains by Mander’s Correlation Coefficient (MCC) analyses. Control 90° and +Nsp6 90° are the MCC measured after rotating one channel 90° relative to the other channel to account for overlap due to chance and pixel density. (E) Images of FL83B cells co-transfected with mCh-KDEL (magenta), Sec61α-mNe, and in the absence (top panels) or presence (bottom panels) of HALO-Nsp6 (blue). (F-H) MCC were measured and graphed as in (B-D) for (E). (I) Images of FL83B cells co-transfected with mCh-KDEL (magenta), SRPRβ-mNe, and in the absence (top panels) or presence (bottom panels) of HALO-Nsp6 (blue). (J-L) MCC were performed as in (B-D) for (I). (M) Images of FL83B cells co-transfected with mCh-KDEL (magenta), WRB-mNe, and in the absence (top panels) or presence (bottom panels) of HALO-Nsp6 (blue). (N-P) MCC were measured and graphed as in (B-D) for (M). 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: 293T cells (ATCC CRL-3216) were used to produce lentiviral particles for generating acGFP-Sec61β FL83B stable cells.

Techniques: Transfection, Control

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: 293T cells (ATCC CRL-3216) were used to produce lentiviral particles for generating acGFP-Sec61β FL83B stable cells.

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

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: Representative time-lapse images of FL83B cells transfected with (A) SRPRβ-mNe (white) or (A) SRPRβ-mNe (white) and HALO-Nsp6 (magenta) imaged before (prebleach) and after photobleaching a region of interest (ROI) in circle shown. Fluorescence recovery was measured within the ROI at times indicated following the photobleaching event and were plotted (on the right as indicated). Statistics measured during fluorescence recovery after photobleaching (FRAP) as indicated. (C-D) Experiments were performed and recorded as in (A-B) for FL83B cells transfected with (C) Sec61α-mNe (white) or (D) Sec61α-mNe (white) and HALO-Nsp6 within the general ER network (Control) or within an Nsp6 domain (Nsp6, magenta), respectively. (E-F) FL83B cells were transfected with (E) SRPRβ-mNe (white) or (F) SRPRβ-mNe (white) and HALO-Nsp6 (magenta) and were bleached in the white circle ROIs beginning at time = 0 and again every 50 seconds. Fluorescence loss in photobleaching (FLIP) was measured in another region of the cell at ROIs (yellow outline) that corresponded to control or Nsp6 domains. FRAP and FLIP curves were fit using a one phase association (FRAP) or one phase decay (FLIP) to extrapolate the mobile fraction, half-time recovery or loss (dashed line, t 1/2 ), recovery or decay rate (k), normalized intensity at t = 0 (I 0 ), and plateau (I ∞ ). Means for each time-point are shown with green (control) or magenta (+Nsp6) circles (3 replicates, n = 5 cells per replicate, error bars = ± standard deviation). Scale bars = 5 µm (A-D). or 20 µm (E-H).

Article Snippet: 293T cells (ATCC CRL-3216) were used to produce lentiviral particles for generating acGFP-Sec61β FL83B stable cells.

Techniques: Transfection, Fluorescence, Control, Standard Deviation

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) Cartoon of experimental workflow performed for B-D. (B) FL83B cells or (C and D) FL83B cells stably expressing acGFP-Sec61β were transfected with (B and C) HALO-Nsp6 or (B and D, E) infected with 0.1 MOI MHV JHM IA and collected at time points indicated for (B) qPCR or (C and D) immunostaining analysis. (B) qPCR plot of ΔCt (Ct, cycle threshold) values calculated from the difference between Nsp6 and actin (actb – 5, see Methods) in samples extracted from infected cell culture pellets (green), HALO-Nsp6 transfected (magenta), or mock transfected cells (black). (C and D) Representative 63X objective images of fixed FL83B cells stably expressing acGFP-Sec61β (green) and (C) HALO-Nsp6 transfected (JFX650 conjugated, 500 ng, magenta) or (D) 0.1 MOI MHV JHM IA infected (mock control, 8, 12, and 18 hpi). Infected cells were immuno-stained with dsRNA to identify replication centers (magenta) relative to Sec61β signal (green). Scale bars = 5 µm (C, D).

Article Snippet: 293T cells (ATCC CRL-3216) were used to produce lentiviral particles for generating acGFP-Sec61β FL83B stable cells.

Techniques: Stable Transfection, Expressing, Transfection, Infection, Immunostaining, Cell Culture, Control, Staining

Journal: bioRxiv

Article Title: Mammalian Cells Integrate Endoplasmic Reticulum and Nuclear Envelope signals to time mitotic entry

doi: 10.64898/2025.12.12.693814

Figure Lengend Snippet: ( A ) To visualize ER dynamics during mitosis, U2OS cells stably expressing mEmerald–Sec61β were stained with NucBlue and treated with DMSO or a well-established ER stress inducer, tunicamycin (Tm; 10 μg/mL) for 6 h before spinning-disk confocal time-lapse imaging every 5 min for 16 h. In parallel, U2OS cells expressing GFP–Sec61β were stained with SiR-DNA, treated with DMSO or another ER stress inducer, thapsigargin (Tg; 200 nM) for 6 h, and imaged every 5 min for 10 h. Prophase onset, identified by chromosome morphology, was set as time 0 min. Scale bars, 10 μm. ( B ) Quantification of mitotic timing revealed that ER stress prolongs specific transitions within mitosis. Phase durations were measured from asynchronously dividing cells in three independent time-lapse experiments, analyzing all mitotic cells in each field (n). Data represent mean ± SEM from individual cells, and statistical significance was determined by one-way ANOVA with Sidak’s multiple comparisons test. ( C ) To examine chromosome congression defects, metaphase alignment was scored one frame before anaphase onset. Cells were grouped by alignment phenotype: fully aligned (blue), broad metaphase plate (green), incomplete plate (purple), or scattered plate (red). The frequency of each category is plotted, revealing increased misalignment under ER stress. ( D ) The timing between metaphase chromosome alignment and the onset of anaphase was further measured. Under ER stress, even cells forming a metaphase plate immediately prior to anaphase frequently exhibited broad plates (>4 μm width), indicating partial or unstable alignment. ( E ) ER organization relative to condensed chromosomes was analyzed by line-scan fluorescence profiles along the yellow line in “ metaphase” (or prometaphase/metaphase) frames. Normalized intensity plots of mEmerald–Sec61β (ER, green) and NucBlue (chromosomes, red) illustrate the distance between the chromosome region (red-shaded) and the nearest ER fluorescence peak (>50% of maximum; green dashed lines). Scale bars, 10 μm. ( F ) Quantification of these ER–chromosome distances across all mitotic cells confirmed that ER stress increases the spatial separation between the ER and metaphase chromosomes, reflecting altered ER distribution during mitosis.

Article Snippet: REEP4 cDNA was synthesized and cloned into pcDNA3.1+N-eGFP by GenScript Biotech (Piscataway, NJ). ptdTomato-Sec61β was constructed by subcloning Sec61β into the ptdTomato-C1 vector (Cat# 632533; Clontech).

Techniques: Stable Transfection, Expressing, Staining, Imaging, Fluorescence

Journal: bioRxiv

Article Title: Mammalian Cells Integrate Endoplasmic Reticulum and Nuclear Envelope signals to time mitotic entry

doi: 10.64898/2025.12.12.693814

Figure Lengend Snippet: ( A, B ) Time-lapse imaging of ER distribution around MTOCs in U2OS cells under control conditions (DMSO, A) or ER stress (Tm, B). Cells stably expressing GFP–α-tubulin and tdTomato–Sec61β were stained with SiR–DNA, treated with DMSO or Tm (10 µg/mL) for 6 h, and imaged over time, with prophase onset defined as time 0 min. Arrowheads mark MTOCs, and enlarged views highlight ER organization before and after MTOC division, accompanied by line-scan fluorescence intensity profiles of ER (red) around MTOCs. Scale bars, 10 µm. ( C, D ) Effect of CLIMP63(1–192) overexpression on ER organization around MTOCs in the presence or absence of ER stress. U2OS cells co-expressing GFP–α-tubulin and mCherry–CLIMP63(1–192) were treated with DMSO (C) or Tm (D) and imaged by time-lapse microscopy to visualize how CLIMP63(1–192) modulates ER distribution surrounding MTOCs under both unstressed and Tm-treated conditions. Scale bars, 10 µm.

Article Snippet: REEP4 cDNA was synthesized and cloned into pcDNA3.1+N-eGFP by GenScript Biotech (Piscataway, NJ). ptdTomato-Sec61β was constructed by subcloning Sec61β into the ptdTomato-C1 vector (Cat# 632533; Clontech).

Techniques: Imaging, Control, Stable Transfection, Expressing, Staining, Fluorescence, Over Expression, Time-lapse Microscopy

Journal: bioRxiv

Article Title: Mammalian Cells Integrate Endoplasmic Reticulum and Nuclear Envelope signals to time mitotic entry

doi: 10.64898/2025.12.12.693814

Figure Lengend Snippet: ( A ) Time-lapse imaging of U2OS cells stably expressing GFP–Sec61β reveals that low-dose MG132 (0.2 µM) prolongs metaphase while still permitting anaphase onset, creating a window to visualize how the ER remodels under sustained metaphase tension. Prophase onset, marked by chromosome condensation, was designated as time 0 min. Scale bars, 10 µm. ( B ) Quantitative analysis of mitotic phase durations under these conditions shows that even partial inhibition of proteasome-dependent APC/C activity is sufficient to extend metaphase. U2OS cells expressing GFP–Sec61β or CLIMP63(1–192) were pretreated with DMSO or MG132 (0.2 µM) as indicated. Data represent mean ± SEM from individual cells. ( C ) Representative images of early and late metaphase (as defined in ) illustrate that ER–chromosome relationships become progressively distorted when metaphase is prolonged. Corresponding line-scan profiles of normalized GFP–Sec61β (green) and SiR–DNA (red) intensities highlight a loss of the tight ER association with condensed chromosomes seen in control cells. Scale bars, 5 µm. ( D ) Quantification of ER-chromosome distances in cells expressing GFP–Sec61β demonstrates that low-dose MG132 consistently decreased ER-chromosome distances, linking altered ER architecture to a defined change in mitotic timing. Data represent mean ± SEM from individual cells. ( E ) To determine whether this mitotic perturbation engages ER stress signaling, IRE1 activity was monitored by IRE1–mNeonGreen oligomerization. Cells treated with DMSO, Tm, or MG132 for the indicated times show an increase in the fraction of cells with IRE1 foci under Tm but not by MG132. Scale bars, 20 µm. ( F ) ATF6 pathway engagement was assessed in parallel by immunoblotting for ATF6 cleavage. U2OS cells treated with DMSO or MG132 (0.2 µM) for 6 h did not exhibit a detectable increase in the cleaved (∼50 kDa) ATF6 fraction relative to full-length (∼100 kDa) species. Tm treatment generated cleaved ATF6, showing the robust activation as expected. Fold changes in cleaved ATF6 were normalized to DMSO controls, with GAPDH as a loading control. ( G ) PERK signaling branch activation under low-dose MG132 was evaluated by monitoring eIF2α phosphorylation at Ser51. MG132 (0.2 µM) did not induce a time-dependent increase in phospho-eIF2α while Tm (10 µg/mL) treatment induced robust levels of phospho-eIF2α. Phospho-eIF2α levels were normalized to total eIF2α and expressed relative to time 0, and P values were determined by one-way ANOVA with Sidak’s multiple comparisons test. In (B) and (D), data represent mean ± SEM from individual cells from three independent time lapse experiments; in (E–G), mean ± SEM from three independent experiments

Article Snippet: REEP4 cDNA was synthesized and cloned into pcDNA3.1+N-eGFP by GenScript Biotech (Piscataway, NJ). ptdTomato-Sec61β was constructed by subcloning Sec61β into the ptdTomato-C1 vector (Cat# 632533; Clontech).

Techniques: Imaging, Stable Transfection, Expressing, Inhibition, Activity Assay, Control, Western Blot, Generated, Activation Assay, Phospho-proteomics

Journal: EMBO Reports

Article Title: Sec61β maintains cytoplasmic proteostasis via ARIH1-mediated translational repression upon ER stress

doi: 10.1038/s44319-026-00690-y

Figure Lengend Snippet: ( A ) Gene Ontology (GO) analysis of Derlins-interacting proteins by thapsigargin (Tg) treatment. The bar graph shows the top 10 GO molecular function terms with a false discovery rate of <0.05 calculated from the DAVID online tool. P values were calculated using the modified Fisher’s exact test implemented in DAVID; 37 proteins identified as RNA binding in terms of molecular function are listed in Dataset . ( B ) Interactions of Derlins with Sec61β. HEK293 cells transfected with indicated plasmids and treated with or without 50 nM Tg and 200 nM MG132 for 16 h were immunoprecipitated (IPed) with an anti-Flag antibody and immunoblotted with indicated antibodies. ( C ) Endogenous interaction of Derlin-1 with Sec61β. Immunoprecipitation (IP) with anti-Sec61β antibody or control (Ctrl) IgG using Protein G Sepharose and immunoblotting (IB) with indicated antibodies in HepG2 cells treated with or without 200 nM Tg and/or 500 nM MG132 for 16 h. ( D – F ) IB of ERpQC substrates in HEK293 cells transfected with indicated siRNAs and plasmids and treated with or without 50 nM Tg and 200 nM MG132 for 16 h. All samples were immunoblotted with indicated antibodies. Black arrowhead, signal peptide-uncleaved NHK QQQ ( S NHK QQQ ); white arrowhead, signal peptide-cleaved NHK QQQ ( C NHK QQQ ). ( G ) IB of ERpQC substrate in wild-type (WT) or Derlin-1, -2 , and -3 triple knockout (TKO) HEK293 cells transfected with indicated siRNAs and plasmid for NHK QQQ and treated with or without 50 nM Tg and 200 nM MG132 for 16 h. All samples were immunoblotted with indicated antibodies. Expression levels of S NHK QQQ were calculated and shown as the percentage of S NHK QQQ out of the total amount of NHK QQQ ( S NHK QQQ and C NHK QQQ ). Black arrowhead, S NHK QQQ ; white arrowhead, C NHK QQQ . .

Article Snippet: Wild-type and Derlin-1 , Derlin-2 , and Derlin-3 triple knockout HEK293 cells (Kadowaki et al, ) and 3× Flag-tagged Sec61β knock-in HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque; 08459-64) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin solution (Nacalai Tesque; 09367-34).

Techniques: Modification, RNA Binding Assay, Transfection, Immunoprecipitation, Control, Western Blot, Triple Knockout, Plasmid Preparation, Expressing

Journal: EMBO Reports

Article Title: Sec61β maintains cytoplasmic proteostasis via ARIH1-mediated translational repression upon ER stress

doi: 10.1038/s44319-026-00690-y

Figure Lengend Snippet: ( A ) Gene Ontology (GO) analysis of Derlins-interacting proteins by thapsigargin (Tg) and MG132 treatment. The bar graph shows the top 10 GO molecular function terms with a false discovery rate of <0.05 calculated from the DAVID online tool. P values were calculated using the modified Fisher’s exact test implemented in DAVID. eIF4A1 and Sec61β are included among 43 proteins identified as RNA binding in terms of molecular function. Related to Fig. . ( B ) Interactions of exogenous Derlins with endogenous eIF4A1 and eIF4E. HEK293 cells transfected with indicated plasmids and treated with or without 50 nM Tg and 200 nM MG132 for 16 h were immunoprecipitated (IPed) with an anti-Flag antibody and immunoblotted with indicated antibodies. ( C , D ) Endogenous interactions of Derlin-1 ( C ) or Derlin-2 ( D ) with eIF4A1 ( C , D ) and eIF4E ( D ). HepG2 cells treated with or without 200 nM Tg and 500 nM MG132 for 16 h were IPed with an anti-Derlin-1 or an anti-Derlin-2 antibody and immunoblotted with indicated antibodies. ( E ) Interaction of endogenous Derlin-1 with exogenous eIF4E. HEK293 cells transfected with Flag-eIF4E and treated with or without 50 nM Tg and 200 nM MG132 for 16 h were IPed with an anti-Flag antibody and immunoblotted with indicated antibodies.

Article Snippet: Wild-type and Derlin-1 , Derlin-2 , and Derlin-3 triple knockout HEK293 cells (Kadowaki et al, ) and 3× Flag-tagged Sec61β knock-in HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque; 08459-64) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin solution (Nacalai Tesque; 09367-34).

Techniques: Modification, RNA Binding Assay, Transfection, Immunoprecipitation

Journal: EMBO Reports

Article Title: Sec61β maintains cytoplasmic proteostasis via ARIH1-mediated translational repression upon ER stress

doi: 10.1038/s44319-026-00690-y

Figure Lengend Snippet: ( A ) Volcano plots of the quantitative proteomic analysis of ER stress-induced Sec61β interactome. Plots indicate the fold change in the abundance of identified proteins in Tg and MG132-treated samples compared with that in DMSO-treated samples (log2 fold change [Tg_MG132/DMSO], x axis) against its significance (−log10 P value, y axis) ( n = 4). Orange, significant fold change (Tg_MG132/DMSO) ≥ 2, P value ≤ 0.05; red, ARIH1. See also Dataset . ( B ) Domain structures of human ARIH1 and truncated forms with or without mutation. UBA-L, ubiquitin-associated domain-like; RING1, RING domain 1; IBR, in-between RING domain; RING2, RING domain 2; C357S (CS), catalytically inactive serine mutant of Cys357, the active site of Ub ligase activity; ΔAri, mutant lacking inhibitory Ariadne domain. ( C ) Endogenous interaction of Sec61β with ARIH1 during ER stress. IP with an anti-Flag antibody and IB with indicated antibodies in WT or 3× Flag-tagged Sec61β knock-in HEK293 cells and treated with or without 50 nM Tg and/or 200 nM MG132 for 16 h. ( D ) Interaction of endogenous Sec61β and Derlin-1 with exogenous ARIH1 during ER stress. IP with an anti-Flag antibody and IB with indicated antibodies in HEK293 cells transfected with Flag-ARIH1 and treated with or without 50 nM Tg and 200 nM MG132 for 16 h. The amounts of co-IPed proteins with Flag-ARIH1 were normalized by the amount of input for each protein and shown as fold increases relative to the control lane. ( E ) Endogenous interaction of Sec61β and ARIH1 with Derlin-1 during ER stress. Wild-type C57BL/6 J mice (12 to 14-week-old), matched for sex, were given a single 2 μg/gram body weight intraperitoneal injection of a 0.1 mg/ml suspension of tunicamycin (Tun) in PBS or vehicle (PBS) alone. After 20 h, mice were deeply anesthetized and transcardially perfused with PBS. Whole cell lysates were prepared by homogenizing livers for 60 s × five times in lysis buffer. Cell lysates were IPed with anti-Derlin-1 antibody or control IgG using Protein G Sepharose. All samples were immunoblotted with indicated antibodies. ( F , G ) Interaction of endogenous ARIH1 ( F ), Sec61β ( F , G ), or Derlin-1 ( F , G ) with exogenous 4EHP in HEK293 cells ( F ) and ARIH1-deficient HEK293 cells ( G ). IP with an anti-Flag antibody and IB with indicated antibodies in HEK293 cells transfected with indicated siRNAs ( G ) and Flag-4EHP ( F , G ) and treated with or without 50 nM Tg and 200 nM MG132 for 16 h. The amounts of co-IPed proteins with Flag-4EHP were normalized by the amount of input for each protein and shown as fold changes relative to the control lane. ( H , I ) Interaction of endogenous Derlin-1 with exogenous eIF4E in ARIH1- ( H ) or Sec61β- ( I ) deficient cells. IP with an anti-Flag antibody and IB with indicated antibodies in HEK293 cells transfected with indicated siRNAs and Flag-eIF4E and treated with 50 nM Tg and 200 nM MG132 for 16 h. The amount of co-IPed Derlin-1 with Flag-eIF4E was normalized by the amount of input Derlin-1 and shown as fold increase relative to the control lane. .

Article Snippet: Wild-type and Derlin-1 , Derlin-2 , and Derlin-3 triple knockout HEK293 cells (Kadowaki et al, ) and 3× Flag-tagged Sec61β knock-in HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque; 08459-64) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin solution (Nacalai Tesque; 09367-34).

Techniques: Mutagenesis, Ubiquitin Proteomics, Activity Assay, Knock-In, Transfection, Control, Injection, Suspension, Lysis

Journal: EMBO Reports

Article Title: Sec61β maintains cytoplasmic proteostasis via ARIH1-mediated translational repression upon ER stress

doi: 10.1038/s44319-026-00690-y

Figure Lengend Snippet: ( A – D ) Representative fluorescence images of interaction between Sec61β and ARIH1 ( A , B ) or 4EHP ( C , D ) detected by a proximity ligation assay (PLA). HepG2 cells transfected with GFP-KDEL, Flag-Sec61β and HA-ARIH1 ( A , B ) or HA-4EHP ( C , D ) were treated with 50 nM Tg and 200 nM MG132 for 16 h. PLA was performed using anti-Flag and anti-HA antibodies. GFP-KDEL (green, ER marker), PLA signal (red, Flag/HA) and DAPI (blue, nuclei) are shown. Scale bars, 3–10 μm. ( E – G ) RNA immunoprecipitation (RIP) of Flag-4EHP with DUSP6 mRNA ( E ) and ERpQC substrate TTR mRNA ( F ). RIP of Flag-4EHP with TTR mRNA in Sec61β-deficient cells ( G ). HepG2 cells were transfected with Flag-4EHP alone ( E , F ) or together with siCtrl or siSec61β ( G ) and treated with or without 50 nM Tg and 200 nM MG132 for 16 h ( E , F ) or with 50 nM Tg and 200 nM MG132 for 16 h ( G ). Flag-4EHP was IPed using an anti-Flag antibody, and the levels of the indicated mRNAs (normalized to input) in Flag-4EHP-bound mRNA were analyzed by RT-qPCR ( E , F ; n = 3, G ; n = 4). ( H , I ) Interaction of exogenous ( H ) or endogenous ( I ) 4EHP and endogenous Sec61β with ARIH1 mutants upon ER stress. IP with an anti-Flag antibody and IB with indicated antibodies in HEK293 cells transfected with indicated plasmids and treated with 50 nM Tg and 200 nM MG132 for 16 h. The amounts of co-IPed proteins with Flag-ARIH1 were normalized by the amount of IPed Flag-ARIH1 and the input amount of each protein and shown as fold changes relative to the intensity observed in IP with Flag-ARIH1(CC) retaining its E3 ligase activity. Details regarding the ARIH1 mutants are described in Fig. . ΔAri, mutant lacking the inhibitory Ariadne domain; CS, catalytically inactive serine mutant of Cys357; S427D, phospho-mimetic mutant of Ser427 to Asp on the Ariadne domain. ( J ) IB of ERpQC substrate in HEK293 cells transfected with indicated siRNAs and plasmids and then treated with or without 50 nM Tg and 200 nM MG132 for 16 h; samples were immunoblotted with indicated antibodies. 4KR, non-ISGylatable 4EHP mutant (Lys121/130/134/222 to Arg). Data are means ± SEM. * P < 0.05, ** P < 0.01; n.s., not significant. Two-tailed unpaired t test for DMSO vs Tg+MG ( E , F ) and for siCtrl vs siSec61β ( G ).

Article Snippet: Wild-type and Derlin-1 , Derlin-2 , and Derlin-3 triple knockout HEK293 cells (Kadowaki et al, ) and 3× Flag-tagged Sec61β knock-in HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque; 08459-64) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin solution (Nacalai Tesque; 09367-34).

Techniques: Fluorescence, Proximity Ligation Assay, Transfection, Marker, RNA Immunoprecipitation, Quantitative RT-PCR, Activity Assay, Mutagenesis, Two Tailed Test

Journal: EMBO Reports

Article Title: Sec61β maintains cytoplasmic proteostasis via ARIH1-mediated translational repression upon ER stress

doi: 10.1038/s44319-026-00690-y

Figure Lengend Snippet: ( A , B ) IB of ERpQC substrate in HEK293 cells transfected with siRNAs against ARIH1 ( A ), 4EHP ( B ), or Sec61β ( A , B ) and Flag-NHK QQQ and treated with or without 50 nM Tg and 200 nM MG132 for 16 h; samples were immunoblotted with indicated antibodies. Bar graph: Ratio of the expression level of S NHK QQQ to the total amount of NHK QQQ was calculated and shown ( n = 3). ( C ) IB of ERpQC substrates in HEK293 cells transfected with siRNA against 4EHP and cDNAs for Flag-NHK QQQ and HA-4EHP and treated with or without 50 nM Tg and 200 nM MG132 for 16 h; samples were immunoblotted with indicated antibodies. Bar graph: Ratio of expression level of S NHK QQQ to the total amount of NHK QQQ was calculated and shown ( n = 3). ( D , E ) RIP of Flag-4EHP with ERpQC substrate mRNA, α1AT . HepG2 cells were transfected with Flag-4EHP alone ( D ) or together with siCtrl or siSec61β ( E ) and treated with or without 50 nM Tg and 200 nM MG132 for 16 h ( D ) or with 50 nM Tg and 200 nM MG132 for 16 h ( E ). Flag-4EHP was IPed using an anti-Flag antibody, and the levels of the indicated mRNAs (normalized to input) in Flag-4EHP-bound mRNA were analyzed by RT-qPCR ( D ; n = 3, E ; n = 4). ( F ) IB of ERpQC substrates in HEK293 cells transfected with siRNA against ARIH1 UTR and cDNAs for Flag-NHK QQQ and HA-ARIH1ΔAri(CC) or (CS) and treated with or without 50 nM Tg and 200 nM MG132 for 16 h; samples were immunoblotted with indicated antibodies. Bar graph: Ratio of expression level of S NHK QQQ to the total amount of NHK QQQ was calculated and shown ( n = 3). Data are means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; n.s., not significant. Two-tailed unpaired t test for siCtrl vs siARIH1 ( A ), siCtrl vs si4EHP ( B ) or siCtrl vs siSec61β ( A , B ); one-way ANOVA with Tukey’s multiple comparisons test ( C ). Two-tailed unpaired t test for DMSO vs Tg+MG ( D ) or siCtrl vs siSec61β ( E ); one-way ANOVA with Tukey’s multiple comparisons test ( F ). For ( A – C , F ), black arrowhead, S NHK QQQ ; white arrowhead, C NHK QQQ . .

Article Snippet: Wild-type and Derlin-1 , Derlin-2 , and Derlin-3 triple knockout HEK293 cells (Kadowaki et al, ) and 3× Flag-tagged Sec61β knock-in HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque; 08459-64) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin solution (Nacalai Tesque; 09367-34).

Techniques: Transfection, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: EMBO Reports

Article Title: Sec61β maintains cytoplasmic proteostasis via ARIH1-mediated translational repression upon ER stress

doi: 10.1038/s44319-026-00690-y

Figure Lengend Snippet: ( A – D ) IB of ERpQC substrate in HEK293 cells transfected with indicated siRNAs and plasmids and then treated with or without 50 nM Tg and 200 nM MG132 for 16 h; samples were immunoblotted with indicated antibodies. Bar graph: Ratio of the expression level of S NHK QQQ to the total amount of NHK QQQ ( n = 3 for A , C , D and n = 4 for B ). ( E ) Domain structure of human Sec61β and its truncated form. IDR intrinsically disordered region, TM transmembrane domain. ( F ) Interaction of endogenous ARIH1 and 4EHP with exogenous Sec61β. IP with an anti-Flag antibody and IB with indicated antibodies in HEK293 cells transfected with indicated plasmids and treated with 50 nM Tg and 200 nM MG132 for 16 h. Two endogenous 4EHP bands were detected in the input, and both interacted with Sec61βWT (arrows). These bands may reflect post-translational modified 4EHP, including previously reported ubiquitination or ISGylation, in addition to the possibility of splicing products. Arrows, 4EHP; asterisk, a non-specific band unrelated to 4EHP that appears due to overexpression of 3× Flag-Sec61β. ( G ) IB of ERpQC substrate in HEK293 cells transfected with Sec61β UTR siRNA and indicated plasmids treated with or without 50 nM Tg and 200 nM MG132 for 16 h. For exogenous Flag-Sec61β, lysates were IPed with an anti-Flag antibody and immunoblotted with an anti-Flag antibody; input samples were immunoblotted with indicated antibodies. Bar graph: ratio of the expression level of S NHK QQQ to the total amount of NHK QQQ was calculated and shown ( n = 3). Data are means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; n.s., not significant. One-way ANOVA with Tukey’s multiple comparisons test. For ( A – D , G ), black arrowhead, S NHK QQQ ; white arrowhead, C NHK QQQ . .

Article Snippet: Wild-type and Derlin-1 , Derlin-2 , and Derlin-3 triple knockout HEK293 cells (Kadowaki et al, ) and 3× Flag-tagged Sec61β knock-in HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque; 08459-64) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin solution (Nacalai Tesque; 09367-34).

Techniques: Transfection, Expressing, Modification, Ubiquitin Proteomics, Over Expression

Journal: EMBO Reports

Article Title: Sec61β maintains cytoplasmic proteostasis via ARIH1-mediated translational repression upon ER stress

doi: 10.1038/s44319-026-00690-y

Figure Lengend Snippet: ( A ) Proteasome chymotrypsin-like peptidase activity of cell extracts from HepG2 cells transfected with siCtrl or siSec61β (#1, #2, or #3) and treated with 200 nM Tg for 16 h was measured using Suc-LLVY-AMC as a substrate. Fluorescence intensity was normalized to cell viability in each condition. Proteasome activity is shown as fold decrease relative to that of siCtrl-transfected cells ( n = 3). ( B ) Degradation of the cytoplasmic protein CL1 degron after a 15-min pulse of [ 35 S]-methionine/cysteine metabolic labeling, followed by the indicated chase periods. Lysates of HEK293 cells transfected with indicated siRNAs and Venus-CL1-Flag and stimulated with 50 nM Tg for 16 h were IPed with an anti-Flag antibody and resolved via SDS-PAGE. The relative radioactivity of Venus-CL1-Flag at different chase times was calculated and shown as fold decreases relative to the intensity observed at 0 h chase ( n = 3). ( C ) Representative fluorescence images of HepG2 cells transfected with siCtrl or siSec61β and stimulated with 50 nM Tg for 10 h, followed by staining with ProteoStat (green, protein aggregation) and DAPI (blue, nuclei). Scale bars, 25 μm. ( D , E ) Quantification of protein aggregation in Sec61β-deficient HepG2 cells using ProteoStat and flow cytometry. Mean ProteoStat fluorescence in HepG2 cells transfected with siCtrl or siSec61β and stimulated with 50 nM Tg for 4 h ( D ) and relative mean ProteoStat fluorescence in HepG2 cells transfected with siSec61β and indicated cDNAs and stimulated with 50 nM Tg for 4 h ( E ) were analyzed by flow cytometry and software. Boxes represent the 25th–75th percentiles with the median indicated; whiskers represent the minimum and maximum values ( n = 8 for D or n = 3 for E ). ( F ) Superimposed images of tail movements at 3 dpf and a scheme of the quantitative analysis of head-tail angle. Compared with larvae injected with water or control Sec61β-atg-5mis MO, Sec61β-deficient zebrafish exhibited impaired swimming behavior. ( G ) Histogram showing the maximum head-tail angles at 3 dpf. Sec61β-deficient zebrafish ( n = 17) showed reduced maximum head-tail angles compared to control animals ( n = 17 for water injection or n = 18 for Sec61β-atg-5mis MO injection). ( H ) Histogram showing the distribution of phenotype scores of zebrafish at 4 dpf. The degree of morphological and swimming abnormalities was scored from 0 to 3 as shown in Fig. . Sec61β-deficient zebrafish ( n = 49) showed greater abnormality scores than control animals ( n = 44 for water injection or n = 52 for Sec61β-atg-5mis MO injection). ( I ) Histogram showing the rescue of abnormal phenotype in Sec61β-deficient zebrafish by ARIH1 at 4 dpf. Exogenous expression of human ARIH1ΔAri(CC) ( n = 124), but not ARIH1ΔAri(CS) ( n = 113), reduced the abnormal phenotype score in Sec61β-deficient zebrafish ( n = 104). Data are means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; n.s., not significant. Two-tailed unpaired t test for siCtrl vs siSec61β ( A , B ); two-tailed paired t test for siCtrl vs siSec61β ( D ); two-tailed unpaired t test for Sec61β WT vs Sec61β ΔIDR ( E ); one-way ANOVA with Tukey’s multiple comparisons test ( G ); Fisher’s exact test followed by Bonferroni’s post hoc test ( H , I ). .

Article Snippet: Wild-type and Derlin-1 , Derlin-2 , and Derlin-3 triple knockout HEK293 cells (Kadowaki et al, ) and 3× Flag-tagged Sec61β knock-in HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque; 08459-64) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin solution (Nacalai Tesque; 09367-34).

Techniques: Activity Assay, Transfection, Fluorescence, Labeling, SDS Page, Radioactivity, Staining, Flow Cytometry, Software, Injection, Control, Expressing, Two Tailed Test

Journal: EMBO Reports

Article Title: Sec61β maintains cytoplasmic proteostasis via ARIH1-mediated translational repression upon ER stress

doi: 10.1038/s44319-026-00690-y

Figure Lengend Snippet: ( A ) Proteasome chymotrypsin-like peptidase activity of cell extracts from HepG2 cells transfected with siCtrl or siSec61β (#1, #2, or #3) and treated with 2 μg/ml tunicamycin (Tun) for 16 h was measured using Suc-LLVY-AMC as a substrate. Fluorescence intensity was normalized to cell viability in each condition. Proteasome activity is shown as fold decrease relative to that of siCtrl-transfected cells ( n = 7, 7, 6, and 7 from left to right, respectively). ( B ) Degradation of CL1 degron chased for the indicated periods after 15 min pulse of [ 35 S]-methionine/cysteine metabolic labeling shown in Fig. . Cell lysates of HEK293 cells transfected with indicated siRNAs and Venus-CL1-Flag and stimulated with 50 nM Tg for 16 h were IPed with an anti-Flag antibody, resolved by SDS-PAGE and analyzed by autoradiography. ( C ) Representative fluorescence images of HepG2 cells transfected with siSec61β and stimulated with 50 nM Tg for 6 h, followed by staining with ProteoStat (red, protein aggregation), calnexin (green, ER membranes) and DAPI (blue, nuclei). Scale bars, 25 μm. ( D ) Reduced expression of Sec61β in zebrafish by injection of antisense morpholino oligonucleotide (MO). Antisense MO was designed as described in Methods. MOs were injected into zebrafish embryos at 1- to 2-cell stages. The expression of Sec61β in zebrafish at 3 dpf was analyzed by IB using a polyclonal antibody against a peptide against zebrafish Sec61β (SAGTGGMWRFYTEDSPGLKV) raised in rabbits. Lysates were prepared by homogenizing 3 dpf fish for 60 s in lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EGTA, and 1% Triton X-100) supplemented with 5 μg/mL leupeptin (Nacalai Tesque; 43449-62) on ice using a Micro Smash (TOMY; MS-100) (4500 rpm, 4 °C). Lysates were resolved by SDS-PAGE and blotted onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk in TBS-T (50 mM Tris-HCl pH 8.0, 150 mM NaCl, and 0.05% Tween-20), the membranes were probed with antibody against to zebrafish Sec61β (1/1000) or actin (1/5000) diluted in 5% BSA in TBS-T overnight at 4 °C. Secondary antibodies [IRDye 800CW Donkey anti-Rabbit IgG (H + L) (1/10,000) and IRDye 680RD Donkey anti-Mouse IgG (H + L) (1/10,000)] were diluted in 5% skim milk in TBS-T, and membranes were incubated 2 h at room temperature. Images were revealed and analyzed using Odyssey CLx (LICOR) and Empiria Studio software 3.0 (LICOR). ( E ) Representative images showing the typical morphology of zebrafish larvae injected with water, Sec61β-atg MO or Sec61β-5mis MO at 3 dpf. Arrowhead indicates the abnormal morphology observed in rare Sec61β-deficient zebrafish. ( F ) Histogram showing the average body length of zebrafish larvae relative to that of water-injected controls at 3 dpf. Sec61β-deficient zebrafish ( n = 13) showed a tendency toward reduced body length compared to control groups ( n = 10 for water injection or n = 24 for Sec61β-atg-5mis MO injection). ( G ) The behavior of zebrafish was observed at 4 dpf, and a phenotype scores were recorded manually. The scoring criteria were defined as follows: no obvious abnormality (0), abnormal swimming with head shaking and slightly smaller size (1), abnormal swimming and morphology (2), and no swimming and abnormal morphology (3). ( H ) Exogenous expression of ARIH1 mutants in zebrafish by injection of synthesized mRNAs. Synthesized mRNAs for human ARIHΔAri(CC) or (CS) were co-injected into zebrafish embryos at 1- to 2-cell stages. Lysates were prepared by homogenizing 3 dpf 10 fish for 60 s in lysis buffer due to weak expression of exogenous proteins. The expression of HA-ARIHΔAri was analyzed by IB using a rat monoclonal antibody against HA (clone 3F10) and secondary antibody (HRP-linked anti-rat IgG antibody). The membranes were detected by an ECL system, and images were revealed and analyzed using ChemiDoc Touch (BioRad). Data are means ± SEM. * P < 0.05, ** P < 0.01, **** P < 0.0001; n.s., not significant. Two-tailed unpaired t test for siCtrl vs siSec61β ( A ); Kruskal–Wallis rank-sum test ( F ).

Article Snippet: Wild-type and Derlin-1 , Derlin-2 , and Derlin-3 triple knockout HEK293 cells (Kadowaki et al, ) and 3× Flag-tagged Sec61β knock-in HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque; 08459-64) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin solution (Nacalai Tesque; 09367-34).

Techniques: Activity Assay, Transfection, Fluorescence, Labeling, SDS Page, Autoradiography, Staining, Expressing, Injection, Lysis, Blocking Assay, Incubation, Software, Control, Synthesized, Two Tailed Test

Journal: EMBO Reports

Article Title: Sec61β maintains cytoplasmic proteostasis via ARIH1-mediated translational repression upon ER stress

doi: 10.1038/s44319-026-00690-y

Figure Lengend Snippet: Under ER stress conditions, chaperones such as BiP and PDI continue to be synthesized by cotranslational translocation into the ER lumen (upper left). The signal sequence of nascent polypeptides emerging from the ribosome is recognized by SRP, and RNC–SRP complex docks onto SRα/β. When the SRP dissociates from the RNC, the nascent polypeptide is delivered into the ER, where it undergoes proper folding. By contrast, in ERpQC (upper right), some secretory proteins are degraded by the proteasome without translocation into the ER. ERpQC is triggered by recruitment of Derlins to the Sec61 translocon and SR in response to ER stress. Derlins interact with SRP54 on the RNC through their C-terminus, thereby rerouting the nascent polypeptide from the ER translocation pathway to the cytoplasmic degradation pathway (rerouting). Sec61β bound to Derlins recruits the E3 ligase ARIH1 via its IDR upon ER stress. ARIH1, guided in the vicinity of the ER membrane, enables 4EHP to associate with the 5ʹ cap structure of the ERpQC substrate mRNAs captured by Derlins, thereby causing translational repression of ERpQC substrates (translational repression). Rerouted ERpQC substrates that have already initiated translation are ubiquitinated by HRD1 and effectively transported to the proteasome by p97 and Bag6 (proteasomal degradation). Derlins–Sec61β–ARIH1 complex-mediated translational repression contributes to reduced proteasomal degradation load of cytosolic unfolded proteins, leading to inhibition of aggresome formation and maintenance of cytoplasmic proteostasis. Conversely, Sec61β deficiency allows eIF4F recruitment to the mRNA 5ʹ cap structure of the Derlins-bound RNC and polysome formation, resulting in the overproduction of ERpQC substrates and disruption of cytosolic proteostasis (lower).

Article Snippet: Wild-type and Derlin-1 , Derlin-2 , and Derlin-3 triple knockout HEK293 cells (Kadowaki et al, ) and 3× Flag-tagged Sec61β knock-in HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque; 08459-64) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin solution (Nacalai Tesque; 09367-34).

Techniques: Synthesized, Translocation Assay, Sequencing, Membrane, Inhibition, Disruption

Journal: EMBO Reports

Article Title: Sec61β maintains cytoplasmic proteostasis via ARIH1-mediated translational repression upon ER stress

doi: 10.1038/s44319-026-00690-y

Figure Lengend Snippet: ( A ) Gene Ontology (GO) analysis of Derlins-interacting proteins by thapsigargin (Tg) treatment. The bar graph shows the top 10 GO molecular function terms with a false discovery rate of <0.05 calculated from the DAVID online tool. P values were calculated using the modified Fisher’s exact test implemented in DAVID; 37 proteins identified as RNA binding in terms of molecular function are listed in Dataset . ( B ) Interactions of Derlins with Sec61β. HEK293 cells transfected with indicated plasmids and treated with or without 50 nM Tg and 200 nM MG132 for 16 h were immunoprecipitated (IPed) with an anti-Flag antibody and immunoblotted with indicated antibodies. ( C ) Endogenous interaction of Derlin-1 with Sec61β. Immunoprecipitation (IP) with anti-Sec61β antibody or control (Ctrl) IgG using Protein G Sepharose and immunoblotting (IB) with indicated antibodies in HepG2 cells treated with or without 200 nM Tg and/or 500 nM MG132 for 16 h. ( D – F ) IB of ERpQC substrates in HEK293 cells transfected with indicated siRNAs and plasmids and treated with or without 50 nM Tg and 200 nM MG132 for 16 h. All samples were immunoblotted with indicated antibodies. Black arrowhead, signal peptide-uncleaved NHK QQQ ( S NHK QQQ ); white arrowhead, signal peptide-cleaved NHK QQQ ( C NHK QQQ ). ( G ) IB of ERpQC substrate in wild-type (WT) or Derlin-1, -2 , and -3 triple knockout (TKO) HEK293 cells transfected with indicated siRNAs and plasmid for NHK QQQ and treated with or without 50 nM Tg and 200 nM MG132 for 16 h. All samples were immunoblotted with indicated antibodies. Expression levels of S NHK QQQ were calculated and shown as the percentage of S NHK QQQ out of the total amount of NHK QQQ ( S NHK QQQ and C NHK QQQ ). Black arrowhead, S NHK QQQ ; white arrowhead, C NHK QQQ . .

Article Snippet: Rabbit polyclonal anti-Sec61β (IB: 1:500) , Proteintech , Cat. #15087-1-AP; RRID: AB_2186411.

Techniques: Modification, RNA Binding Assay, Transfection, Immunoprecipitation, Control, Western Blot, Triple Knockout, Plasmid Preparation, Expressing