memerald sec61β  (Addgene inc)

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