Journal: bioRxiv

Article Title: ER-associated protein degradation initiates by retrotranslocation from the ER quality control compartment

doi: 10.64898/2026.02.04.703732

Figure Lengend Snippet: a. Illustration of the ER membrane topology of the model ERAD substrate H2a-BAP-SV5 together with cytosolic BirA and the luminal sec-BirA, highlighting three proposed retrotranslocation mechanisms that allow cytosolic BAP biotinylation. b. Western blot of HEK 293 cells expressing H2a-BAP alone or together with BirA or sec-BirA after incubation with Biotin (100µM, 30 min). Biotinylated H2a-BAP was detected with StAv-HRP and total H2a-BAP with α-SV5. c, d. Immunofluorescence of U2OS cells expressing H2a-BAP without BirA untreated or upon bortezomib (Bz, 2.5µM, 3 hours) treatment, stained with α-SV5 and StAv-Cy2 to visualize total and background biotin signal; yellow dashed ROIs mark the ERQC and white boxes indicate zoomed regions, shown on the right. e, f. Localization of total and biotinylated H2a-BAP in U2OS cells expressing the H2a-BAP – BirA system under basal and Bz-treated conditions. g. Quantification of total and biotinylated H2a-BAP in the ERQC relative to whole-cell signal (mean ± SD, n=28; unpaired t-test with Welch’s correction). h-k. Colocalization of biotinylated H2a-BAP with endogenous ER/ERQC markers, Sec61β and calreticulin (CRT). l-n . Colocalization of biotinylated H2a-BAP with ERAD components, endogenous HRD1 and transfected HERP-FLAG. Scale bars= 10 µm. o. Pearson’s R values (Coloc2) for colocalization of biotinylated H2a-BAP with total-H2a-BAP, Sec61β, CRT, HRD1, BiP, and GalT (BiP and GalT from Suppl. Fig. 1) in untreated and Bz-treated cells (unpaired T-test with Welch’s correction).

Article Snippet: Streptavidin-HRP (Jackson ImmunoResearch-016-030-084; 1:5000-7500; WB), Rabbit-α-SV5 (CellSignalling-D3H8Q; 1:2000 (WB), 1:750 (IF), 1:250 (ExM)), Mouse-α-SV5 (GeneScript-A01724-100; 1:1000 (WB), 1:750 (IF), 1:250 (ExM)), Rabbit-α-SYVN (Hrd1) (CellSignaling-14773; 1:1000 (WB), 1:100 (IF)), Rabbit-α-H2a (against N-terminus of H2a as described in ) Rabbit-α-Calnexin (Abcam-92573; 1:5000 (WB)), Rabbit-α-RPL26 (CellSignaling-5400; 1:1000 (WB)), Mouse-α-eIF2α (MBL-AT6031; 1:1000 (WB)), Rabbit-α-C9 (20S) (Abcam-ab118902; 1:1000 (WB)), Rabbit-α-Calnexin (SigmaAldrich - C4731; 1:1000 (WB), 1:500 (IF)), Rabbit-α-Calreticulin (CellSignaling-D3E6; 1:400 (IF)).

Techniques: Membrane, Western Blot, Expressing, Incubation, Immunofluorescence, Staining, Transfection

Journal: bioRxiv

Article Title: ER-associated protein degradation initiates by retrotranslocation from the ER quality control compartment

doi: 10.64898/2026.02.04.703732

Figure Lengend Snippet: a. HEK 293 cells expressing H2a-BAP - BirA were left untreated or treated for 3 hours with Bz (2.5µM), NB-DNJ (200µM) or Kifunensine (Kif, 100µM), alone or with Bz, and lysates were analyzed for total (α-SV5) and biotinylated H2a-BAP (StAv-HRP) or subjected to StAv-agarose pulldown to detect precipitated (α-SV5) and ubiquitylated H2a-BAP (α-Ubiquitin). Deglycosylated species are indicated by red arrows. b, c. Biotinylated H2a-BAP and polyubiquitylated biotinylated H2a-BAP were quantified and normalized to total or StAv-precipitated H2a-BAP, respectively. d. HEK 293 cells co-expressing H2a-BAP - BirA with dominant-negative HRD1 (HRD1-DN) were analyzed under basal or Bz-treated conditions using the same biochemical readouts as in (a). e, f. Quantification of biotinylated and polyubiquitylated H2a-BAP (from d). Statistics were performed using ordinary two-way ANOVA with Tukey’s multiple-comparisons test. g-i. U2OS cells co-expressing H2a-BAP - BirA with HRD1-WT or HRD1-DN, with or without Bz, were stained for total and biotinylated H2a-BAP and HRD1/HRD1-DN (α-Myc) to visualize the impact of HRD1 activity and proteasome inhibition on H2a-BAP localization. j. Pearson’s R values (Coloc2) for colocalization of biotinylated H2a-BAP with HRD1-DN under untreated and Bz-treated cells (unpaired T-test with Welch’s correction). Scale bars= 10 µm.

Article Snippet: Streptavidin-HRP (Jackson ImmunoResearch-016-030-084; 1:5000-7500; WB), Rabbit-α-SV5 (CellSignalling-D3H8Q; 1:2000 (WB), 1:750 (IF), 1:250 (ExM)), Mouse-α-SV5 (GeneScript-A01724-100; 1:1000 (WB), 1:750 (IF), 1:250 (ExM)), Rabbit-α-SYVN (Hrd1) (CellSignaling-14773; 1:1000 (WB), 1:100 (IF)), Rabbit-α-H2a (against N-terminus of H2a as described in ) Rabbit-α-Calnexin (Abcam-92573; 1:5000 (WB)), Rabbit-α-RPL26 (CellSignaling-5400; 1:1000 (WB)), Mouse-α-eIF2α (MBL-AT6031; 1:1000 (WB)), Rabbit-α-C9 (20S) (Abcam-ab118902; 1:1000 (WB)), Rabbit-α-Calnexin (SigmaAldrich - C4731; 1:1000 (WB), 1:500 (IF)), Rabbit-α-Calreticulin (CellSignaling-D3E6; 1:400 (IF)).

Techniques: Expressing, Ubiquitin Proteomics, Dominant Negative Mutation, Staining, Activity Assay, Inhibition

Journal: bioRxiv

Article Title: ER-associated protein degradation initiates by retrotranslocation from the ER quality control compartment

doi: 10.64898/2026.02.04.703732

Figure Lengend Snippet: a, b. H2a-BAP – BirA expressing HEK 293 cells, left untreated (a) or treated with Bz (2.5 µM, 3 hours) (b), were homogenized and post-nuclear supernatants were layered on iodixanol step gradients (10, 16, 22, 28 and 34%) and centrifuged at 24,000 r.p.m. for 16 hours. 1 mL fractions were collected from the top and analyzed by Western blot with StAv-HRP to detect biotinylated H2a-BAP and with α-SV5 (or α-Myc) for total H2a-BAP, together with organelle markers α-CNX, α-HRD1, α-Sec61β, α-20S, α-RPL26 and α-Cab45; the deglycosylated H2a-BAP species is indicated by a red arrow. c . H2a-Myc lacking the BAP tag was co-expressed with BirA as a negative control for biotinylation. d. For each protein, the signal in every fraction was expressed as a percentage of total and plotted versus fraction number; one representative experiment of three is shown. Yellow shading marks fractions enriched in membrane-bound organelles, with fractions 1–2 containing soluble proteins and low-density organelles (Golgi), and green shading marks the ERQC peak. e. HEK 293 cells expressing H2a-BAP - BirA were treated with the p97 inhibitor CB5083 (2.5 µM, 3 hours), alone or with Bz (2.5µM, 3 hours), and analyzed by Western blot. f. Glycosylated and deglycosylated biotinylated H2a-BAP species (from (e)) were quantified, normalized to total H2a-BAP (α-SV5) and expressed relative to untreated cells (mean of 5 experiments ± SD; ordinary two-way ANOVA with Tukey’s multiple-comparisons test).

Article Snippet: Streptavidin-HRP (Jackson ImmunoResearch-016-030-084; 1:5000-7500; WB), Rabbit-α-SV5 (CellSignalling-D3H8Q; 1:2000 (WB), 1:750 (IF), 1:250 (ExM)), Mouse-α-SV5 (GeneScript-A01724-100; 1:1000 (WB), 1:750 (IF), 1:250 (ExM)), Rabbit-α-SYVN (Hrd1) (CellSignaling-14773; 1:1000 (WB), 1:100 (IF)), Rabbit-α-H2a (against N-terminus of H2a as described in ) Rabbit-α-Calnexin (Abcam-92573; 1:5000 (WB)), Rabbit-α-RPL26 (CellSignaling-5400; 1:1000 (WB)), Mouse-α-eIF2α (MBL-AT6031; 1:1000 (WB)), Rabbit-α-C9 (20S) (Abcam-ab118902; 1:1000 (WB)), Rabbit-α-Calnexin (SigmaAldrich - C4731; 1:1000 (WB), 1:500 (IF)), Rabbit-α-Calreticulin (CellSignaling-D3E6; 1:400 (IF)).

Techniques: Expressing, Western Blot, Negative Control, Membrane

Journal: bioRxiv

Article Title: ER-associated protein degradation initiates by retrotranslocation from the ER quality control compartment

doi: 10.64898/2026.02.04.703732

Figure Lengend Snippet: H2a-BAP – BirA expressing HEK 293 cells were processed as in but using iodixanol gradients optimized to achieve higher resolution of low and medium density fractions (10%, 13%, 16%, 20% and 24%) under three microsome conditions: untreated microsomes (M), SDS/Triton X-100/NaDOC lysis (S) or Na₂CO₃ extraction (C). a. 1-mL fractions were collected and immunoblotted for biotinylated H2a-BAP (StAv-HRP), deglycosylated species are indicated by red arrows), total H2a-BAP (α-SV5) and calnexin (CNX). b. Parallel blots were probed for endogenous HRD1 and cytosolic or cytosol-facing membrane proteins (α-20S, α-RPL26 and α-eIF2α). c-f. Signals for biotinylated, total, glycosylated and deglycosylated H2a-BAP were quantified for each condition (M, S, C), expressed as percentage of total signal and compared between untreated and Bz-treated cells; one representative experiment of three is shown. g. Scheme of alkaline carbonate extraction release of peripherally membrane-associated proteins from microsomes while retaining integral ER membrane proteins.

Article Snippet: Streptavidin-HRP (Jackson ImmunoResearch-016-030-084; 1:5000-7500; WB), Rabbit-α-SV5 (CellSignalling-D3H8Q; 1:2000 (WB), 1:750 (IF), 1:250 (ExM)), Mouse-α-SV5 (GeneScript-A01724-100; 1:1000 (WB), 1:750 (IF), 1:250 (ExM)), Rabbit-α-SYVN (Hrd1) (CellSignaling-14773; 1:1000 (WB), 1:100 (IF)), Rabbit-α-H2a (against N-terminus of H2a as described in ) Rabbit-α-Calnexin (Abcam-92573; 1:5000 (WB)), Rabbit-α-RPL26 (CellSignaling-5400; 1:1000 (WB)), Mouse-α-eIF2α (MBL-AT6031; 1:1000 (WB)), Rabbit-α-C9 (20S) (Abcam-ab118902; 1:1000 (WB)), Rabbit-α-Calnexin (SigmaAldrich - C4731; 1:1000 (WB), 1:500 (IF)), Rabbit-α-Calreticulin (CellSignaling-D3E6; 1:400 (IF)).

Techniques: Expressing, Lysis, Extraction, Membrane