Journal: Nature Communications

Article Title: Ubiquitination by HRD1 is essential for TLR3 trafficking and its innate immune signaling

doi: 10.1038/s41467-025-67219-0

Figure Lengend Snippet: a Comparative analysis of anti-HRD1 IP-MS in WT and HRD1 −/− RAW 264.7 cells to identify HRD1-interacting candidates, and the top ten specific HRD1-interacting ER proteins shown. b Immunoblot analysis of indicated proteins in primary macrophages treated with 50 μg/ml poly(I:C) for the indicated times, representative of three biologically independent repeats. The quantitation of protein levels (normalized to the loading control) is shown below the blot. c Immunoblot analysis of indicated proteins following immunoprecipitation of Flag in HEK293T cells transfected with TLR3-Flag and HRD1-Myc plasmids for 24 h, and subsequently treated with 50 μg/ml poly(I:C) for 3 h. The quantitation of protein levels (normalized to the no poly(I:C) treatment) is shown below the blot. IP, immunoprecipitation. d Immunoblot analysis of indicated proteins following immunoprecipitation of endogenous HRD1 in RAW 264.7 macrophages at various time points following treatment with 50 μg/ml poly(I:C). The quantitation of protein levels (normalized to the 0 h) is shown below the blot. IgG, immunoglobulin G. e – h Diagrams of full-length HRD1 protein domains and various HRD1 truncate mutants ( e ) and TLR3 protein domains and various TLR3 truncate mutants ( g ), along with mapping of TLR3 and HRD1 interacting domains ( f , h ). WT, wild type; TM, transmembrane. LRR, leucine-rich repeat; TIR, cytosolic Toll/interleukin-1 receptor domain. Results showing immunoblot analysis following Myc immunoprecipitation ( f ) or Flag immunoprecipitation ( h ) in HEK293T cells transfected with various plasmids encoding WT and truncated HRD1-Myc or TLR3-Flag proteins, as indicated. The blot data are representative of three biologically independent repeats ( b – d , f , h ). Source data are provided as a Source Data file.

Article Snippet: Antibodies used in this study were: Flag (Sigma F1804; 1:2000); HA (Proteintech 51064-2-AP; 1:4000), HRD1 (Proteintech 13473-1-AP; 1:1000), SEL1L (Abclonal; A12073, 1:2000), IRE1α (Cell Signaling 3294; 1:3000), PERK (Proteintech 20582-1-AP; 1:3000), Eif2α(Cell Signaling 5324; 1:3000), p-Eif2α(Cell Signaling 5324; 1:2000), β-tubulin (Proteintech 10068-1-AP; 1:3000), HSP90 (Proteintech 13171-1-AP; 1:5000), Myc (ABclonal AE010; 1:2000), V5 (ABclonal AE017; 1:2000), phospho-IκBα (ABclonal AP0707; 1:1000), HRS (ABclonal A1790; 1:3000), TSG101 (ABclonal A1692; 1:1000), VPS36 (ABclonal A15157; 1:1000), TLR3 (NOVUS NBP2-24875; 2 μg/ml),, p-TBK1 (Ser172) (Cell Signaling D52C2; 1:1000), TBK1 (Cell Signaling E9H5S; 1:2000), p-IRF3 (Ser396) (Cell Signaling D601M; 1:2000), IRF3 (Cell Signaling D83B9; 1:2000), IκBα (Cell Signaling 9242; 1:2000), β-actin (Cell Signaling 13E5; 1:5000), p65 (Cell Signaling D14E12; 1:2000), phospho-p65 (Cell Signaling 93H1; 1:1000), Ubiquitin (linkage-specific K48) (Cell Signaling D9D5; 1:2000), Ubiquitin (linkage-specific K63) (Cell Signaling D7A11; 1:2000), GOLPH2 (ABclonal A26409PM; 1:1000), EEA1 (ABclonal A5057; 1:2000), Ubiquitin (Santa Cruz P4D1; 1:200), TGN38 (Santa Cruz sc-166594; 1:200), and mouse IgG (Santa Cruz sc-2025), Rabbit IgG (Abclonal; AC005)ERK1/2 (Cell Signaling 4695; 1:2000), p-ERK1/2 (Cell Signaling 9101; 1:2000), STAT1 (Cell Signaling 14994; 1:2000), p-STAT1 (Cell Signaling 9167;1:2000).

Techniques: Protein-Protein interactions, Western Blot, Quantitation Assay, Control, Immunoprecipitation, Transfection

Journal: Nature Communications

Article Title: Ubiquitination by HRD1 is essential for TLR3 trafficking and its innate immune signaling

doi: 10.1038/s41467-025-67219-0

Figure Lengend Snippet: a Immunoblot analysis of indicated proteins in wild-type (scramble) versus Hrd1 −/− RAW 264.7 cells treated with 50 μg/ml poly(I:C) for the indicated times, representative of three independent repeats. gRNA, guide RNA. b Immunoblot analysis of indicated proteins in Hrd1 +/+ and Hrd1 −/− HEK293T cells with stable expression of TLR3, following treatment with 50 μg/ml poly(I:C) for the indicated times. The data are representative of three independent repeats. c Immunoblot analysis of indicated proteins in primary macrophages treated with vehicle or LS-102 (5 μM) for 24 h, and subsequently treated with 50 μg/ml poly(I:C) for the indicated times, representative of three independent repeats. d qPCR analysis of Ifnb1 , Il1b , Tnfa , Il6 , Ccl5 , and Bip in WT versus Hrd1 −/− RAW 264.7 cells treated with vehicle or 50 μg/ml poly(I:C) for the indicated times. n = 4 each, representative of at least three independent repeats. mRNA, messenger RNA. e ELISA analysis of TNFα, IL6 and IFNβ in the culture supernatants of vehicle- or LS-102 (5 μM for 24 h)-treated primary macrophages, followed by poly(I:C) (50 μg/ml) treatment for 6 h. n = 4 each, representative of three independent repeats. f Representative microscopy images showing replication of GFP-tagged HSV-1 in Vero cells under various treatments, with quantitation shown in Supplementary Fig. ). Vero cells were exposed to secretome obtained from poly(I:C)-stimulated WT and Hrd1 −/− RAW 264.7 cells and incubated with 0.5 or 1.0 MOI HSV-1 for 24 h. GFP-HSV, GFP-tagged HSV-1. Quantitation of the ratio of phosphorylated to total protein (p/t) is shown below each blot. Values represent mean ± SEM, by unpaired, two-tailed, Student’s t -test ( d , e ). Source data are provided as a Source Data file.

Article Snippet: Antibodies used in this study were: Flag (Sigma F1804; 1:2000); HA (Proteintech 51064-2-AP; 1:4000), HRD1 (Proteintech 13473-1-AP; 1:1000), SEL1L (Abclonal; A12073, 1:2000), IRE1α (Cell Signaling 3294; 1:3000), PERK (Proteintech 20582-1-AP; 1:3000), Eif2α(Cell Signaling 5324; 1:3000), p-Eif2α(Cell Signaling 5324; 1:2000), β-tubulin (Proteintech 10068-1-AP; 1:3000), HSP90 (Proteintech 13171-1-AP; 1:5000), Myc (ABclonal AE010; 1:2000), V5 (ABclonal AE017; 1:2000), phospho-IκBα (ABclonal AP0707; 1:1000), HRS (ABclonal A1790; 1:3000), TSG101 (ABclonal A1692; 1:1000), VPS36 (ABclonal A15157; 1:1000), TLR3 (NOVUS NBP2-24875; 2 μg/ml),, p-TBK1 (Ser172) (Cell Signaling D52C2; 1:1000), TBK1 (Cell Signaling E9H5S; 1:2000), p-IRF3 (Ser396) (Cell Signaling D601M; 1:2000), IRF3 (Cell Signaling D83B9; 1:2000), IκBα (Cell Signaling 9242; 1:2000), β-actin (Cell Signaling 13E5; 1:5000), p65 (Cell Signaling D14E12; 1:2000), phospho-p65 (Cell Signaling 93H1; 1:1000), Ubiquitin (linkage-specific K48) (Cell Signaling D9D5; 1:2000), Ubiquitin (linkage-specific K63) (Cell Signaling D7A11; 1:2000), GOLPH2 (ABclonal A26409PM; 1:1000), EEA1 (ABclonal A5057; 1:2000), Ubiquitin (Santa Cruz P4D1; 1:200), TGN38 (Santa Cruz sc-166594; 1:200), and mouse IgG (Santa Cruz sc-2025), Rabbit IgG (Abclonal; AC005)ERK1/2 (Cell Signaling 4695; 1:2000), p-ERK1/2 (Cell Signaling 9101; 1:2000), STAT1 (Cell Signaling 14994; 1:2000), p-STAT1 (Cell Signaling 9167;1:2000).

Techniques: Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Microscopy, Quantitation Assay, Incubation, Two Tailed Test

Journal: Nature Communications

Article Title: Ubiquitination by HRD1 is essential for TLR3 trafficking and its innate immune signaling

doi: 10.1038/s41467-025-67219-0

Figure Lengend Snippet: a qPCR analysis of Hrd1 mRNA levels in primary macrophages pre- and post-poly(I:C) (50 μg/ml for 3 h) treatment. n = 4 each. b – c Immunoblot analysis of ERK1/2 phosphorylation levels ( b ) and qPCR analysis of Ets1 and Hrd1 mRNA levels ( c ) in macrophage cells pre-treated with LS-102 (5 μM) or ERK1/2 inhibitor PD98059 for 24 h, and then with poly(I:C) (50 μg/ml) for 3 h. n = 4 each in ( c ). d – e Immunoblot analysis of ERK1/2 phosphorylation levels ( d ) and qPCR analysis of Ets1 and Hrd1 mRNA levels ( e ) in macrophage cells pre-treated with LS-102 (5 μM for 24 h), and stimulate with poly(I:C) (50 μg/ml) and indicated cytokines for 3 h. n = 4 each in ( e ). f Immunoblot analysis of indicated proteins in macrophages treated with poly(I:C), TNFα, IL1β, IL6 or IFNβ for 12 h. g qPCR analysis of Tlr3 mRNA levels in macrophages pre- and post-poly(I:C) (50 μg/ml for 3 h) treatment. n = 4 each. h Immunoblot analysis of indicated proteins in macrophages pre-treated with vehicle or LS-102 for 24 h, followed by poly(I:C) (50 μg/ml) treatment for the indicated times. i – j Immunoblot analysis of STAT1 phosphorylation levels ( i ) and qPCR analysis of Irf1, Irf2 and Tlr3 mRNA levels ( j ) in macrophages pre-treated with LS-102 (5 μM) or STAT1 inhibitor Fludarabine for 24 h, and then stimulated with poly(I:C) (50 μg/ml) for 3 h. n = 4 for ( j ). k – l Immunoblot analysis of STAT1 phosphorylation levels ( k ) and qPCR analysis of Irf1, Irf2 and Tlr3 mRNA levels ( l ) in macrophages pre-treated with LS-102 (5 μM) for 24 h, followed by stimulation with poly(I:C) (50 μg/ml) and IFNβ for 3 h. n = 4 for ( l ). All experiments were repeated for at least three times. Quantitation of the ratio of phosphorylated to total protein (p/t) and indicated protein is shown below each blot. Values represent mean ± SEM, NS, not significant, by unpaired, two-tailed, Student’s t -test ( a , c , e , g , j , l ). Source data are provided as a Source Data file.

Article Snippet: Antibodies used in this study were: Flag (Sigma F1804; 1:2000); HA (Proteintech 51064-2-AP; 1:4000), HRD1 (Proteintech 13473-1-AP; 1:1000), SEL1L (Abclonal; A12073, 1:2000), IRE1α (Cell Signaling 3294; 1:3000), PERK (Proteintech 20582-1-AP; 1:3000), Eif2α(Cell Signaling 5324; 1:3000), p-Eif2α(Cell Signaling 5324; 1:2000), β-tubulin (Proteintech 10068-1-AP; 1:3000), HSP90 (Proteintech 13171-1-AP; 1:5000), Myc (ABclonal AE010; 1:2000), V5 (ABclonal AE017; 1:2000), phospho-IκBα (ABclonal AP0707; 1:1000), HRS (ABclonal A1790; 1:3000), TSG101 (ABclonal A1692; 1:1000), VPS36 (ABclonal A15157; 1:1000), TLR3 (NOVUS NBP2-24875; 2 μg/ml),, p-TBK1 (Ser172) (Cell Signaling D52C2; 1:1000), TBK1 (Cell Signaling E9H5S; 1:2000), p-IRF3 (Ser396) (Cell Signaling D601M; 1:2000), IRF3 (Cell Signaling D83B9; 1:2000), IκBα (Cell Signaling 9242; 1:2000), β-actin (Cell Signaling 13E5; 1:5000), p65 (Cell Signaling D14E12; 1:2000), phospho-p65 (Cell Signaling 93H1; 1:1000), Ubiquitin (linkage-specific K48) (Cell Signaling D9D5; 1:2000), Ubiquitin (linkage-specific K63) (Cell Signaling D7A11; 1:2000), GOLPH2 (ABclonal A26409PM; 1:1000), EEA1 (ABclonal A5057; 1:2000), Ubiquitin (Santa Cruz P4D1; 1:200), TGN38 (Santa Cruz sc-166594; 1:200), and mouse IgG (Santa Cruz sc-2025), Rabbit IgG (Abclonal; AC005)ERK1/2 (Cell Signaling 4695; 1:2000), p-ERK1/2 (Cell Signaling 9101; 1:2000), STAT1 (Cell Signaling 14994; 1:2000), p-STAT1 (Cell Signaling 9167;1:2000).

Techniques: Western Blot, Phospho-proteomics, Quantitation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Ubiquitination by HRD1 is essential for TLR3 trafficking and its innate immune signaling

doi: 10.1038/s41467-025-67219-0

Figure Lengend Snippet: a Immunoblot and quantitation analysis of TLR3 in WT and Hrd1 −/− RAW 264.7 cells. n = 3. b Immunoblot analysis in WT and Hrd1 −/− RAW 264.7 cells treated with CHX (50 µg/ml) for the indicated times, with quantitation from five independent experiments shown. c , Immunoblot analysis of indicated proteins in HEK293T-TLR3 cells transfected with increasing amounts of plasmid expressing HRD1 protein. d qPCR analysis of Xbp1s, Bip , Tnfa , Ifnb1 , Il6 and Il1b in WT and Hrd1 −/− RAW 264.7 cells treated with Eeyarestatin I (5 uM) for 8 h and/or poly(I:C) (50 µg/ml) for 6 h. n = 4 each. e Immunoblot analysis of indicated proteins in WT and Hrd1 −/− RAW 264.7 cells treated with thapsigargin (300 nM) for 1.5 h and/or poly(I:C) (50 µg/ml) for 1 h. Tg, thapsigargin. p-, phosphorylated PERK; n-, non-phosphorylated PERK. f qPCR analysis of Bip , Xbp1s, Il6 , Ccl5 , Chop , Tnfa , Ifnb1 and Il1b in WT and Hrd1 −/− RAW 264.7 cells treated with thapsigargin (300 nM) and/or poly(I:C) (50 µg/ml) for 6 h. n = 4 each. g qPCR analysis of Xbp1u , Xbp1s , Tnfa , Il1b , Ccl5 , Il6 and Ifnb1 in WT and Hrd1 −/− RAW 264.7 cells treated with 4μ8c (0.1 mM) for 30 h and/or poly(I:C) (50 µg/ml) for 6 h. n = 4 each. Quantitation of the ratio of phosphorylated to total protein (p/t) and indicated protein is shown below each blot ( c , e ). All data were representative of at least three independent repeats. Values represent mean ± SEM NS, not significant, by unpaired, two-tailed, Student’s t -test ( a , b , d , f , g ). Source data are provided as a Source Data file.

Article Snippet: Antibodies used in this study were: Flag (Sigma F1804; 1:2000); HA (Proteintech 51064-2-AP; 1:4000), HRD1 (Proteintech 13473-1-AP; 1:1000), SEL1L (Abclonal; A12073, 1:2000), IRE1α (Cell Signaling 3294; 1:3000), PERK (Proteintech 20582-1-AP; 1:3000), Eif2α(Cell Signaling 5324; 1:3000), p-Eif2α(Cell Signaling 5324; 1:2000), β-tubulin (Proteintech 10068-1-AP; 1:3000), HSP90 (Proteintech 13171-1-AP; 1:5000), Myc (ABclonal AE010; 1:2000), V5 (ABclonal AE017; 1:2000), phospho-IκBα (ABclonal AP0707; 1:1000), HRS (ABclonal A1790; 1:3000), TSG101 (ABclonal A1692; 1:1000), VPS36 (ABclonal A15157; 1:1000), TLR3 (NOVUS NBP2-24875; 2 μg/ml),, p-TBK1 (Ser172) (Cell Signaling D52C2; 1:1000), TBK1 (Cell Signaling E9H5S; 1:2000), p-IRF3 (Ser396) (Cell Signaling D601M; 1:2000), IRF3 (Cell Signaling D83B9; 1:2000), IκBα (Cell Signaling 9242; 1:2000), β-actin (Cell Signaling 13E5; 1:5000), p65 (Cell Signaling D14E12; 1:2000), phospho-p65 (Cell Signaling 93H1; 1:1000), Ubiquitin (linkage-specific K48) (Cell Signaling D9D5; 1:2000), Ubiquitin (linkage-specific K63) (Cell Signaling D7A11; 1:2000), GOLPH2 (ABclonal A26409PM; 1:1000), EEA1 (ABclonal A5057; 1:2000), Ubiquitin (Santa Cruz P4D1; 1:200), TGN38 (Santa Cruz sc-166594; 1:200), and mouse IgG (Santa Cruz sc-2025), Rabbit IgG (Abclonal; AC005)ERK1/2 (Cell Signaling 4695; 1:2000), p-ERK1/2 (Cell Signaling 9101; 1:2000), STAT1 (Cell Signaling 14994; 1:2000), p-STAT1 (Cell Signaling 9167;1:2000).

Techniques: Western Blot, Quantitation Assay, Transfection, Plasmid Preparation, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Ubiquitination by HRD1 is essential for TLR3 trafficking and its innate immune signaling

doi: 10.1038/s41467-025-67219-0

Figure Lengend Snippet: a Immunoblot analysis of TLR3 polyubiquitination following TLR3-Flag immunoprecipitation in HEK293T cells transfected with the specified plasmids, including tagged Ub, HRD1 and TLR3. b Immunoblot analysis of TLR3 polyubiquitination in vitro. Arrows indicated HRD1 and HRD1ΔTM proteins. c Immunoblot analysis of polyubiquitination following immunoprecipitation of endogenous TLR3 in WT and Hrd1 −/− RAW 264.7 cells. Ub, ubiquitin. d Immunoblot analysis of polyubiquitination following immunoprecipitation of endogenous TLR3 in RAW 264.7 cells treated with 50 μg/ml poly(I:C) for the indicated times. e Immunoblot analysis of TLR3 polyubiquitination following TLR3-Flag immunoprecipitation in HEK293T cells that were transfected with the indicated plasmids for 18 h and treated with vehicle or 10 μg/ml Brefeldin A for 8 h. BFA, Brefeldin A. Immunoblot data of the input are shown in Supplementary Fig. . f Immunoblot analysis of TLR3 polyubiquitination following TLR3-Flag immunoprecipitation in HEK293T cells transfected with the indicated plasmids. C2A, HRD1-dead variant; ΔRING, RING domain-deleted truncations; ΔTM, transmembrane domain-deleted truncation. Immunoblot data of the input are shown in Supplementary Fig. . g Immunoblot analysis of TLR3 polyubiquitination following TLR3-Flag immunoprecipitation in HEK293T cells transfected with the indicated plasmids for 18 h, and sequentially treated with or without LS-102 (5 μM) for 8 h. Immunoblot data of the input are shown in Supplementary Fig. . h Immunoblot analysis following immunoprecipitation of TLR3-Flag in the lysates of HEK293T cells transfected with TLR3-Flag, HRD1-Myc, and HA-Ub (WT and K-loss mutants). K-R, Lys mutated to Arg. i Immunoblot analysis of the indicated proteins following immunoprecipitation of TLR3-Flag or VCP-V5 in the lysates of HEK293T cells transfected with TLR3-Flag, HRD1-Myc, VCP-V5 and HA-Ub. Immunoblot data of the input are shown in Supplementary Fig. . j Immunoblot analysis of the indicated proteins following immunoprecipitation of TLR3-Flag in the lysates of Hrd1 +/+ and Hrd1 −/− HEK293T cells transfected with TLR3-Flag. All blot data were representative of at least three independent repeats. Source data are provided as a Source Data file.

Article Snippet: Antibodies used in this study were: Flag (Sigma F1804; 1:2000); HA (Proteintech 51064-2-AP; 1:4000), HRD1 (Proteintech 13473-1-AP; 1:1000), SEL1L (Abclonal; A12073, 1:2000), IRE1α (Cell Signaling 3294; 1:3000), PERK (Proteintech 20582-1-AP; 1:3000), Eif2α(Cell Signaling 5324; 1:3000), p-Eif2α(Cell Signaling 5324; 1:2000), β-tubulin (Proteintech 10068-1-AP; 1:3000), HSP90 (Proteintech 13171-1-AP; 1:5000), Myc (ABclonal AE010; 1:2000), V5 (ABclonal AE017; 1:2000), phospho-IκBα (ABclonal AP0707; 1:1000), HRS (ABclonal A1790; 1:3000), TSG101 (ABclonal A1692; 1:1000), VPS36 (ABclonal A15157; 1:1000), TLR3 (NOVUS NBP2-24875; 2 μg/ml),, p-TBK1 (Ser172) (Cell Signaling D52C2; 1:1000), TBK1 (Cell Signaling E9H5S; 1:2000), p-IRF3 (Ser396) (Cell Signaling D601M; 1:2000), IRF3 (Cell Signaling D83B9; 1:2000), IκBα (Cell Signaling 9242; 1:2000), β-actin (Cell Signaling 13E5; 1:5000), p65 (Cell Signaling D14E12; 1:2000), phospho-p65 (Cell Signaling 93H1; 1:1000), Ubiquitin (linkage-specific K48) (Cell Signaling D9D5; 1:2000), Ubiquitin (linkage-specific K63) (Cell Signaling D7A11; 1:2000), GOLPH2 (ABclonal A26409PM; 1:1000), EEA1 (ABclonal A5057; 1:2000), Ubiquitin (Santa Cruz P4D1; 1:200), TGN38 (Santa Cruz sc-166594; 1:200), and mouse IgG (Santa Cruz sc-2025), Rabbit IgG (Abclonal; AC005)ERK1/2 (Cell Signaling 4695; 1:2000), p-ERK1/2 (Cell Signaling 9101; 1:2000), STAT1 (Cell Signaling 14994; 1:2000), p-STAT1 (Cell Signaling 9167;1:2000).

Techniques: Western Blot, Immunoprecipitation, Transfection, In Vitro, Ubiquitin Proteomics, Variant Assay

Journal: Nature Communications

Article Title: Ubiquitination by HRD1 is essential for TLR3 trafficking and its innate immune signaling

doi: 10.1038/s41467-025-67219-0

Figure Lengend Snippet: a – f Confocal microscopic images of TLR3 co-stained with DAPI and ER marker KDEL ( a ), trans-Golgi network marker TGN38 ( b ) and lysosome marker LAMP1 ( c ) in WT and Hrd1 −/− RAW 264.7 cells with or without poly(I:C) (50 µg/ml) treatment. Quantitation of the fraction of TLR3 in the ER ( d , from left to right, n = 60, 58, 60, 60, 60, 60), trans-Golgi network ( e , n = 60 for all groups), and lysosomes ( f , n = 60 for all groups) in these macrophages were shown. Mander’s overlap coefficient is used for measurement of colocalization. g – j Confocal microscopic images of TLR3-Flag co-stained with DAPI and ER marker Calnexin ( g ), Golgi marker GM130 ( h ), early endosome marker Rab5 ( i ) and late endosome marker Rab7 ( j ) in WT and Hrd1 −/− HEK293T cells transfected with TLR3-Flag plasmid and with or without poly(I:C) treatment. k – m Immunoblot analysis of full-length (FL) and cleaved TLR3 (CL) in WT and Hrd1 −/− HEK293T cells transfected with TLR3-Flag plasmid and treated with 50 μg/ml poly(I:C) for 12 h ( k ), LS-102 (5 μM) for 8 h ( l ), and with Endo H or PNGase F ( m ). Arrow, Endo H-sensitive; Red box, Endo H-resistant. n Immunoblot analysis of indicated proteins following the isolation of the ER, Golgi and endosomes from WT and Hrd1 −/− HEK293T cells transfected with TLR3-Flag for 24 h, followed by 50 µg/ml poly(I:C) stimulation for 1 h. Relative quantitation of indicated TLR3 bands shown below the blot. o – q Immunoblot analysis of indicated proteins following immunoprecipitation of TLR3-Flag in the lysates of HEK293T cells transfected with TLR3-Flag, HRD1-myc (WT, C2A, and ΔRING), and HRS-V5 ( o ), or TGS101-V5 ( p ), or VPS36-V5 ( q ). C2A, HRD1-dead variant; ΔRING, RING domain-deleted truncate. Quantitation of the protein level is shown below the blot. All confocal image and blot data were representative of at least three independent repeats ( a – q ). Values represent mean ± SEM, by unpaired, two-tailed, Student’s t -test ( d – f ). Source data are provided as a Source Data file.

Article Snippet: Antibodies used in this study were: Flag (Sigma F1804; 1:2000); HA (Proteintech 51064-2-AP; 1:4000), HRD1 (Proteintech 13473-1-AP; 1:1000), SEL1L (Abclonal; A12073, 1:2000), IRE1α (Cell Signaling 3294; 1:3000), PERK (Proteintech 20582-1-AP; 1:3000), Eif2α(Cell Signaling 5324; 1:3000), p-Eif2α(Cell Signaling 5324; 1:2000), β-tubulin (Proteintech 10068-1-AP; 1:3000), HSP90 (Proteintech 13171-1-AP; 1:5000), Myc (ABclonal AE010; 1:2000), V5 (ABclonal AE017; 1:2000), phospho-IκBα (ABclonal AP0707; 1:1000), HRS (ABclonal A1790; 1:3000), TSG101 (ABclonal A1692; 1:1000), VPS36 (ABclonal A15157; 1:1000), TLR3 (NOVUS NBP2-24875; 2 μg/ml),, p-TBK1 (Ser172) (Cell Signaling D52C2; 1:1000), TBK1 (Cell Signaling E9H5S; 1:2000), p-IRF3 (Ser396) (Cell Signaling D601M; 1:2000), IRF3 (Cell Signaling D83B9; 1:2000), IκBα (Cell Signaling 9242; 1:2000), β-actin (Cell Signaling 13E5; 1:5000), p65 (Cell Signaling D14E12; 1:2000), phospho-p65 (Cell Signaling 93H1; 1:1000), Ubiquitin (linkage-specific K48) (Cell Signaling D9D5; 1:2000), Ubiquitin (linkage-specific K63) (Cell Signaling D7A11; 1:2000), GOLPH2 (ABclonal A26409PM; 1:1000), EEA1 (ABclonal A5057; 1:2000), Ubiquitin (Santa Cruz P4D1; 1:200), TGN38 (Santa Cruz sc-166594; 1:200), and mouse IgG (Santa Cruz sc-2025), Rabbit IgG (Abclonal; AC005)ERK1/2 (Cell Signaling 4695; 1:2000), p-ERK1/2 (Cell Signaling 9101; 1:2000), STAT1 (Cell Signaling 14994; 1:2000), p-STAT1 (Cell Signaling 9167;1:2000).

Techniques: Staining, Marker, Quantitation Assay, Transfection, Plasmid Preparation, Western Blot, Isolation, Immunoprecipitation, Variant Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Ubiquitination by HRD1 is essential for TLR3 trafficking and its innate immune signaling

doi: 10.1038/s41467-025-67219-0

Figure Lengend Snippet: a Immunoblot analysis of TLR3 polyubiquitination following TLR3-Flag immunoprecipitation in HEK293T cells transfected with HA-Ub, HRD1-myc, and TLR3-Flag (WT and truncations). FL, full length; LRR, leucine-rich repeat truncation; TM + TIR, transmembrane domain and cytosolic Toll/interleukin-1 receptor domain truncation. b Immunoblot analysis of TLR3 polyubiquitination following immunoprecipitation of TLR3-Flag in the lysates of HEK293T cells transfected with HRD1-myc, HA-Ub (WT and K48-, K63-only mutants), and TLR3-Flag (WT and K813-loss mutant). K813R, K813-loss. c Immunoblot analysis of FL and cleaved TLR3 in MEF cells stably expressing WT TLR3-Flag or K813R mutant TLR3-Flag and treated with 50 μg/ml poly(I:C) for the indicated times. d Immunoblot analysis of TLR3-Flag in MEF cell lysates treated with or without Endo H or PNGase F. The cells stably expressed WT TLR3-Flag or K813R mutant TLR3-Flag and treated with 50 μg/ml poly(I:C) for 12 h before preparing the cell lysates. Arrow, Endo H-sensitive; Red box, Endo H-resistant. e Immunoblot analysis of the indicated proteins following the isolation of the ER, Golgi and endosomes from MEF cells stably expressing WT TLR3-Flag or K813R mutant TLR3-Flag and treated with 50 μg/ml poly(I:C) for 60 minutes. Relative quantitation of TLR3 bands in indicated organelles shown below the gel. f Immunoblot analysis of indicated proteins in HEK293T cells stably expressing WT TLR3 or K813R TLR3 and treated with 50 μg/ml poly(I:C) for the indicated times. Quantitation of the ratio of phosphorylated to total protein (p/t) is shown below each blot. All data were representative of at least three independent repeats ( a – f ). Source data are provided as a Source Data file.

Article Snippet: Antibodies used in this study were: Flag (Sigma F1804; 1:2000); HA (Proteintech 51064-2-AP; 1:4000), HRD1 (Proteintech 13473-1-AP; 1:1000), SEL1L (Abclonal; A12073, 1:2000), IRE1α (Cell Signaling 3294; 1:3000), PERK (Proteintech 20582-1-AP; 1:3000), Eif2α(Cell Signaling 5324; 1:3000), p-Eif2α(Cell Signaling 5324; 1:2000), β-tubulin (Proteintech 10068-1-AP; 1:3000), HSP90 (Proteintech 13171-1-AP; 1:5000), Myc (ABclonal AE010; 1:2000), V5 (ABclonal AE017; 1:2000), phospho-IκBα (ABclonal AP0707; 1:1000), HRS (ABclonal A1790; 1:3000), TSG101 (ABclonal A1692; 1:1000), VPS36 (ABclonal A15157; 1:1000), TLR3 (NOVUS NBP2-24875; 2 μg/ml),, p-TBK1 (Ser172) (Cell Signaling D52C2; 1:1000), TBK1 (Cell Signaling E9H5S; 1:2000), p-IRF3 (Ser396) (Cell Signaling D601M; 1:2000), IRF3 (Cell Signaling D83B9; 1:2000), IκBα (Cell Signaling 9242; 1:2000), β-actin (Cell Signaling 13E5; 1:5000), p65 (Cell Signaling D14E12; 1:2000), phospho-p65 (Cell Signaling 93H1; 1:1000), Ubiquitin (linkage-specific K48) (Cell Signaling D9D5; 1:2000), Ubiquitin (linkage-specific K63) (Cell Signaling D7A11; 1:2000), GOLPH2 (ABclonal A26409PM; 1:1000), EEA1 (ABclonal A5057; 1:2000), Ubiquitin (Santa Cruz P4D1; 1:200), TGN38 (Santa Cruz sc-166594; 1:200), and mouse IgG (Santa Cruz sc-2025), Rabbit IgG (Abclonal; AC005)ERK1/2 (Cell Signaling 4695; 1:2000), p-ERK1/2 (Cell Signaling 9101; 1:2000), STAT1 (Cell Signaling 14994; 1:2000), p-STAT1 (Cell Signaling 9167;1:2000).

Techniques: Western Blot, Immunoprecipitation, Transfection, Mutagenesis, Stable Transfection, Expressing, Isolation, Quantitation Assay

Journal: Frontiers in Veterinary Science

Article Title: The H protein of attenuated canine distemper virus is degraded via endoplasmic reticulum-associated protein degradation

doi: 10.3389/fvets.2023.1214318

Figure Lengend Snippet: The degradation of CDV 851H protein via ERAD. (A) The inhibition of ERAD by Eevarestatin I increased the CDV 851H protein level. 293T cells were transfected with Flag-CDV H for 24 h, then were treated with Eevarestatin I (4 μM) for 4 h. The cell lysates were analyzed by Western blot using anti-Flag and anti-GAPDH antibodies. The gray values of protein bands were analyzed by ImageJ software and the ratio of target protein gray values to GAPDH was calculated. (B) Knockdown of Hrd1 increased the CDV 851H protein level. After transfecting 293T cells with siRNA for Hrd1 or si-NC for 24 h, the cells were transfected with H protein of CDV 851 and CDV NJ(11)2 for another 24 h. Western blot was used to detect the CDV H protein level and confirm the silencing efficacy of Hrd1-targeted siRNA. The gray values of protein bands were analyzed by ImageJ software and the ratio of target protein gray values to GAPDH was calculated. All results are presented as means ± SD obtained from at least three independent sample preparations. * p < 0.05, ** p < 0.01.

Article Snippet: The antibodies were obtained commercially: anti-Flag mouse monoclonal antibody (F1804, Sigma), anti-ATF6 rabbit polyclonal antibody (DF6009, Affinity), anti-Hrd1 rabbit polyclonal antibody (A2605, Abclonal), anti-HA mouse monoclonal antibody (BD-PM2095, Biodragon), anti-GAPDH mouse monoclonal antibody (60004-1-Ig, Proteintech), HRP-conjugated goat anti-mouse IgG (BF03001, Biodragon), and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (A0568, Beyotime).

Techniques: Inhibition, Transfection, Western Blot, Software, Knockdown

Journal: Frontiers in cardiovascular medicine

Article Title: Pak2 Regulation of Nrf2 Serves as a Novel Signaling Nexus Linking ER Stress Response and Oxidative Stress in the Heart.

doi: 10.3389/fcvm.2022.851419

Figure Lengend Snippet: FIGURE 5 | Pak2-dependent IRE-XBP1 activation leads to enhanced Hrd1-mediated Nrf2 ubiquitination and degradation. (A) Hrd1 reporter luciferase activity was detected in Pak2 knockdown (Ad-shPak2) H9C2 cells in response to Tunicamycin in XBP1s expressing (Flag-XBP1) cardiomyocytes (n = 4). (B) The effect of active Pak2 overexpression and Hrd1 knockdown (siHrd1) (C) on Nrf2 ubiquitination was determined by HA-ubiquitin (HA-ub) immunoprecipitation and Nrf2 immunoblot (n = 3). Association of endogenous Nrf2 with Hrd1 was observed by immunoprecipitation in H9C2 cells under basal conditions (D) and in Tunicamycin stressed cells (E). The effect of tunicamycin in Hrd1 and Nrf2 binding is expressed as the change in IP/input ratio for Hrd1 and Nrf2 (n = 3). Nrf2 degradation in cardiomyocytes was assessed by cyclohexamide (CHX, 100 µg/mL) chases. Immunoblots showed that Flag-Pak2-T402E (F) and Myc-Hrd1 (G) enhance the clearance of Nrf2 in ARCMs (n = 3). (H) Immunoblots showing the effect of Keap1 knockdown (Keap1 siRNA) on Pak2-mediated Nrf2 downregulation (n = 3). Student’s t-test or 2-way ANOVA with Bonferroni correction for post-hoc comparisons were used for analyses. Data presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Two milligram of the protein extract was immunoprecipitated with antibodies against Nrf2 (Ab137550, Abcam), HA (2367S, Cell Signaling), Hrd1 (Nb100-2526, Novus), or IgG (Cell Signaling, 2729), respectively.

Techniques: Activation Assay, Ubiquitin Proteomics, Luciferase, Activity Assay, Knockdown, Expressing, Over Expression, Immunoprecipitation, Western Blot, Binding Assay

Journal: Frontiers in cardiovascular medicine

Article Title: Pak2 Regulation of Nrf2 Serves as a Novel Signaling Nexus Linking ER Stress Response and Oxidative Stress in the Heart.

doi: 10.3389/fcvm.2022.851419

Figure Lengend Snippet: FIGURE 6 | Pak2 activation alleviates ER stress-induced cell death in human cardiomyocites via Nrf2 downregulation. (A) Immunoblots and quantification of Nrf2, Hrd1 and cell death markers in tunicamycin stressed human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) infected with adenovirus expressing active Pak2 (Ad-Pak2) (n = 3). (B) Cell viability of iPSC-CMs was determined by MTT assay (n = 4). Immunoblots and quantification of Nrf2, cell death markers (C), ATR1, Angiotensinogen, and Mas1 (E) in tunicamycin stressed Pak2 knockdown iPSC-CMs under Nrf2 inhibition with ML385 (n = 3). (D) The effect on cell viability was determined by MTT assay (n = 4). (F) Immunoblots and quantification of Pak2 and Nrf2 in heart samples from healthy donors and transplantation patients with dilated heart disease (n = 5). Student’s t-test or 2-way ANOVA with Bonferroni correction for post-hoc comparisons were used for analyses. Data presented as mean ± SEM. (G) Proposed model for Pak2-mediated regulation of Nrf2 in response to ER stress. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Two milligram of the protein extract was immunoprecipitated with antibodies against Nrf2 (Ab137550, Abcam), HA (2367S, Cell Signaling), Hrd1 (Nb100-2526, Novus), or IgG (Cell Signaling, 2729), respectively.

Techniques: Activation Assay, Western Blot, Derivative Assay, Infection, Expressing, MTT Assay, Knockdown, Inhibition, Transplantation Assay

Journal: PLoS pathogens

Article Title: Cytolethal distending toxins require components of the ER-associated degradation pathway for host cell entry.

doi: 10.1371/journal.ppat.1004295

Figure Lengend Snippet: Figure 3. Hrd1 is required for CDT intoxication. (a) Co-immunoprecipitation of Derl2 and Hrd1. Derl2 was immunoprecipitated as in figure 2i and samples were analyzed for Hrd1 by western blot. (b) CRISPR mediated deletion of Hrd1 (DHrd1) results in decreased expression as judged by western blot of Hrd1 from a-Hrd1 immunoprecipitated protein from normalized cell lysates. (c) Co-immunoprecipitation of Derl2 with Hrd1. Hrd1 was immunoprecipitated and samples were analyzed for Derl2 by western blot. (d–g) Wild type 293 and DHrd1 cells were intoxicated with Aa-CDT (d), Hd- CDT (e), Ec-CDT (f) and Cj-CDT (g) similar to figure 1. Percent viability is normalized to unintoxicated controls and error bars indicate standard error. (h–j) Retrograde trafficking of Hd-CDT in DHrd1 cells is blocked at the endoplasmic reticulum. pDsRed2-ER (red) transfected 293 cells and DHrd1 cells were incubated with Hd-CDT on ice, washed and incubated at 37uC for 240 minutes. Cells were then fixed and stained with DAPI (nuclei, blue) and a- Hd-CdtB (green) antibody. White scale bars indicate 5 mm. (i,j) Quantification of microscopy results comparing the percentage of cells with at least one green puncta localized to the nucleus (i), or Pearson’s coefficient values indicating colocalization of the Hd-CdtB signal with the ER (j). Images and quantitation are representative of those collected from a total of 30 randomly chosen cells analyzed during two independent experiments and error bars represent standard deviations. Unless otherwise noted, data are representative of at least three independent experiments. doi:10.1371/journal.ppat.1004295.g003

Article Snippet: Membranes were probed with either rabbit antiDerl2 antibody (Sigma Aldrich) or rabbit anti-Hrd1 polyclonal antibody (Novus Biologicals) at a 1:2000 dilution followed by HRP conjugated a-rabbit antibody (Invitrogen) to allow detection.

Techniques: Immunoprecipitation, Western Blot, CRISPR, Expressing, Transfection, Incubation, Staining, Microscopy, Quantitation Assay

Journal: PLoS pathogens

Article Title: Cytolethal distending toxins require components of the ER-associated degradation pathway for host cell entry.

doi: 10.1371/journal.ppat.1004295

Figure Lengend Snippet: Figure 6. Derl2 and Hrd1 contribute to sensitivity to Ricin, independent of the Derl2 WR motif and the interaction of Derl2 with p97. (a) Derl2 deficiency causes resistance to ricin. A745TKR cells, CHO-CDTRC1 cells, and CHO-CDTRC1 cells expressing Derl2 were seeded in a 384- well plate (16103 cells/well) and allowed to adhere overnight, followed by 48 hour intoxication with ricin and quantitation of viability using ATPlite 1- step reagent (Perkin Elmer). Ricin LD50 values were calculated from three independent experiments and paired t-test was performed to calculate two tailed p-values. (b) CRISPR mediated Hrd1 deletion in 293 cells causes resistance to ricin. Wildtype and Hrd1-deleted 293 cells were intoxicated with ricin, similar to figure (a). (c) Derl2DC complements the resistance to ricin. CHO-CDTRC1 cells expressing empty vector, Derl2 and Derl2DC were intoxicated similar to (a). (d) The Derl2 WR motif is not required for intoxication by ricin. CHO-CDTRC1 cells expressing empty vector, wildtype Derl2, Derl2 Q53A, Derl2 W55A and Derl2 T59A were intoxicated similar to (a). Data are representative of at least three independent experiments performed in triplicate, percent viability is normalized to unintoxicated controls and error bars indicate standard error. doi:10.1371/journal.ppat.1004295.g006

Article Snippet: Membranes were probed with either rabbit antiDerl2 antibody (Sigma Aldrich) or rabbit anti-Hrd1 polyclonal antibody (Novus Biologicals) at a 1:2000 dilution followed by HRP conjugated a-rabbit antibody (Invitrogen) to allow detection.

Techniques: Expressing, Quantitation Assay, Two Tailed Test, CRISPR, Plasmid Preparation

Journal: Oncogene

Article Title: TRC8 suppresses tumorigenesis through targeting heme oxygenase-1 for ubiquitination and degradation.

doi: 10.1038/onc.2012.244

Figure Lengend Snippet: Figure 2. TRC8 promotes HO-1 ubiquitination. (a) HEK293T cells were transfected for 24 h with HA-tagged ubiquitin cDNA (HA-Ub) alone or together with His-tagged HO-1 cDNA (His-HO-1) alone or together with wild-type TRC8 cDNA (Wt-TRC8) or mutant TRC8 cDNA (Mu-TRC8) as indicated, then His-HO-1 protein was isolated by metal affinity resin and subjected to western blot analysis with antibodies against the HA tag (top panel) or HO-1 (middle panel). Expression of HO-1, TRC8 or b-actin (internal control) in whole-cell lysates (WCLs) was also examined by western blot analysis (bottom panel). (b) HEK293T cells were transfected with TRC8 cDNA alone or together with HA-Ub cDNA and cDNA encoding either His-tagged HO-1 or His-tagged truncated HO-1 lacking the C-terminal 24 residues (His-t-HO-1). After 24 h, the cells were lysed and His-tagged HO-1 or the truncated HO-1 isolated and subjected to western blot analysis with antibodies against the HA tag (top panel) or HO-1 (bottom panel). (c) HEK293T cells were transfected for 24 h with HA-Ub cDNA and His-HO-1cDNA with or without cDNA encoding TRC8, AMFR or SYVN1 as indicated, then His-HO-1 was isolated and subjected to western blot analysis as described above (top and middle panels). Expression of TRC8, AMFR and SYVN1 in the WCL was also examined by western blot analysis using specific antibodies (bottom panel).

Article Snippet: Plasmids The ubiquitin genome-wide full-length cDNA (GFC)-transfection array and plasmids containing human TRC8, AMFR or SYVN1 cDNA were obtained from OriGene (Rockville, MD, USA).

Techniques: Ubiquitin Proteomics, Transfection, Mutagenesis, Isolation, Western Blot, Expressing, Control