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: 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: bioRxiv

Article Title: Intramembrane protease SPP defines a cholesterol-regulated switch of the mevalonate pathway

doi: 10.1101/2021.07.19.452877

Figure Lengend Snippet: (A) Stability of endogenous SQS in Hek293T wt and Hek293TΔSPP cells assessed by cycloheximide (CHX) chase assay. Actin was used as loading control. Mean ± SEM, n=3 . (B) SQS degradation assay in Hek293T wt, ΔTRC8, ΔMARCH6, and ΔTRC8ΔMARCH6 (ΔTΔM) double-deficient Hek293T cells. Mean ± SEM, n=3 . (C) SQS degradation assay in ΔTΔM cells transfected with non-targeting (nt) or Hrd1 siRNA. Mean ± SEM, n=3 .

Article Snippet: The following antibodies were used in this study: rabbit polyclonal anti-SPP (kind gift of C. Schaller), mouse monoclonal anti-FLAG (M2, Sigma-Aldrich, #F1804), rat monoclonal anti-HA (3F10, Sigma-Aldrich, #11867423001), mouse monoclonal anti-CLIMP63 (G1/296, Enzo Life Sciences, #ENZ-ABS669-0100), mouse monoclonal anti-β actin (AC-15, Sigma-Aldrich, #A1978), mouse monoclonal anti-ubiquitin (P4D1, Santa Cruz Biotechnology, #sc-8017), rabbit polyclonal anti-HO1 (Enzo Life Sciences, #ADI-SPA-896-F), rabbit polyclonal anti-SQLE (Proteintech, #12544-1-AP), rabbit monoclonal anti-SQS (Abcam, #ab109723), rabbit monoclonal anti-SQS (Abcam, #ab195046), mouse monoclonal anti-GFP (Sigma-Aldrich, #11814460001), mouse monoclonal anti-HMGCR (Merck, #MABS1233), rabbit polyclonal anti-Synoviolin (Hrd1) (Bethyl Laboratories, #A302-946A), AlexaFluor488 goat anti-mouse IgG (H+L) (Invitrogen, #A-11029), mouse monoclonal S-peptide epitope antibody (Thermo Fischer # MA1-981).

Techniques: Control, Degradation Assay, Transfection

Journal: Cellular and Molecular Life Sciences

Article Title: S100A16 promotes acute kidney injury by activating HRD1-induced ubiquitination and degradation of GSK3β and CK1α

doi: 10.1007/s00018-022-04213-5

Figure Lengend Snippet: HRD1 involves in the activation of Wnt/β-catenin signaling pathway and is regulated by S100A16 in injured renal fibroblasts. a Representative micrographs in the corticomedullary junction of mice kidneys showed the expression of HRD1 in WT mice and S100A16 +/− mice at 1 day after IRI, as determined by immunohistochemical staining. Scale bar, 50 μm, 20 μm (Enlarged). b HRD1, GSK3β and CK1α expressions were tested using kidney tissues from WT mice and S100A16 +/− mice at 1 day after IRI compared with sham mice by western blot assays. c Quantitation of immunoblot data for HRD1, GSK3β and CK1α proteins as in b . **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05. n = 3; each point represents the expression in a sample pooled from two mice. d Western blot analyses showed that ICG-001 blocked the increased HRD1 expression induced by H/R in NRK-49F cells, and ICG-001 also recovered the expressions of GSK3β and CK1α. Cell lysates after various treatments, as indicated, were immunoblotted with antibodies against HRD1, GSK3β, CK1α and β-actin. e Quantitation of western blot data for HRD1, GSK3β and CK1α proteins as in d . *** P < 0.001, ** P < 0.01, versus control; ## P < 0.01, # P < 0.05, n.s. not significant, versus H/R alone. n = 3. f Western blot analyses showed that knockdown of S100A16 inhibited the HRD1 expression and enhanced the expressions of GSK3β and CK1α in normal or hypoxia condition. g Quantitation of western blot data for HRD1, GSK3β and CK1α proteins as in f . *** P < 0.001, ** P < 0.01, versus scrambled shRNA; ## P < 0.01, versus H/R + scrambled shRNA. n = 3. h Western blots showed that overexpression of S100A16 increased the HRD1 expression, but it was impeded by ICG-001 in NRK-49F cells. The GSK3β and CK1α expressions were opposite to HRD1. i Quantified HRD1, GSK3β and CK1α protein levels in h . ** P < 0.01, versus pcDNA3.1; # P < 0.05, versus S100A16 OE. n = 3

Article Snippet: The corresponding primary antibody against HRD1 (13473-1-AP; Proteintech, Chicago, IL, USA), GSK3β (9832S; Cell Signaling Technology, Danvers, MA, USA), or CK1α (ab206652; Abcam, Cambridge, UK) was added to about 500 μg cell proteins respectively, and then protein-antibody complex was incubated at 4 °C, 70 rpm (revolutions per minute) overnight.

Techniques: Activation Assay, Expressing, Immunohistochemical staining, Staining, Western Blot, Quantitation Assay, Control, Knockdown, shRNA, Over Expression

Journal: Cellular and Molecular Life Sciences

Article Title: S100A16 promotes acute kidney injury by activating HRD1-induced ubiquitination and degradation of GSK3β and CK1α

doi: 10.1007/s00018-022-04213-5

Figure Lengend Snippet: HRD1 degrades both GSK3β and CK1α via the ubiquitin–proteasome pathway in NRK-49F cells. a The interaction between HRD1 and either GSK3β or CK1α was detected in the co-IP analysis in NRK-49F cells. b A cycloheximide chase was performed to establish the time course of GSK3β or CK1α biogenesis. NRK-49F cells were infected with or without Ad-HRD1 for 48 h, and then the cells were treated with CHX (100 μg/ml) for 0, 2,4 or 6 h. The expressions of GSK3β and CK1α in whole-cell lysates were measured by western blotting. c Quantitation of western blot data for GSK3β and CK1α proteins as in b . * P < 0.05. n = 3. d The presence of MG132 (20 µM) increased the expressions of GSK3β and CK1α with or without Ad-HRD1 infection compared with controls in NRK-49F cells. e Quantification of GSK3β and CK1α protein expressions from experiments as shown in d . *** P < 0.001, ** P < 0.01, * P < 0.05. n = 3. f More ubiquitin conjugated to GSK3β was detected in the cells overexpressing HRD1 compared with no HRD1 transfection. g More ubiquitin conjugated to CK1α was detected in the cells overexpressing HRD1 compared with no HRD1 transfection. h Quantification of ubiquitin conjugated to GSK3β as in f , normalized to GAPDH expression. *** P < 0.001. n = 3. i Quantification of ubiquitin conjugated to CK1α as in g , normalized to GAPDH expression. ** P < 0.01. n = 3

Article Snippet: The corresponding primary antibody against HRD1 (13473-1-AP; Proteintech, Chicago, IL, USA), GSK3β (9832S; Cell Signaling Technology, Danvers, MA, USA), or CK1α (ab206652; Abcam, Cambridge, UK) was added to about 500 μg cell proteins respectively, and then protein-antibody complex was incubated at 4 °C, 70 rpm (revolutions per minute) overnight.

Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Infection, Western Blot, Quantitation Assay, Transfection, Expressing

Journal: Cellular and Molecular Life Sciences

Article Title: S100A16 promotes acute kidney injury by activating HRD1-induced ubiquitination and degradation of GSK3β and CK1α

doi: 10.1007/s00018-022-04213-5

Figure Lengend Snippet: S100A16 down-regulates the expressions of both GSK3β and CK1α by HRD1 to affect downstream HGF in NRK-49F cells. a Western blot analyses showed that overexpression of S100A16 aggravated the downregulated expressions of GSK3β, CK1α, and HGF induced by Ad-HRD1 in NRK-49F cells. b Quantitation of western blot data for GSK3β, CK1α, and HGF proteins, as in a . * P < 0.05, versus pcDNA3.1; ## P < 0.01, versus pcDNA3.1 + Ad-HRD1. n = 3. c Real-time qPCR demonstrated that overexpression of S100A16 aggravated the reduction of relative HGF/β-actin mRNA level induced by Ad-HRD1 compared with the Ad-HRD1 treatment alone in NRK-49F cells. ** P < 0.01, versus pcDNA3.1; # P < 0.05, versus pcDNA3.1 + Ad-HRD1. n = 3. d Western blot analyses showed that knockdown S100A16 recovered Ad-HRD1-induced the down-regulated expressions of GSK3β, CK1α, and HGF. e Quantitation of western blot data for GSK3β, CK1α and HGF proteins, as in d . ** P < 0.01, * P < 0.05, versus scrambled shRNA; ## P < 0.01, versus scrambled shRNA + Ad-HRD1. n = 3. f Representative images showed the accumulation of β-catenin fluorescence in the nucleus of HRD1-overexpressing NRK-49F cells. The β-catenin fluorescence was amplified after overexpressing S100A16 in the HRD1-overexpressing NRK-49F cells. Scale bar, 20 μm

Article Snippet: The corresponding primary antibody against HRD1 (13473-1-AP; Proteintech, Chicago, IL, USA), GSK3β (9832S; Cell Signaling Technology, Danvers, MA, USA), or CK1α (ab206652; Abcam, Cambridge, UK) was added to about 500 μg cell proteins respectively, and then protein-antibody complex was incubated at 4 °C, 70 rpm (revolutions per minute) overnight.

Techniques: Western Blot, Over Expression, Quantitation Assay, Knockdown, shRNA, Fluorescence, Amplification

Journal: Cellular and Molecular Life Sciences

Article Title: S100A16 promotes acute kidney injury by activating HRD1-induced ubiquitination and degradation of GSK3β and CK1α

doi: 10.1007/s00018-022-04213-5

Figure Lengend Snippet: The Wnt/β-catenin signaling activation induced by S100A16–HRD1–GSK3β/CK1α pathway in renal fibroblasts under IRI condition promotes the occurrence of AKI. Schematic diagram shows the increased expression of S100A16 in fibroblasts during renal ischemia and hypoxia. The expression of the E3 ubiquitin ligase HRD1 is elevated, and the members of β-catenin degradation complex, GSK3β and CK1α, are destroyed via the ubiquitin–proteasome pathway, thereby promoting the accumulation of β-catenin and its transfer to the cell nucleus. Activation of the Wnt/β-catenin signaling pathway then inhibits the transcription of the downstream HGF that secreted by fibroblasts, and this procedure eventually leads to unrepairable kidney injury and renal dysfunction

Article Snippet: The corresponding primary antibody against HRD1 (13473-1-AP; Proteintech, Chicago, IL, USA), GSK3β (9832S; Cell Signaling Technology, Danvers, MA, USA), or CK1α (ab206652; Abcam, Cambridge, UK) was added to about 500 μg cell proteins respectively, and then protein-antibody complex was incubated at 4 °C, 70 rpm (revolutions per minute) overnight.

Techniques: Activation Assay, Expressing, Ubiquitin Proteomics

Journal: International Journal of Molecular Sciences

Article Title: USP19-Mediated Deubiquitination Facilitates the Stabilization of HRD1 Ubiquitin Ligase

doi: 10.3390/ijms17111829

Figure Lengend Snippet: USP19 interacts with HRD1. The 293T cells were transfected with HRD1-FLAG ( A ) or FLAG-Nixin ( B ) along with either a pcDNA3 vector (lanes 1 and 3) or Myc-USP19 (lanes 2 and 4). The cells were lysed and then subjected to immunoprecipitation (IP) with an anti-Myc antibody. The lysates (10% of the input; lanes 1 and 2) and the immunoprecipitates (50% of the eluates; lanes 3 and 4) were analyzed by Western blotting with antibodies against FLAG ( top panels), Myc ( middle panels) and α-tubulin (an internal loading control; bottom panels).

Article Snippet: The following polyclonal and monoclonal antibodies were purchased: anti-USP19 antibody (A301-587A; Bethyl Laboratories, Montgomery, TX, USA); anti-HRD1 antibody (Novus Biologicals, Littleton, CO, USA); anti-α-tubulin and anti-FLAG M2 antibodies (Sigma-Aldrich); anti-c-Myc ant-HA antibodies (Roche, Indianapolis, IN, USA); anti-neomycin phosphotransferase II antibody (NPT II; clone AC113; Merck Millipore, Billerica, MA, USA); anti-K48-linked ubiquitin antibodies (clone Apu2; Merck Millipore); anti-syntaxin 6 antibody (BD Transduction Laboratories, San Jose, CA, USA) and anti-MHC class I antibody (clone EMR8-5; Hokudo, Sapporo, Japan).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: USP19-Mediated Deubiquitination Facilitates the Stabilization of HRD1 Ubiquitin Ligase

doi: 10.3390/ijms17111829

Figure Lengend Snippet: USP19 deubiquitinates HRD1. ( A ) HRD1-FLAG was transfected into 293T cells along with either pcDNA3 (lanes 1, 4 and 7), Myc-USP19 (lanes 2, 5 and 8) or Myc-USP19 C548S (lanes 3, 6 and 9). After epoxomicin treatment for 7.5 h, the cells were lysed and then subjected to immunoprecipitation (IP) with an anti-FLAG antibody under denaturing conditions. The immunoprecipitates (50% of the eluates) were analyzed by Western blotting with antibodies against K48-linked ubiquitin (lanes 1–3) and FLAG (lanes 4–6). The lysates (50 μg of protein; lanes 7–9) were analyzed by Western blotting with antibodies against Myc ( top panel), α-tubulin ( middle panel) and NPT II (the Neo r gene product as a transfection control; bottom panel); ( B ) 293T cells were transfected with control siRNA (lanes 1, 3 and 5) or USP19 -specific siRNA duplexes (lanes 2, 4 and 6). Two days after transfection, the cells were further transfected with HRD1-FLAG. After epoxomicin treatment for 6 h, the cells were lysed and then subjected to immunoprecipitation (IP) with an anti-FLAG antibody under denaturing conditions. The immunoprecipitates (50% of the eluates) were analyzed by Western blotting with antibodies against K48-linked ubiquitin (lanes 1 and 2) and FLAG (lanes 3 and 4). The lysates (50 μg of protein; lanes 5 and 6) were analyzed by Western blotting with antibodies against USP19 ( top panel), α-tubulin ( middle panel) and NPT II ( bottom panel).

Article Snippet: The following polyclonal and monoclonal antibodies were purchased: anti-USP19 antibody (A301-587A; Bethyl Laboratories, Montgomery, TX, USA); anti-HRD1 antibody (Novus Biologicals, Littleton, CO, USA); anti-α-tubulin and anti-FLAG M2 antibodies (Sigma-Aldrich); anti-c-Myc ant-HA antibodies (Roche, Indianapolis, IN, USA); anti-neomycin phosphotransferase II antibody (NPT II; clone AC113; Merck Millipore, Billerica, MA, USA); anti-K48-linked ubiquitin antibodies (clone Apu2; Merck Millipore); anti-syntaxin 6 antibody (BD Transduction Laboratories, San Jose, CA, USA) and anti-MHC class I antibody (clone EMR8-5; Hokudo, Sapporo, Japan).

Techniques: Transfection, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Control

Journal: International Journal of Molecular Sciences

Article Title: USP19-Mediated Deubiquitination Facilitates the Stabilization of HRD1 Ubiquitin Ligase

doi: 10.3390/ijms17111829

Figure Lengend Snippet: Both overexpression and knockdown of USP19 affect endogenous HRD1 expression. ( A ) The 293T cells were transfected with either a pcDNA3 vector (lane 1), Myc-USP19 (lane 2) or Myc-USP19 C548S (lane 3). Whole cell lysates (20 μg of protein) were analyzed by Western blotting with antibodies against HRD1 ( top panel), Myc ( second top panel), α-tubulin ( third top panel) and NPT II ( bottom panel); ( B ) the 293T cells were transfected with control siRNA (lane 1) or USP19 -specific siRNA duplexes (lanes 2 and 3). Three days after transfection, whole cell lysates (30 μg of protein) were analyzed by Western blotting with antibodies against HRD1 ( top panel), USP19 ( middle panel) and α-tubulin ( bottom panel). The bar graphs show the relative expression of HRD1 normalized to α-tubulin expression from at least three independent experiments (mean ± SEM). **, *** statistically significant (one-way ANOVA, post-hoc test, ** p < 0.01 and *** p < 0.001, respectively).

Article Snippet: The following polyclonal and monoclonal antibodies were purchased: anti-USP19 antibody (A301-587A; Bethyl Laboratories, Montgomery, TX, USA); anti-HRD1 antibody (Novus Biologicals, Littleton, CO, USA); anti-α-tubulin and anti-FLAG M2 antibodies (Sigma-Aldrich); anti-c-Myc ant-HA antibodies (Roche, Indianapolis, IN, USA); anti-neomycin phosphotransferase II antibody (NPT II; clone AC113; Merck Millipore, Billerica, MA, USA); anti-K48-linked ubiquitin antibodies (clone Apu2; Merck Millipore); anti-syntaxin 6 antibody (BD Transduction Laboratories, San Jose, CA, USA) and anti-MHC class I antibody (clone EMR8-5; Hokudo, Sapporo, Japan).

Techniques: Over Expression, Knockdown, Expressing, Transfection, Plasmid Preparation, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: USP19-Mediated Deubiquitination Facilitates the Stabilization of HRD1 Ubiquitin Ligase

doi: 10.3390/ijms17111829

Figure Lengend Snippet: USP19 stabilizes HRD1. HRD1-FLAG ( A ) and FLAG-Nixin ( B ) were transfected into 293T cells along with either a pcDNA3 vector, Myc-USP19 (wild type; WT) or Myc-USP19 C548S (C548S). The cells were pulse labeled with 35 S for 30 min and chased for the indicated periods of time. The cell lysates were immunoprecipitated with an anti-FLAG antibody and then analyzed by SDS-PAGE followed by autoradiography. The data were plotted as a percentage of the remaining proteins relative to time zero from at least three independent experiments (mean ± SEM). ** statistically significant (Student’s t -test, ** p < 0.01).

Article Snippet: The following polyclonal and monoclonal antibodies were purchased: anti-USP19 antibody (A301-587A; Bethyl Laboratories, Montgomery, TX, USA); anti-HRD1 antibody (Novus Biologicals, Littleton, CO, USA); anti-α-tubulin and anti-FLAG M2 antibodies (Sigma-Aldrich); anti-c-Myc ant-HA antibodies (Roche, Indianapolis, IN, USA); anti-neomycin phosphotransferase II antibody (NPT II; clone AC113; Merck Millipore, Billerica, MA, USA); anti-K48-linked ubiquitin antibodies (clone Apu2; Merck Millipore); anti-syntaxin 6 antibody (BD Transduction Laboratories, San Jose, CA, USA) and anti-MHC class I antibody (clone EMR8-5; Hokudo, Sapporo, Japan).

Techniques: Transfection, Plasmid Preparation, Labeling, Immunoprecipitation, SDS Page, Autoradiography