Journal: The Journal of Clinical Investigation

Article Title: ER-associated degradation pathway protein SEL1L plays an evolutionarily conserved role in platelet adhesion

doi: 10.1172/JCI191433

Figure Lengend Snippet: Genotypes of the larger population of 114 phenotyped TBs for ( A ) SEL1L c.1810A>G p.Ile604Val and ( B ) VIPAS39:g.22685398_22685470del . The y axis is percent difference in fibrinogen binding compared with control horses. The 35% cutoff line characterizing affected horses is indicated by a dashed line. **** P < 0.0001 by 1-way ANOVA.

Article Snippet: Washed equine platelets ( SEL1L A>G : n = 7 WT, 5 heterozygous, and 2 homozygous alternate) were fixed in 1% paraformaldehyde, permeabilized in 0.1% NP-40, and labeled with a monoclonal SEL1L antibody conjugated to AlexaFluor488 (Santa Cruz Biotechnology; sc-377350) and a monoclonal P selectin antibody conjugated to APC (Invitrogen; 17-0626-82).

Techniques: Binding Assay, Control

Journal: The Journal of Clinical Investigation

Article Title: ER-associated degradation pathway protein SEL1L plays an evolutionarily conserved role in platelet adhesion

doi: 10.1172/JCI191433

Figure Lengend Snippet: SEL1L protein expression from flow cytometry in ( A ) resting permeabilized platelets and ( B ) on the surface of thrombin-activated platelets. The median for each horse and the SD are shown. n = 7 Ref/Ref, 6 Ref/Alt, and 2 Alt/Alt. The percent platelets that were positive for SEL1L was decreased in the Alt/Alt horses compared with the Ref/Ref horses in both resting and activated platelets.

Article Snippet: Washed equine platelets ( SEL1L A>G : n = 7 WT, 5 heterozygous, and 2 homozygous alternate) were fixed in 1% paraformaldehyde, permeabilized in 0.1% NP-40, and labeled with a monoclonal SEL1L antibody conjugated to AlexaFluor488 (Santa Cruz Biotechnology; sc-377350) and a monoclonal P selectin antibody conjugated to APC (Invitrogen; 17-0626-82).

Techniques: Expressing, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: ER-associated degradation pathway protein SEL1L plays an evolutionarily conserved role in platelet adhesion

doi: 10.1172/JCI191433

Figure Lengend Snippet: ( A ) TBTs using horses that were Ref/Ref or Alt/Alt for the SEL1L c.1810A>G p.Ile604Val variant found no significant difference between genotypes by 2-tailed Student’s t test. n = 10 Ref/Ref and 3 Alt/Alt. ( B ) SEL1L (red) localization in relation to P selectin (green) in (top) resting permeabilized platelets and (bottom) thrombin-activated platelet surface of horses that are Ref/Ref for SEL1L c.1810A>G p.Ile604Val. SEL1L did not localize to the surface in resting permeabilized platelets (top) but did localize to the surface in thrombin-activated platelets (bottom). n = 2. Original magnification, ×100. ( C ) Collagen spreading assay comparing median platelet area to genotype. There was a substantial difference between homozygous Ref and Alt genotypes. n = 11 Ref/Ref, 5 Ref/Alt, and 2 Alt/Alt.

Article Snippet: Washed equine platelets ( SEL1L A>G : n = 7 WT, 5 heterozygous, and 2 homozygous alternate) were fixed in 1% paraformaldehyde, permeabilized in 0.1% NP-40, and labeled with a monoclonal SEL1L antibody conjugated to AlexaFluor488 (Santa Cruz Biotechnology; sc-377350) and a monoclonal P selectin antibody conjugated to APC (Invitrogen; 17-0626-82).

Techniques: Variant Assay

Journal: The Journal of Clinical Investigation

Article Title: ER-associated degradation pathway protein SEL1L plays an evolutionarily conserved role in platelet adhesion

doi: 10.1172/JCI191433

Figure Lengend Snippet: ( A ) Flow cytometry analyses of human megakaryocytic surface markers from differentiated cord blood. CD61 hi CD42b hi megakaryocytes are observed after 2 weeks of differentiation. ( B ) Phase contrast microscopy of mature human megakaryocytes and ( C ) proplatelets. Scale bars: 50 μm. ( D ) Representative Western blot analysis of SEL1L during megakaryopoiesis and in platelets from peripheral blood. ( E ) Densitometric analyses of SEL1L isoforms ( n = 7, results are presented as mean ± SD). ** P < 0.01, *** P < 0.001, and **** P < 0.0001 by 1-way ANOVA with post hoc pairwise comparisons. ( F and G ) Immunofluorescence microscopy of megakaryocytes extending proplatelets. Scale bars: 30 μm. ( H ) Analysis of SEL1L fluorescence intensity in the megakaryocytic cell body and proplatelet tips. **** P < 0.0001 by 2-tailed Student’s t test.

Article Snippet: Washed equine platelets ( SEL1L A>G : n = 7 WT, 5 heterozygous, and 2 homozygous alternate) were fixed in 1% paraformaldehyde, permeabilized in 0.1% NP-40, and labeled with a monoclonal SEL1L antibody conjugated to AlexaFluor488 (Santa Cruz Biotechnology; sc-377350) and a monoclonal P selectin antibody conjugated to APC (Invitrogen; 17-0626-82).

Techniques: Flow Cytometry, Microscopy, Western Blot, Immunofluorescence, Fluorescence

Journal: The Journal of Clinical Investigation

Article Title: ER-associated degradation pathway protein SEL1L plays an evolutionarily conserved role in platelet adhesion

doi: 10.1172/JCI191433

Figure Lengend Snippet: ( A ) Representative images of murine platelets (green) adhering to a collagen-coated chamber after perfusion of whole blood from both a control Mx1-cre – Sel1l fl/fl and a knockdown Mx1-cre + Sel1l fl/fl mouse. Scale bars: 10 μm. ( B ) Quantification of the change in MFI between the beginning and end of the whole blood perfusion across collagen was normalized to the control for each pair. n = 6 cre + /cre – sibling pairs. * P < 0.05 by Wilcoxon’s matched-pairs signed-rank test; a significant decrease in MFI change was found between control and knockdown groups.

Article Snippet: Washed equine platelets ( SEL1L A>G : n = 7 WT, 5 heterozygous, and 2 homozygous alternate) were fixed in 1% paraformaldehyde, permeabilized in 0.1% NP-40, and labeled with a monoclonal SEL1L antibody conjugated to AlexaFluor488 (Santa Cruz Biotechnology; sc-377350) and a monoclonal P selectin antibody conjugated to APC (Invitrogen; 17-0626-82).

Techniques: Control, Knockdown

Journal: The Journal of Clinical Investigation

Article Title: ER-associated degradation pathway protein SEL1L plays an evolutionarily conserved role in platelet adhesion

doi: 10.1172/JCI191433

Figure Lengend Snippet: TEG was performed on control Mx1-cre − Sel1l fl/fl ( n = 7) and knockdown Mx1-cre + Sel1l fl/fl ( n = 6) mice. The results showed ( A ) no significant difference in reaction time, ( B ) a significant decrease in maximum amplitude, ( C ) a significant increase in the time for the clot to reach a strength of 20 mm (K time), ( D ) a significant decrease in the rate of clot formation (α-angle), and ( E ) a significant decrease in maximal rate of thrombus generation. ( F ) Representative tracings and velocity curves show the difference between the control in black and the KO in gray. * P < 0.05 and *** P < 0.001 by 2-tailed Student’s t test.

Article Snippet: Washed equine platelets ( SEL1L A>G : n = 7 WT, 5 heterozygous, and 2 homozygous alternate) were fixed in 1% paraformaldehyde, permeabilized in 0.1% NP-40, and labeled with a monoclonal SEL1L antibody conjugated to AlexaFluor488 (Santa Cruz Biotechnology; sc-377350) and a monoclonal P selectin antibody conjugated to APC (Invitrogen; 17-0626-82).

Techniques: Control, Knockdown

Journal: The Journal of Clinical Investigation

Article Title: ER-associated degradation pathway protein SEL1L plays an evolutionarily conserved role in platelet adhesion

doi: 10.1172/JCI191433

Figure Lengend Snippet: Human phenotypes associated with SEL1L variants

Article Snippet: Washed equine platelets ( SEL1L A>G : n = 7 WT, 5 heterozygous, and 2 homozygous alternate) were fixed in 1% paraformaldehyde, permeabilized in 0.1% NP-40, and labeled with a monoclonal SEL1L antibody conjugated to AlexaFluor488 (Santa Cruz Biotechnology; sc-377350) and a monoclonal P selectin antibody conjugated to APC (Invitrogen; 17-0626-82).

Techniques:

Journal: Burns & Trauma

Article Title: Tripartite motif 13 orchestrates endoplasmic reticulum-associated degradation and endoplasmic reticulum-phagy to modulate dendritic cell-mediated immune responses in sepsis

doi: 10.1093/burnst/tkaf077

Figure Lengend Snippet: TRIM13 promotes SEL1L-HRD1 endoplasmic reticulum-associated degradation (ERAD) and ER-selective autophagy (ER-phagy) in DCs upon LPS stimulation. (a) Immunoblot analysis of ER-phagy-related protein expression in WT and Trim13 Cd11c DCs at the indicated post-CLP time points ( n = 3). The quantitative results are shown on the right. (b) Immunoblot analysis of ERAD-related protein expression in WT and Trim13 Cd11c DCs at the indicated post-CLP time points (n = 3) ( n = 3). The quantitative results are shown on the right. (c) Immunoblot analysis of ER-phagy-related protein expression in Trim13 KD and Trim13 OE DC2.4 cells following stimulation with LPS (1 µg/ml, 24 h) (n =3). The quantitative results are shown on the right. (d) Immunoblot analysis of ERAD-related protein expression in Trim13 KD and Trim13 OE DC2.4 cells following stimulation with LPS (1 μg/ml, 24 h) ( n = 3). The quantitative results are shown below. (e) Immunoblot analysis of STING ubiquitination following STING immunoprecipitation in Trim13 OE DC2.4 cells treated with MG-132 (10 μm, 12 h) or Hrd1 knockdown. Data are expressed as means ± SD. ns = not significant; * P < 0.05; ** P < 0.01; *** P < 0.001. DCs dendritic cells, SD standard deviation, ER endoplasmic reticulum, WT wild-type, TRIM13 tripartite motif 13, CLP cecal ligation and puncture, STING stimulator of interferon genes

Article Snippet: HRD1/SYVN1 (13473–1-AP) and SEL1L (29801-1-AP) antibodies were purchased from Proteintech (Wuhan, China).

Techniques: Western Blot, Expressing, Ubiquitin Proteomics, Immunoprecipitation, Knockdown, Standard Deviation, Ligation