274 rabbit anti rnf20 Search Results


anti rnf20  (Novus Biologicals)
92
Novus Biologicals anti rnf20
Anti Rnf20, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/274+rabbit+anti+rnf20/RNF20+Antibody/pmc02559905-353-0-1
Average 92 stars, based on 1 article reviews
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anti rnf20  (Cell Signaling Technology Inc)
94
Cell Signaling Technology Inc anti rnf20
Anti Rnf20, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/274+rabbit+anti+rnf20/RNF20+XP+Rabbit+mAb/pmc10873513-329-134-135
Average 94 stars, based on 1 article reviews
anti rnf20 - by Bioz Stars, 2026-10
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rabbit anti rnf20 antibody  (Cell Signaling Technology Inc)
93
Cell Signaling Technology Inc rabbit anti rnf20 antibody
A. Images of tumorspheres from 3 lung adenocarcinoma tissues (Samples 1, 2, and 3). B. Western Blot analysis of CD133 (as a surface biomarker of CICs), CD44 (as another surface biomarker of CICs), USP22, <t>RNF20,</t> and H2Bub1, shows enriched USP22 protein in tumorspheres compared to original cancer tissues. C. Flow cytometry analysis of CD133 expression demonstrates a strong CD133 expression in about 30% of tumorsphere cells. D. Western Blot analysis demonstrates USP22 is markedly elevated, while H2Bub1 were significantly deceased in CD133+ cancer cells compared to CD133− cells sorted from tumorspheres. E. Flow cytometry analysis of CD44 expression demonstrates a strong CD44 expression in about 12% to 44% of tumorsphere cells. F. Western Blot analysis demonstrates USP22 and CD133 are markedly elevated, while H2Bub1 and RNF20 were markedly or moderately deceased in CD44+ cancer cells compared to CD44− cells sorted from tumorspheres. G. Mouse xenografts generated by both CD133− and CD133+ cancer cells isolated from tumorspheres, indicating CD133+ cells may represent CICs with stem cell characteristics in tumorspheres.
Rabbit Anti Rnf20 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/274+rabbit+anti+rnf20/RNF20+Rabbit+mAb/pmc06030467-92-0-7
Average 93 stars, based on 1 article reviews
rabbit anti rnf20 antibody - by Bioz Stars, 2026-10
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resource source identifier antibodies rabbit anti rnf20 proteintech  (Proteintech)
93
Proteintech resource source identifier antibodies rabbit anti rnf20 proteintech
Figure 1. <t>RNF20</t> is correlated with DNA damage during cerebrovascular development (A) Graphic representation of the mouse cerebral cortex and its vascularization pattern. The boxed area represented the location of cerebral cortex. MGE, medial ganglionic eminence; LGE, lateral ganglionic eminence; VZ, ventricular zone. (B) Immunofluorescence staining for cerebral cortex sections of E13.5 labeled for IB4, gH2AX, and DAPI. Scale bars, 20 mm. The right insets indicated higher- magnification images of the delineated areas. Scale bars, 5 mm. (C) Representative images of cerebrovascular development at E13.5, E16, and E18. IB4 was used to label endothelial cells (ECs) and gH2AX was used to label DNA damage. The arrows in (C) showed ECs with DNA damage. Scale bars, 5 mm. (D) The graph shows the ratio of gH2AX+ ECs/total ECs in the brain area at E13.5, E16, and E18. n = 4 independent experiments. (E) Immunostaining for IB4, Sox2, and DAPI at the E13.5 cerebral cortex. 3D reconstruction of confocal microscopy from E13.5 cerebral cortex sections stained for IB4 (red) and Sox2 (green). Scale bars, 20 mm. (F) RNF20 mRNA levels were detected in ECs, neurons, astrocytes, microglia, and NPCs. b-Actin was an internal reference gene. NPCs, neural precursor cells. n = 3 independent experiments. (G) Relative mRNA levels of RNF20 in brain ECs at E13.5, E16, E18, and P0. n = 3 independent experiments.
Resource Source Identifier Antibodies Rabbit Anti Rnf20 Proteintech, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/274+rabbit+anti+rnf20/RNF20+Antibody/pm36103829-226-2-8
Average 93 stars, based on 1 article reviews
resource source identifier antibodies rabbit anti rnf20 proteintech - by Bioz Stars, 2026-10
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anti rnf20  (Santa Cruz Biotechnology)
90
Santa Cruz Biotechnology anti rnf20
(A) IFN-β luciferase reporter assay of HEK293T cells transiently transfected with HA-tagged RIG-I, MDA5, MAVS, STING, or TBK1 (100 ng) together with either <t>RNF20-Flag</t> expression plasmid (100 ng) or an empty control vector, followed by infection with VSV-GFP (MOI = 1.0) for 12h . (B, C) HEK293T cells were transiently co-transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), and HA-tagged RIG-I or MDA5, together with increasing doses of RNF20-Flag expression plasmid (0, 50, 100, 200, 300, or 400 ng). (D) HEK293T cells were transiently co-transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), poly(I:C), and Flag-RIG-I (100 ng), together with RNF20-interfering plasmid. (E, F) HEK293T cells were co-transfected with Flag-RNF20 and either Myc-tagged RIG-I or Myc-tagged MDA5, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Flag antibody and analyzed by immunoblotting with the indicated antibodies. (G, H) Confocal microscopy analysis of HeLa cells infected with NDV for 24 h, followed by immunostaining with <t>anti-RNF20,</t> anti-RIG-I, or anti-MDA5 primary antibodies, and Alexa Fluor 488-conjugated goat anti-mouse IgG (green) or Cy3-conjugated goat anti-rat IgG (red) secondary antibodies. (I, J) Schematic representation of RIG-I and MDA5 structures and their truncated mutants. (K) HEK293T cells were co-transfected with Myc-tagged MDA5 or its mutants together with Flag-RNF20, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Myc antibody and analyzed by immunoblotting with the indicated antibodies. (L, M) HEK293T cells were co-transfected with Myc-tagged MDA5 or RIG-I (wild-type or dCARD mutants) together with Flag-RNF20, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Myc antibody and analyzed by immunoblotting with the indicated antibodies. Data are presented as mean ± SD; *** p < 0.001; Scale bars = 10 µm. The results were repeated in three independent experiments with three replicas each. (Note: UI denotes uninfected virus; pcDNA3.1: empty vector control; shNC: non-targeting shRNA control).
Anti Rnf20, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/274+rabbit+anti+rnf20/RNF20+Antibody/pmc12948124-224-75-76
Average 90 stars, based on 1 article reviews
anti rnf20 - by Bioz Stars, 2026-10
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rnf20 anti rabbit bio rad hrp conjugate  (Bio-Rad)
99
Bio-Rad rnf20 anti rabbit bio rad hrp conjugate
(A) IFN-β luciferase reporter assay of HEK293T cells transiently transfected with HA-tagged RIG-I, MDA5, MAVS, STING, or TBK1 (100 ng) together with either <t>RNF20-Flag</t> expression plasmid (100 ng) or an empty control vector, followed by infection with VSV-GFP (MOI = 1.0) for 12h . (B, C) HEK293T cells were transiently co-transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), and HA-tagged RIG-I or MDA5, together with increasing doses of RNF20-Flag expression plasmid (0, 50, 100, 200, 300, or 400 ng). (D) HEK293T cells were transiently co-transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), poly(I:C), and Flag-RIG-I (100 ng), together with RNF20-interfering plasmid. (E, F) HEK293T cells were co-transfected with Flag-RNF20 and either Myc-tagged RIG-I or Myc-tagged MDA5, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Flag antibody and analyzed by immunoblotting with the indicated antibodies. (G, H) Confocal microscopy analysis of HeLa cells infected with NDV for 24 h, followed by immunostaining with <t>anti-RNF20,</t> anti-RIG-I, or anti-MDA5 primary antibodies, and Alexa Fluor 488-conjugated goat anti-mouse IgG (green) or Cy3-conjugated goat anti-rat IgG (red) secondary antibodies. (I, J) Schematic representation of RIG-I and MDA5 structures and their truncated mutants. (K) HEK293T cells were co-transfected with Myc-tagged MDA5 or its mutants together with Flag-RNF20, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Myc antibody and analyzed by immunoblotting with the indicated antibodies. (L, M) HEK293T cells were co-transfected with Myc-tagged MDA5 or RIG-I (wild-type or dCARD mutants) together with Flag-RNF20, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Myc antibody and analyzed by immunoblotting with the indicated antibodies. Data are presented as mean ± SD; *** p < 0.001; Scale bars = 10 µm. The results were repeated in three independent experiments with three replicas each. (Note: UI denotes uninfected virus; pcDNA3.1: empty vector control; shNC: non-targeting shRNA control).
Rnf20 Anti Rabbit Bio Rad Hrp Conjugate, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/274+rabbit+anti+rnf20/Goat+Anti-Rabbit+IgG+(H+%2B+L)-HRP+Conjugate/pmc05895630__41467_2018_3895_MOESM1_ESM-126-99-101
Average 99 stars, based on 1 article reviews
rnf20 anti rabbit bio rad hrp conjugate - by Bioz Stars, 2026-10
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rabbit anti rnf20  (Bethyl)
92
Bethyl rabbit anti rnf20
(A) IFN-β luciferase reporter assay of HEK293T cells transiently transfected with HA-tagged RIG-I, MDA5, MAVS, STING, or TBK1 (100 ng) together with either <t>RNF20-Flag</t> expression plasmid (100 ng) or an empty control vector, followed by infection with VSV-GFP (MOI = 1.0) for 12h . (B, C) HEK293T cells were transiently co-transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), and HA-tagged RIG-I or MDA5, together with increasing doses of RNF20-Flag expression plasmid (0, 50, 100, 200, 300, or 400 ng). (D) HEK293T cells were transiently co-transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), poly(I:C), and Flag-RIG-I (100 ng), together with RNF20-interfering plasmid. (E, F) HEK293T cells were co-transfected with Flag-RNF20 and either Myc-tagged RIG-I or Myc-tagged MDA5, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Flag antibody and analyzed by immunoblotting with the indicated antibodies. (G, H) Confocal microscopy analysis of HeLa cells infected with NDV for 24 h, followed by immunostaining with <t>anti-RNF20,</t> anti-RIG-I, or anti-MDA5 primary antibodies, and Alexa Fluor 488-conjugated goat anti-mouse IgG (green) or Cy3-conjugated goat anti-rat IgG (red) secondary antibodies. (I, J) Schematic representation of RIG-I and MDA5 structures and their truncated mutants. (K) HEK293T cells were co-transfected with Myc-tagged MDA5 or its mutants together with Flag-RNF20, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Myc antibody and analyzed by immunoblotting with the indicated antibodies. (L, M) HEK293T cells were co-transfected with Myc-tagged MDA5 or RIG-I (wild-type or dCARD mutants) together with Flag-RNF20, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Myc antibody and analyzed by immunoblotting with the indicated antibodies. Data are presented as mean ± SD; *** p < 0.001; Scale bars = 10 µm. The results were repeated in three independent experiments with three replicas each. (Note: UI denotes uninfected virus; pcDNA3.1: empty vector control; shNC: non-targeting shRNA control).
Rabbit Anti Rnf20, supplied by Bethyl, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/274+rabbit+anti+rnf20/RNF20+Antibody/pmc04274264-275-14-17
Average 92 stars, based on 1 article reviews
rabbit anti rnf20 - by Bioz Stars, 2026-10
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monoclonal anti rabbit igg  (Bethyl)
93
Bethyl monoclonal anti rabbit igg
(A) IFN-β luciferase reporter assay of HEK293T cells transiently transfected with HA-tagged RIG-I, MDA5, MAVS, STING, or TBK1 (100 ng) together with either <t>RNF20-Flag</t> expression plasmid (100 ng) or an empty control vector, followed by infection with VSV-GFP (MOI = 1.0) for 12h . (B, C) HEK293T cells were transiently co-transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), and HA-tagged RIG-I or MDA5, together with increasing doses of RNF20-Flag expression plasmid (0, 50, 100, 200, 300, or 400 ng). (D) HEK293T cells were transiently co-transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), poly(I:C), and Flag-RIG-I (100 ng), together with RNF20-interfering plasmid. (E, F) HEK293T cells were co-transfected with Flag-RNF20 and either Myc-tagged RIG-I or Myc-tagged MDA5, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Flag antibody and analyzed by immunoblotting with the indicated antibodies. (G, H) Confocal microscopy analysis of HeLa cells infected with NDV for 24 h, followed by immunostaining with <t>anti-RNF20,</t> anti-RIG-I, or anti-MDA5 primary antibodies, and Alexa Fluor 488-conjugated goat anti-mouse IgG (green) or Cy3-conjugated goat anti-rat IgG (red) secondary antibodies. (I, J) Schematic representation of RIG-I and MDA5 structures and their truncated mutants. (K) HEK293T cells were co-transfected with Myc-tagged MDA5 or its mutants together with Flag-RNF20, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Myc antibody and analyzed by immunoblotting with the indicated antibodies. (L, M) HEK293T cells were co-transfected with Myc-tagged MDA5 or RIG-I (wild-type or dCARD mutants) together with Flag-RNF20, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Myc antibody and analyzed by immunoblotting with the indicated antibodies. Data are presented as mean ± SD; *** p < 0.001; Scale bars = 10 µm. The results were repeated in three independent experiments with three replicas each. (Note: UI denotes uninfected virus; pcDNA3.1: empty vector control; shNC: non-targeting shRNA control).
Monoclonal Anti Rabbit Igg, supplied by Bethyl, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/274+rabbit+anti+rnf20/RNF40+Antibody/pmc10371241__jciinsight___8___164995___s114-33-45-70
Average 93 stars, based on 1 article reviews
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mouse anti-uh2b mm-0029 nro3  (MediMabs Inc)
90
MediMabs Inc mouse anti-uh2b mm-0029 nro3
(A) IFN-β luciferase reporter assay of HEK293T cells transiently transfected with HA-tagged RIG-I, MDA5, MAVS, STING, or TBK1 (100 ng) together with either <t>RNF20-Flag</t> expression plasmid (100 ng) or an empty control vector, followed by infection with VSV-GFP (MOI = 1.0) for 12h . (B, C) HEK293T cells were transiently co-transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), and HA-tagged RIG-I or MDA5, together with increasing doses of RNF20-Flag expression plasmid (0, 50, 100, 200, 300, or 400 ng). (D) HEK293T cells were transiently co-transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), poly(I:C), and Flag-RIG-I (100 ng), together with RNF20-interfering plasmid. (E, F) HEK293T cells were co-transfected with Flag-RNF20 and either Myc-tagged RIG-I or Myc-tagged MDA5, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Flag antibody and analyzed by immunoblotting with the indicated antibodies. (G, H) Confocal microscopy analysis of HeLa cells infected with NDV for 24 h, followed by immunostaining with <t>anti-RNF20,</t> anti-RIG-I, or anti-MDA5 primary antibodies, and Alexa Fluor 488-conjugated goat anti-mouse IgG (green) or Cy3-conjugated goat anti-rat IgG (red) secondary antibodies. (I, J) Schematic representation of RIG-I and MDA5 structures and their truncated mutants. (K) HEK293T cells were co-transfected with Myc-tagged MDA5 or its mutants together with Flag-RNF20, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Myc antibody and analyzed by immunoblotting with the indicated antibodies. (L, M) HEK293T cells were co-transfected with Myc-tagged MDA5 or RIG-I (wild-type or dCARD mutants) together with Flag-RNF20, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Myc antibody and analyzed by immunoblotting with the indicated antibodies. Data are presented as mean ± SD; *** p < 0.001; Scale bars = 10 µm. The results were repeated in three independent experiments with three replicas each. (Note: UI denotes uninfected virus; pcDNA3.1: empty vector control; shNC: non-targeting shRNA control).
Mouse Anti Uh2b Mm 0029 Nro3, supplied by MediMabs Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/274+rabbit+anti+rnf20/antiubiquityl+h2b/pmc04274264-275-21-25
Average 90 stars, based on 1 article reviews
mouse anti-uh2b mm-0029 nro3 - by Bioz Stars, 2026-10
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anti top2a  (OriGene)
91
OriGene anti top2a
(A) IFN-β luciferase reporter assay of HEK293T cells transiently transfected with HA-tagged RIG-I, MDA5, MAVS, STING, or TBK1 (100 ng) together with either <t>RNF20-Flag</t> expression plasmid (100 ng) or an empty control vector, followed by infection with VSV-GFP (MOI = 1.0) for 12h . (B, C) HEK293T cells were transiently co-transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), and HA-tagged RIG-I or MDA5, together with increasing doses of RNF20-Flag expression plasmid (0, 50, 100, 200, 300, or 400 ng). (D) HEK293T cells were transiently co-transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), poly(I:C), and Flag-RIG-I (100 ng), together with RNF20-interfering plasmid. (E, F) HEK293T cells were co-transfected with Flag-RNF20 and either Myc-tagged RIG-I or Myc-tagged MDA5, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Flag antibody and analyzed by immunoblotting with the indicated antibodies. (G, H) Confocal microscopy analysis of HeLa cells infected with NDV for 24 h, followed by immunostaining with <t>anti-RNF20,</t> anti-RIG-I, or anti-MDA5 primary antibodies, and Alexa Fluor 488-conjugated goat anti-mouse IgG (green) or Cy3-conjugated goat anti-rat IgG (red) secondary antibodies. (I, J) Schematic representation of RIG-I and MDA5 structures and their truncated mutants. (K) HEK293T cells were co-transfected with Myc-tagged MDA5 or its mutants together with Flag-RNF20, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Myc antibody and analyzed by immunoblotting with the indicated antibodies. (L, M) HEK293T cells were co-transfected with Myc-tagged MDA5 or RIG-I (wild-type or dCARD mutants) together with Flag-RNF20, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Myc antibody and analyzed by immunoblotting with the indicated antibodies. Data are presented as mean ± SD; *** p < 0.001; Scale bars = 10 µm. The results were repeated in three independent experiments with three replicas each. (Note: UI denotes uninfected virus; pcDNA3.1: empty vector control; shNC: non-targeting shRNA control).
Anti Top2a, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/274+rabbit+anti+rnf20/Topoisomerase+II+alpha+(TOP2A)+Rabbit+Polyclonal+Antibody/pm36738952-113-12-15
Average 91 stars, based on 1 article reviews
anti top2a - by Bioz Stars, 2026-10
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zscan4  (OriGene)
92
OriGene zscan4
(A) IFN-β luciferase reporter assay of HEK293T cells transiently transfected with HA-tagged RIG-I, MDA5, MAVS, STING, or TBK1 (100 ng) together with either <t>RNF20-Flag</t> expression plasmid (100 ng) or an empty control vector, followed by infection with VSV-GFP (MOI = 1.0) for 12h . (B, C) HEK293T cells were transiently co-transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), and HA-tagged RIG-I or MDA5, together with increasing doses of RNF20-Flag expression plasmid (0, 50, 100, 200, 300, or 400 ng). (D) HEK293T cells were transiently co-transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), poly(I:C), and Flag-RIG-I (100 ng), together with RNF20-interfering plasmid. (E, F) HEK293T cells were co-transfected with Flag-RNF20 and either Myc-tagged RIG-I or Myc-tagged MDA5, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Flag antibody and analyzed by immunoblotting with the indicated antibodies. (G, H) Confocal microscopy analysis of HeLa cells infected with NDV for 24 h, followed by immunostaining with <t>anti-RNF20,</t> anti-RIG-I, or anti-MDA5 primary antibodies, and Alexa Fluor 488-conjugated goat anti-mouse IgG (green) or Cy3-conjugated goat anti-rat IgG (red) secondary antibodies. (I, J) Schematic representation of RIG-I and MDA5 structures and their truncated mutants. (K) HEK293T cells were co-transfected with Myc-tagged MDA5 or its mutants together with Flag-RNF20, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Myc antibody and analyzed by immunoblotting with the indicated antibodies. (L, M) HEK293T cells were co-transfected with Myc-tagged MDA5 or RIG-I (wild-type or dCARD mutants) together with Flag-RNF20, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Myc antibody and analyzed by immunoblotting with the indicated antibodies. Data are presented as mean ± SD; *** p < 0.001; Scale bars = 10 µm. The results were repeated in three independent experiments with three replicas each. (Note: UI denotes uninfected virus; pcDNA3.1: empty vector control; shNC: non-targeting shRNA control).
Zscan4, supplied by OriGene, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/274+rabbit+anti+rnf20/ZSCAN4+Rabbit+Polyclonal+Antibody/portney_benjamin_a__2018__the_role_of_zscan4_in_cancer_stemness-1180-11-12
Average 92 stars, based on 1 article reviews
zscan4 - by Bioz Stars, 2026-10
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Image Search Results


A. Images of tumorspheres from 3 lung adenocarcinoma tissues (Samples 1, 2, and 3). B. Western Blot analysis of CD133 (as a surface biomarker of CICs), CD44 (as another surface biomarker of CICs), USP22, RNF20, and H2Bub1, shows enriched USP22 protein in tumorspheres compared to original cancer tissues. C. Flow cytometry analysis of CD133 expression demonstrates a strong CD133 expression in about 30% of tumorsphere cells. D. Western Blot analysis demonstrates USP22 is markedly elevated, while H2Bub1 were significantly deceased in CD133+ cancer cells compared to CD133− cells sorted from tumorspheres. E. Flow cytometry analysis of CD44 expression demonstrates a strong CD44 expression in about 12% to 44% of tumorsphere cells. F. Western Blot analysis demonstrates USP22 and CD133 are markedly elevated, while H2Bub1 and RNF20 were markedly or moderately deceased in CD44+ cancer cells compared to CD44− cells sorted from tumorspheres. G. Mouse xenografts generated by both CD133− and CD133+ cancer cells isolated from tumorspheres, indicating CD133+ cells may represent CICs with stem cell characteristics in tumorspheres.

Journal: Molecular cancer research : MCR

Article Title: Targeting USP22 Suppresses Tumorigenicity and Enhances Cisplatin Sensitivity Through ALDH1A3 Downregulation in Cancer-initiating Cells from Lung Adenocarcinoma

doi: 10.1158/1541-7786.MCR-18-0042

Figure Lengend Snippet: A. Images of tumorspheres from 3 lung adenocarcinoma tissues (Samples 1, 2, and 3). B. Western Blot analysis of CD133 (as a surface biomarker of CICs), CD44 (as another surface biomarker of CICs), USP22, RNF20, and H2Bub1, shows enriched USP22 protein in tumorspheres compared to original cancer tissues. C. Flow cytometry analysis of CD133 expression demonstrates a strong CD133 expression in about 30% of tumorsphere cells. D. Western Blot analysis demonstrates USP22 is markedly elevated, while H2Bub1 were significantly deceased in CD133+ cancer cells compared to CD133− cells sorted from tumorspheres. E. Flow cytometry analysis of CD44 expression demonstrates a strong CD44 expression in about 12% to 44% of tumorsphere cells. F. Western Blot analysis demonstrates USP22 and CD133 are markedly elevated, while H2Bub1 and RNF20 were markedly or moderately deceased in CD44+ cancer cells compared to CD44− cells sorted from tumorspheres. G. Mouse xenografts generated by both CD133− and CD133+ cancer cells isolated from tumorspheres, indicating CD133+ cells may represent CICs with stem cell characteristics in tumorspheres.

Article Snippet: Rabbit anti-RNF20 antibody (#9425) was purchased from Cell Signaling Technology (Beverly, CA, USA).

Techniques: Western Blot, Biomarker Discovery, Flow Cytometry, Expressing, Generated, Isolation

A. Morphology changes of tumorspheres in differentiating culture medium (left panel); Western Blot analysis of USP22, CD133, RNF20 and H2Bub1 in tumorspheres in differentiating conditions (right panel). B. Western Blot analysis of USP22, CD133 and H2Bub1 in GFP-CTRL and USP22-KD cells (left panel); Quantitative analysis of tumorsphere formation upon USP22 knockdown, GFP-CTRL for cells transfected with control shRNA, USP22-KD for specific shRNA targeting USP22 (**P < 0.01, compared with Control, middle panel) and Tumorspheres (right panel). C. In vivo tumorigenesis of GFP-CTRL and USP22-KD tumorsphere cells, demonstrating USP22 knockdown suppressed both tumorsphere formation and tumorigenesis of lung adenocarcinoma CICs.

Journal: Molecular cancer research : MCR

Article Title: Targeting USP22 Suppresses Tumorigenicity and Enhances Cisplatin Sensitivity Through ALDH1A3 Downregulation in Cancer-initiating Cells from Lung Adenocarcinoma

doi: 10.1158/1541-7786.MCR-18-0042

Figure Lengend Snippet: A. Morphology changes of tumorspheres in differentiating culture medium (left panel); Western Blot analysis of USP22, CD133, RNF20 and H2Bub1 in tumorspheres in differentiating conditions (right panel). B. Western Blot analysis of USP22, CD133 and H2Bub1 in GFP-CTRL and USP22-KD cells (left panel); Quantitative analysis of tumorsphere formation upon USP22 knockdown, GFP-CTRL for cells transfected with control shRNA, USP22-KD for specific shRNA targeting USP22 (**P < 0.01, compared with Control, middle panel) and Tumorspheres (right panel). C. In vivo tumorigenesis of GFP-CTRL and USP22-KD tumorsphere cells, demonstrating USP22 knockdown suppressed both tumorsphere formation and tumorigenesis of lung adenocarcinoma CICs.

Article Snippet: Rabbit anti-RNF20 antibody (#9425) was purchased from Cell Signaling Technology (Beverly, CA, USA).

Techniques: Western Blot, Knockdown, Transfection, Control, shRNA, In Vivo

Figure 1. RNF20 is correlated with DNA damage during cerebrovascular development (A) Graphic representation of the mouse cerebral cortex and its vascularization pattern. The boxed area represented the location of cerebral cortex. MGE, medial ganglionic eminence; LGE, lateral ganglionic eminence; VZ, ventricular zone. (B) Immunofluorescence staining for cerebral cortex sections of E13.5 labeled for IB4, gH2AX, and DAPI. Scale bars, 20 mm. The right insets indicated higher- magnification images of the delineated areas. Scale bars, 5 mm. (C) Representative images of cerebrovascular development at E13.5, E16, and E18. IB4 was used to label endothelial cells (ECs) and gH2AX was used to label DNA damage. The arrows in (C) showed ECs with DNA damage. Scale bars, 5 mm. (D) The graph shows the ratio of gH2AX+ ECs/total ECs in the brain area at E13.5, E16, and E18. n = 4 independent experiments. (E) Immunostaining for IB4, Sox2, and DAPI at the E13.5 cerebral cortex. 3D reconstruction of confocal microscopy from E13.5 cerebral cortex sections stained for IB4 (red) and Sox2 (green). Scale bars, 20 mm. (F) RNF20 mRNA levels were detected in ECs, neurons, astrocytes, microglia, and NPCs. b-Actin was an internal reference gene. NPCs, neural precursor cells. n = 3 independent experiments. (G) Relative mRNA levels of RNF20 in brain ECs at E13.5, E16, E18, and P0. n = 3 independent experiments.

Journal: Cell reports

Article Title: Endothelial cells regulated by RNF20 orchestrate the proliferation and differentiation of neural precursor cells during embryonic development.

doi: 10.1016/j.celrep.2022.111350

Figure Lengend Snippet: Figure 1. RNF20 is correlated with DNA damage during cerebrovascular development (A) Graphic representation of the mouse cerebral cortex and its vascularization pattern. The boxed area represented the location of cerebral cortex. MGE, medial ganglionic eminence; LGE, lateral ganglionic eminence; VZ, ventricular zone. (B) Immunofluorescence staining for cerebral cortex sections of E13.5 labeled for IB4, gH2AX, and DAPI. Scale bars, 20 mm. The right insets indicated higher- magnification images of the delineated areas. Scale bars, 5 mm. (C) Representative images of cerebrovascular development at E13.5, E16, and E18. IB4 was used to label endothelial cells (ECs) and gH2AX was used to label DNA damage. The arrows in (C) showed ECs with DNA damage. Scale bars, 5 mm. (D) The graph shows the ratio of gH2AX+ ECs/total ECs in the brain area at E13.5, E16, and E18. n = 4 independent experiments. (E) Immunostaining for IB4, Sox2, and DAPI at the E13.5 cerebral cortex. 3D reconstruction of confocal microscopy from E13.5 cerebral cortex sections stained for IB4 (red) and Sox2 (green). Scale bars, 20 mm. (F) RNF20 mRNA levels were detected in ECs, neurons, astrocytes, microglia, and NPCs. b-Actin was an internal reference gene. NPCs, neural precursor cells. n = 3 independent experiments. (G) Relative mRNA levels of RNF20 in brain ECs at E13.5, E16, E18, and P0. n = 3 independent experiments.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit Anti-RNF20 Proteintech Cat#21625-1-AP; RRID: AB_10734436 Rabbit Anti-CILP2 Proteintech Cat#11813-1-AP; RRID: AB_2877795 Rat Anti-BrdU Abcam Cat#ab6326; RRID: AB_305426 Rabbit Anti-gH2AX Cell Signaling Technology Cat# 9718S; RRID: AB_2118009 Rabbit Anti-PDGFRb Abcam Cat#ab32570; RRID: AB_777165 Rat Anti-CD31 Biolegend Cat#102406; RRID: AB_312901 Rabbit Anti-Collagen IV Abcam Cat#ab6586; RRID: AB_305584 Rabbit Anti-ZO-1 Invitrogen Cat#40–2200; RRID: AB_2533456 Mouse Anti-Claudin5 Invitrogen Cat#352500; RRID: AB_2533200 Mouse Anti-Nestin Millipore Cat#MAB353; RRID: AB_94911 Mouse Anti-Tuj1 Millipore Cat#MAB1637; RRID: AB_2210524 Rabbit Anti-PAX6 Abcam Cat#ab5790; RRID: AB_305110 Rabbit anti-SOX2 Cell Signaling Technologies Cat#3728; RRID: AB_2194037 Rabbit Anti-Ki67 Abcam Cat#ab15580; RRID: AB_443209 Rabbit Anti-pH3 Cell Signaling Technology Cat#3377S; RRID: AB_1549592 Rabbit Anti-TBR2 Abcam Cat#ab23345; RRID: AB_778267 Rat Anti-Ctip2 Abcam Cat#ab18465; RRID: AB_2064130 Mouse Anti-SATB2 Abcam Cat#ab51502; RRID: AB_882455 Rabbit Anti-TBR1 Abcam Cat#ab31940; RRID: AB_2200219 Rabbit Anti-H2Bub1 Cell Signaling Technology Cat# 5546T; RRID: AB_10693452 Mouse Anti-FLAG Sigma Cat# F1804; RRID: AB_262044 Rabbit Anti-HA Abmart Cat#M20003; RRID: AB_2864345 Rabbit Anti-b-Catenin Cell Signaling Technology Cat# 8480s; RRID: AB_11127855 Rabbit Anti-p-b-Catenin Cell Signaling Technology Cat#9561s; RRID: AB_331729 Rabbit Anti-p-GSK3b (ser9) Cell Signaling Technology Cat#5558; RRID: AB_10013750 Rabbit Anti-b-ACTIN Proteintech Cat# 20536-1-AP; RRID: AB_10700003 Chemicals, peptides, and recombinant proteins Papain Worthington Cat# 38P18865 Alexa Fluor 555 Invitrogen Cat# a30677 IB4 Vector Labs Cat# B-1205 TOPFlash Beyotime Cat# D2501 5-bromo-2-deoxyuridine (BrdU) Sigma-Aldrich Cat# B5002-5G CILP2 Recombinant Human Protein Zeye Cat# IC2132402 Recombinant Murine Wnt-3a PeproTech Cat# 315-2010 IWP-2 Selleck Cat# S7085 Critical commercial assays In Situ Cell Death Detection Kit Roche Cat#12156792910 SYBR Green PCR Kit TIANGEN Cat#FP205 CILP2 ELISA Kit Boshen Cat#BS-E8014H1 Deposited data Raw and analyzed data of bulk RNA-seq This paper GEO: GSE192997 Experimental models: Cell lines HEK293 Cell Line ATCC ATCC CRL-1573; RRID: CVCL_0045 (Continued on next page) e1 Cell Reports 40, 111350, September 13, 2022

Techniques: Staining, Labeling, Immunostaining, Confocal Microscopy

Figure 2. RNF20 deficiency leads to DNA damage accumulation in ECs (A) Schematic of the Rnf20 conditional knockout (cKO) mouse construction strategy. LoxP sites were inserted between the second exon and fourth exon in these mice. The Rnf20 gene was knocked out by endothelial-specific Cre recombinase splicing. (B) Western blot analysis showed that the RNF20 expression was reduced in the ECs of Rnf20cKO-Tie2 mice. (C) The bar graph shows the normalized densitometry of RNF20. N = 3 Rnf20fl/flmice and 3 Rnf20cKO-Tie2 mice. (D) Representative confocal images of cerebral vessels in the brain cortex stained with IB4. Scale bars, 100 mm. (E and F) Quantification of the length of the vessels (E) and the number of branch points (F) in the brain cortex. N = 5 Rnf20fl/flmice and 5 Rnf20cKO-Tie2 mice. (G) Platelet-derived growth factor receptor b (PDGFRb) labeling of pericytes (green) showed similar coverage along cerebral vessels (red) in Rnf20fl/fland Rnf20cKO-Tie2 brain cortices. The right insets indicated higher magnification images of the delineated areas. Scale bars, 20 mm. (H) Quantification of PDGFRb+ pericyte coverage along cerebral vessels.N = 6 Rnf20fl/flmice and 6 Rnf20cKO-Tie2 mice. (I) RNF20 conditional KO decreased the protein levels of H2Bub1, while the protein levels of gH2AX were increased in the ECs. (J) Quantification of the H2Bub1 and gH2AX protein levels. N = 4 Rnf20fl/flmice and 4 Rnf20cKO-Tie2 mice. (K) The loss of endothelial RNF20 increased the fluorescence intensity of gH2AX. Scale bars, 10 mm. (L) Quantification of the gH2AX fluorescence intensity in Rnf20fl/flmice and Rnf20cKO-Tie2 mice. N = 5 Rnf20fl/flmice and 5 Rnf20cKO-Tie2 mice. Error bars represent means ± SEMs; 2-tailed unpaired t test; *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Journal: Cell reports

Article Title: Endothelial cells regulated by RNF20 orchestrate the proliferation and differentiation of neural precursor cells during embryonic development.

doi: 10.1016/j.celrep.2022.111350

Figure Lengend Snippet: Figure 2. RNF20 deficiency leads to DNA damage accumulation in ECs (A) Schematic of the Rnf20 conditional knockout (cKO) mouse construction strategy. LoxP sites were inserted between the second exon and fourth exon in these mice. The Rnf20 gene was knocked out by endothelial-specific Cre recombinase splicing. (B) Western blot analysis showed that the RNF20 expression was reduced in the ECs of Rnf20cKO-Tie2 mice. (C) The bar graph shows the normalized densitometry of RNF20. N = 3 Rnf20fl/flmice and 3 Rnf20cKO-Tie2 mice. (D) Representative confocal images of cerebral vessels in the brain cortex stained with IB4. Scale bars, 100 mm. (E and F) Quantification of the length of the vessels (E) and the number of branch points (F) in the brain cortex. N = 5 Rnf20fl/flmice and 5 Rnf20cKO-Tie2 mice. (G) Platelet-derived growth factor receptor b (PDGFRb) labeling of pericytes (green) showed similar coverage along cerebral vessels (red) in Rnf20fl/fland Rnf20cKO-Tie2 brain cortices. The right insets indicated higher magnification images of the delineated areas. Scale bars, 20 mm. (H) Quantification of PDGFRb+ pericyte coverage along cerebral vessels.N = 6 Rnf20fl/flmice and 6 Rnf20cKO-Tie2 mice. (I) RNF20 conditional KO decreased the protein levels of H2Bub1, while the protein levels of gH2AX were increased in the ECs. (J) Quantification of the H2Bub1 and gH2AX protein levels. N = 4 Rnf20fl/flmice and 4 Rnf20cKO-Tie2 mice. (K) The loss of endothelial RNF20 increased the fluorescence intensity of gH2AX. Scale bars, 10 mm. (L) Quantification of the gH2AX fluorescence intensity in Rnf20fl/flmice and Rnf20cKO-Tie2 mice. N = 5 Rnf20fl/flmice and 5 Rnf20cKO-Tie2 mice. Error bars represent means ± SEMs; 2-tailed unpaired t test; *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit Anti-RNF20 Proteintech Cat#21625-1-AP; RRID: AB_10734436 Rabbit Anti-CILP2 Proteintech Cat#11813-1-AP; RRID: AB_2877795 Rat Anti-BrdU Abcam Cat#ab6326; RRID: AB_305426 Rabbit Anti-gH2AX Cell Signaling Technology Cat# 9718S; RRID: AB_2118009 Rabbit Anti-PDGFRb Abcam Cat#ab32570; RRID: AB_777165 Rat Anti-CD31 Biolegend Cat#102406; RRID: AB_312901 Rabbit Anti-Collagen IV Abcam Cat#ab6586; RRID: AB_305584 Rabbit Anti-ZO-1 Invitrogen Cat#40–2200; RRID: AB_2533456 Mouse Anti-Claudin5 Invitrogen Cat#352500; RRID: AB_2533200 Mouse Anti-Nestin Millipore Cat#MAB353; RRID: AB_94911 Mouse Anti-Tuj1 Millipore Cat#MAB1637; RRID: AB_2210524 Rabbit Anti-PAX6 Abcam Cat#ab5790; RRID: AB_305110 Rabbit anti-SOX2 Cell Signaling Technologies Cat#3728; RRID: AB_2194037 Rabbit Anti-Ki67 Abcam Cat#ab15580; RRID: AB_443209 Rabbit Anti-pH3 Cell Signaling Technology Cat#3377S; RRID: AB_1549592 Rabbit Anti-TBR2 Abcam Cat#ab23345; RRID: AB_778267 Rat Anti-Ctip2 Abcam Cat#ab18465; RRID: AB_2064130 Mouse Anti-SATB2 Abcam Cat#ab51502; RRID: AB_882455 Rabbit Anti-TBR1 Abcam Cat#ab31940; RRID: AB_2200219 Rabbit Anti-H2Bub1 Cell Signaling Technology Cat# 5546T; RRID: AB_10693452 Mouse Anti-FLAG Sigma Cat# F1804; RRID: AB_262044 Rabbit Anti-HA Abmart Cat#M20003; RRID: AB_2864345 Rabbit Anti-b-Catenin Cell Signaling Technology Cat# 8480s; RRID: AB_11127855 Rabbit Anti-p-b-Catenin Cell Signaling Technology Cat#9561s; RRID: AB_331729 Rabbit Anti-p-GSK3b (ser9) Cell Signaling Technology Cat#5558; RRID: AB_10013750 Rabbit Anti-b-ACTIN Proteintech Cat# 20536-1-AP; RRID: AB_10700003 Chemicals, peptides, and recombinant proteins Papain Worthington Cat# 38P18865 Alexa Fluor 555 Invitrogen Cat# a30677 IB4 Vector Labs Cat# B-1205 TOPFlash Beyotime Cat# D2501 5-bromo-2-deoxyuridine (BrdU) Sigma-Aldrich Cat# B5002-5G CILP2 Recombinant Human Protein Zeye Cat# IC2132402 Recombinant Murine Wnt-3a PeproTech Cat# 315-2010 IWP-2 Selleck Cat# S7085 Critical commercial assays In Situ Cell Death Detection Kit Roche Cat#12156792910 SYBR Green PCR Kit TIANGEN Cat#FP205 CILP2 ELISA Kit Boshen Cat#BS-E8014H1 Deposited data Raw and analyzed data of bulk RNA-seq This paper GEO: GSE192997 Experimental models: Cell lines HEK293 Cell Line ATCC ATCC CRL-1573; RRID: CVCL_0045 (Continued on next page) e1 Cell Reports 40, 111350, September 13, 2022

Techniques: Knock-Out, Western Blot, Expressing, Staining, Derivative Assay, Labeling

Figure 3. Endothelial RNF20 cKO mice show autism-like behaviors (A) The open field test showed that the total distance was similar in Rnf20fl/flmice and Rnf20cKO-Tie2 mice. N = 17 Rnf20fl/flmice and 17 Rnf20cKO-Tie2 mice. (B) The open field test showed that time spent in the center was reduced in Rnf20cKO-Tie2 mice. N = 12 Rnf20fl/flmice and 12 Rnf20cKO-Tie2 mice. (C and D) Compared with Rnf20fl/flmice, the Rnf20cKO-Tie2 mice spent less time in the open arms (C). The Rnf20cKO-Tie2 mice spent much more time in the closed arms (D). N = 12 Rnf20fl/flmice and 12 Rnf20cKO-Tie2 mice. (E and F) The representative tracks of the 3-chamber social interaction test. (G) The 3-chamber social interaction test showed that Rnf20cKO-Tie2 mice spent a similar amount of time in the chamber with a stranger 1 and in the empty chamber. Rnf20fl/flmice spent more time with stranger 1. N = 16 Rnf20fl/flmice and 11 Rnf20cKO-Tie2 mice. (H) Rnf20cKO-Tie2 mice spent a similar amount of time in the chamber with a stranger 1 and in the opposite chamber with stranger 2. Rnf20fl/flmice spent more time with stranger 2. N = 15 Rnf20fl/flmice and 8 Rnf20cKO-Tie2 mice. (I–L) Representative USV spectrogram in isolated pups (I). Compared with Rnf20fl/flmice, the number of calls (J), the call duration (K), and the total duration (L) were reduced in Rnf20cKO-Tie2 mice. N = 14 Rnf20fl/flmice and 14 Rnf20cKO-Tie2 mice. (M) In the marble-burying test, Rnf20cKO-Tie2 mice buried more marbles than Rnf20fl/flmice. N = 10 Rnf20fl/flmice and 10 Rnf20cKO-Tie2 mice. Error bars represent means ± SEMs; 2-tailed unpaired t test; *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Journal: Cell reports

Article Title: Endothelial cells regulated by RNF20 orchestrate the proliferation and differentiation of neural precursor cells during embryonic development.

doi: 10.1016/j.celrep.2022.111350

Figure Lengend Snippet: Figure 3. Endothelial RNF20 cKO mice show autism-like behaviors (A) The open field test showed that the total distance was similar in Rnf20fl/flmice and Rnf20cKO-Tie2 mice. N = 17 Rnf20fl/flmice and 17 Rnf20cKO-Tie2 mice. (B) The open field test showed that time spent in the center was reduced in Rnf20cKO-Tie2 mice. N = 12 Rnf20fl/flmice and 12 Rnf20cKO-Tie2 mice. (C and D) Compared with Rnf20fl/flmice, the Rnf20cKO-Tie2 mice spent less time in the open arms (C). The Rnf20cKO-Tie2 mice spent much more time in the closed arms (D). N = 12 Rnf20fl/flmice and 12 Rnf20cKO-Tie2 mice. (E and F) The representative tracks of the 3-chamber social interaction test. (G) The 3-chamber social interaction test showed that Rnf20cKO-Tie2 mice spent a similar amount of time in the chamber with a stranger 1 and in the empty chamber. Rnf20fl/flmice spent more time with stranger 1. N = 16 Rnf20fl/flmice and 11 Rnf20cKO-Tie2 mice. (H) Rnf20cKO-Tie2 mice spent a similar amount of time in the chamber with a stranger 1 and in the opposite chamber with stranger 2. Rnf20fl/flmice spent more time with stranger 2. N = 15 Rnf20fl/flmice and 8 Rnf20cKO-Tie2 mice. (I–L) Representative USV spectrogram in isolated pups (I). Compared with Rnf20fl/flmice, the number of calls (J), the call duration (K), and the total duration (L) were reduced in Rnf20cKO-Tie2 mice. N = 14 Rnf20fl/flmice and 14 Rnf20cKO-Tie2 mice. (M) In the marble-burying test, Rnf20cKO-Tie2 mice buried more marbles than Rnf20fl/flmice. N = 10 Rnf20fl/flmice and 10 Rnf20cKO-Tie2 mice. Error bars represent means ± SEMs; 2-tailed unpaired t test; *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit Anti-RNF20 Proteintech Cat#21625-1-AP; RRID: AB_10734436 Rabbit Anti-CILP2 Proteintech Cat#11813-1-AP; RRID: AB_2877795 Rat Anti-BrdU Abcam Cat#ab6326; RRID: AB_305426 Rabbit Anti-gH2AX Cell Signaling Technology Cat# 9718S; RRID: AB_2118009 Rabbit Anti-PDGFRb Abcam Cat#ab32570; RRID: AB_777165 Rat Anti-CD31 Biolegend Cat#102406; RRID: AB_312901 Rabbit Anti-Collagen IV Abcam Cat#ab6586; RRID: AB_305584 Rabbit Anti-ZO-1 Invitrogen Cat#40–2200; RRID: AB_2533456 Mouse Anti-Claudin5 Invitrogen Cat#352500; RRID: AB_2533200 Mouse Anti-Nestin Millipore Cat#MAB353; RRID: AB_94911 Mouse Anti-Tuj1 Millipore Cat#MAB1637; RRID: AB_2210524 Rabbit Anti-PAX6 Abcam Cat#ab5790; RRID: AB_305110 Rabbit anti-SOX2 Cell Signaling Technologies Cat#3728; RRID: AB_2194037 Rabbit Anti-Ki67 Abcam Cat#ab15580; RRID: AB_443209 Rabbit Anti-pH3 Cell Signaling Technology Cat#3377S; RRID: AB_1549592 Rabbit Anti-TBR2 Abcam Cat#ab23345; RRID: AB_778267 Rat Anti-Ctip2 Abcam Cat#ab18465; RRID: AB_2064130 Mouse Anti-SATB2 Abcam Cat#ab51502; RRID: AB_882455 Rabbit Anti-TBR1 Abcam Cat#ab31940; RRID: AB_2200219 Rabbit Anti-H2Bub1 Cell Signaling Technology Cat# 5546T; RRID: AB_10693452 Mouse Anti-FLAG Sigma Cat# F1804; RRID: AB_262044 Rabbit Anti-HA Abmart Cat#M20003; RRID: AB_2864345 Rabbit Anti-b-Catenin Cell Signaling Technology Cat# 8480s; RRID: AB_11127855 Rabbit Anti-p-b-Catenin Cell Signaling Technology Cat#9561s; RRID: AB_331729 Rabbit Anti-p-GSK3b (ser9) Cell Signaling Technology Cat#5558; RRID: AB_10013750 Rabbit Anti-b-ACTIN Proteintech Cat# 20536-1-AP; RRID: AB_10700003 Chemicals, peptides, and recombinant proteins Papain Worthington Cat# 38P18865 Alexa Fluor 555 Invitrogen Cat# a30677 IB4 Vector Labs Cat# B-1205 TOPFlash Beyotime Cat# D2501 5-bromo-2-deoxyuridine (BrdU) Sigma-Aldrich Cat# B5002-5G CILP2 Recombinant Human Protein Zeye Cat# IC2132402 Recombinant Murine Wnt-3a PeproTech Cat# 315-2010 IWP-2 Selleck Cat# S7085 Critical commercial assays In Situ Cell Death Detection Kit Roche Cat#12156792910 SYBR Green PCR Kit TIANGEN Cat#FP205 CILP2 ELISA Kit Boshen Cat#BS-E8014H1 Deposited data Raw and analyzed data of bulk RNA-seq This paper GEO: GSE192997 Experimental models: Cell lines HEK293 Cell Line ATCC ATCC CRL-1573; RRID: CVCL_0045 (Continued on next page) e1 Cell Reports 40, 111350, September 13, 2022

Techniques: Isolation

Figure 4. Endothelial RNF20 regulates brain neural progenitor cells proliferation (A) Confocal images of BrdU staining at E13.5 cerebral cortex. BrdU was injected intraperitoneally to pregnant mice at E13.5 for 2 h. Scale bars, 20 mm. (B) Statistic of BrdU+ cells in Rnf20fl/flmice and Rnf20cKO-Tie2 mice. n = 5 independent experiments. (C) Confocal images showed that the marker of proliferation Ki67 was increased at E13.5 cerebral cortex in Rnf20cKO-Tie2 mice. Scale bars, 20 mm. (D) Quantitative analyses of the Ki67+ cells at E13.5 cerebral cortex. n = 5 independent experiments. (E) Immunostaining for the mitotic marker pH3 in Rnf20fl/flmice and Rnf20cKO-Tie2 mice cortices at E13.5. Scale bars, 20 mm. (F) Quantification of pH3+ mitotic cells in the ventricular zone and subventricular zone (VZ/SVZ). n = 5 independent experiments. (G) Western blot analysis of Pax6 and Tbr2 protein levels in E13.5 Rnf20fl/flmice and Rnf20cKO-Tie2 mice. (H) The bar graph showed Pax6 and Tbr2 normalized densitometry. n = 5 independent experiments. (I) Confocal images showed that the Pax6+ cells were increased at E13.5 cerebral cortex in Rnf20cKO-Tie2 mice. Scale bars, 20 mm. (legend continued on next page)

Journal: Cell reports

Article Title: Endothelial cells regulated by RNF20 orchestrate the proliferation and differentiation of neural precursor cells during embryonic development.

doi: 10.1016/j.celrep.2022.111350

Figure Lengend Snippet: Figure 4. Endothelial RNF20 regulates brain neural progenitor cells proliferation (A) Confocal images of BrdU staining at E13.5 cerebral cortex. BrdU was injected intraperitoneally to pregnant mice at E13.5 for 2 h. Scale bars, 20 mm. (B) Statistic of BrdU+ cells in Rnf20fl/flmice and Rnf20cKO-Tie2 mice. n = 5 independent experiments. (C) Confocal images showed that the marker of proliferation Ki67 was increased at E13.5 cerebral cortex in Rnf20cKO-Tie2 mice. Scale bars, 20 mm. (D) Quantitative analyses of the Ki67+ cells at E13.5 cerebral cortex. n = 5 independent experiments. (E) Immunostaining for the mitotic marker pH3 in Rnf20fl/flmice and Rnf20cKO-Tie2 mice cortices at E13.5. Scale bars, 20 mm. (F) Quantification of pH3+ mitotic cells in the ventricular zone and subventricular zone (VZ/SVZ). n = 5 independent experiments. (G) Western blot analysis of Pax6 and Tbr2 protein levels in E13.5 Rnf20fl/flmice and Rnf20cKO-Tie2 mice. (H) The bar graph showed Pax6 and Tbr2 normalized densitometry. n = 5 independent experiments. (I) Confocal images showed that the Pax6+ cells were increased at E13.5 cerebral cortex in Rnf20cKO-Tie2 mice. Scale bars, 20 mm. (legend continued on next page)

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit Anti-RNF20 Proteintech Cat#21625-1-AP; RRID: AB_10734436 Rabbit Anti-CILP2 Proteintech Cat#11813-1-AP; RRID: AB_2877795 Rat Anti-BrdU Abcam Cat#ab6326; RRID: AB_305426 Rabbit Anti-gH2AX Cell Signaling Technology Cat# 9718S; RRID: AB_2118009 Rabbit Anti-PDGFRb Abcam Cat#ab32570; RRID: AB_777165 Rat Anti-CD31 Biolegend Cat#102406; RRID: AB_312901 Rabbit Anti-Collagen IV Abcam Cat#ab6586; RRID: AB_305584 Rabbit Anti-ZO-1 Invitrogen Cat#40–2200; RRID: AB_2533456 Mouse Anti-Claudin5 Invitrogen Cat#352500; RRID: AB_2533200 Mouse Anti-Nestin Millipore Cat#MAB353; RRID: AB_94911 Mouse Anti-Tuj1 Millipore Cat#MAB1637; RRID: AB_2210524 Rabbit Anti-PAX6 Abcam Cat#ab5790; RRID: AB_305110 Rabbit anti-SOX2 Cell Signaling Technologies Cat#3728; RRID: AB_2194037 Rabbit Anti-Ki67 Abcam Cat#ab15580; RRID: AB_443209 Rabbit Anti-pH3 Cell Signaling Technology Cat#3377S; RRID: AB_1549592 Rabbit Anti-TBR2 Abcam Cat#ab23345; RRID: AB_778267 Rat Anti-Ctip2 Abcam Cat#ab18465; RRID: AB_2064130 Mouse Anti-SATB2 Abcam Cat#ab51502; RRID: AB_882455 Rabbit Anti-TBR1 Abcam Cat#ab31940; RRID: AB_2200219 Rabbit Anti-H2Bub1 Cell Signaling Technology Cat# 5546T; RRID: AB_10693452 Mouse Anti-FLAG Sigma Cat# F1804; RRID: AB_262044 Rabbit Anti-HA Abmart Cat#M20003; RRID: AB_2864345 Rabbit Anti-b-Catenin Cell Signaling Technology Cat# 8480s; RRID: AB_11127855 Rabbit Anti-p-b-Catenin Cell Signaling Technology Cat#9561s; RRID: AB_331729 Rabbit Anti-p-GSK3b (ser9) Cell Signaling Technology Cat#5558; RRID: AB_10013750 Rabbit Anti-b-ACTIN Proteintech Cat# 20536-1-AP; RRID: AB_10700003 Chemicals, peptides, and recombinant proteins Papain Worthington Cat# 38P18865 Alexa Fluor 555 Invitrogen Cat# a30677 IB4 Vector Labs Cat# B-1205 TOPFlash Beyotime Cat# D2501 5-bromo-2-deoxyuridine (BrdU) Sigma-Aldrich Cat# B5002-5G CILP2 Recombinant Human Protein Zeye Cat# IC2132402 Recombinant Murine Wnt-3a PeproTech Cat# 315-2010 IWP-2 Selleck Cat# S7085 Critical commercial assays In Situ Cell Death Detection Kit Roche Cat#12156792910 SYBR Green PCR Kit TIANGEN Cat#FP205 CILP2 ELISA Kit Boshen Cat#BS-E8014H1 Deposited data Raw and analyzed data of bulk RNA-seq This paper GEO: GSE192997 Experimental models: Cell lines HEK293 Cell Line ATCC ATCC CRL-1573; RRID: CVCL_0045 (Continued on next page) e1 Cell Reports 40, 111350, September 13, 2022

Techniques: BrdU Staining, Injection, Marker, Immunostaining, Western Blot

Figure 6. Endothelial RNF20 influences NPCs proliferation and differentiation by secreting CILP2 (A and B) Gene Ontology (GO) analysis for downregulated or upregulated genes in the RNF20-depleted ECs at E13.5 cerebral cortex. (C) Heatmap shows global genes were sorted by fold change. (D) Volcano plots illustrated the downregulated (blue) and upregulated (yellow) genes in the ECs of Rnf20fl/flmice and Rnf20cKO-Tie2 mice. The Cilp2 gene indicated by the red dots highlighted the significant reduction in downregulated genes. (E) Heatmaps were further analyzed. The Cilp2 gene was one of the significantly differentially expressed genes. (F) Six significantly expressed genes were analyzed by qPCR in ECs of Rnf20fl/flmice and Rnf20cKO-Tie2 mice, of which Cilp2 has the highest expression. n = 3 independent experiments. (G) Western blot analysis of CILP2 and gH2AX protein levels in ECs from Rnf20fl/flmice and Rnf20cKO-Tie2 mice. (H) The bar graph shows CILP2 and gH2AX normalized densitometry. n = 3 independent experiments. (I) The Cilp2 mRNA levels were reduced in E13.5 ECs from Rnf20cKO-Tie2 mice. N =3Rnf20fl/flmice and 4 Rnf20cKO-Tie2 mice. (J) CILP2 secretion from the cultured ECs was measured by an ELISA kit. n = 4 independent experiments. (K) Western blot analysis showed that endothelial supernatant after Cilp2 knockdown affected NPC proliferation and differentiation. (L) Quantitative analyses of relative protein abundance in (K). Error bars represent means ± SEMs; 2-tailed unpaired t test; *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Journal: Cell reports

Article Title: Endothelial cells regulated by RNF20 orchestrate the proliferation and differentiation of neural precursor cells during embryonic development.

doi: 10.1016/j.celrep.2022.111350

Figure Lengend Snippet: Figure 6. Endothelial RNF20 influences NPCs proliferation and differentiation by secreting CILP2 (A and B) Gene Ontology (GO) analysis for downregulated or upregulated genes in the RNF20-depleted ECs at E13.5 cerebral cortex. (C) Heatmap shows global genes were sorted by fold change. (D) Volcano plots illustrated the downregulated (blue) and upregulated (yellow) genes in the ECs of Rnf20fl/flmice and Rnf20cKO-Tie2 mice. The Cilp2 gene indicated by the red dots highlighted the significant reduction in downregulated genes. (E) Heatmaps were further analyzed. The Cilp2 gene was one of the significantly differentially expressed genes. (F) Six significantly expressed genes were analyzed by qPCR in ECs of Rnf20fl/flmice and Rnf20cKO-Tie2 mice, of which Cilp2 has the highest expression. n = 3 independent experiments. (G) Western blot analysis of CILP2 and gH2AX protein levels in ECs from Rnf20fl/flmice and Rnf20cKO-Tie2 mice. (H) The bar graph shows CILP2 and gH2AX normalized densitometry. n = 3 independent experiments. (I) The Cilp2 mRNA levels were reduced in E13.5 ECs from Rnf20cKO-Tie2 mice. N =3Rnf20fl/flmice and 4 Rnf20cKO-Tie2 mice. (J) CILP2 secretion from the cultured ECs was measured by an ELISA kit. n = 4 independent experiments. (K) Western blot analysis showed that endothelial supernatant after Cilp2 knockdown affected NPC proliferation and differentiation. (L) Quantitative analyses of relative protein abundance in (K). Error bars represent means ± SEMs; 2-tailed unpaired t test; *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit Anti-RNF20 Proteintech Cat#21625-1-AP; RRID: AB_10734436 Rabbit Anti-CILP2 Proteintech Cat#11813-1-AP; RRID: AB_2877795 Rat Anti-BrdU Abcam Cat#ab6326; RRID: AB_305426 Rabbit Anti-gH2AX Cell Signaling Technology Cat# 9718S; RRID: AB_2118009 Rabbit Anti-PDGFRb Abcam Cat#ab32570; RRID: AB_777165 Rat Anti-CD31 Biolegend Cat#102406; RRID: AB_312901 Rabbit Anti-Collagen IV Abcam Cat#ab6586; RRID: AB_305584 Rabbit Anti-ZO-1 Invitrogen Cat#40–2200; RRID: AB_2533456 Mouse Anti-Claudin5 Invitrogen Cat#352500; RRID: AB_2533200 Mouse Anti-Nestin Millipore Cat#MAB353; RRID: AB_94911 Mouse Anti-Tuj1 Millipore Cat#MAB1637; RRID: AB_2210524 Rabbit Anti-PAX6 Abcam Cat#ab5790; RRID: AB_305110 Rabbit anti-SOX2 Cell Signaling Technologies Cat#3728; RRID: AB_2194037 Rabbit Anti-Ki67 Abcam Cat#ab15580; RRID: AB_443209 Rabbit Anti-pH3 Cell Signaling Technology Cat#3377S; RRID: AB_1549592 Rabbit Anti-TBR2 Abcam Cat#ab23345; RRID: AB_778267 Rat Anti-Ctip2 Abcam Cat#ab18465; RRID: AB_2064130 Mouse Anti-SATB2 Abcam Cat#ab51502; RRID: AB_882455 Rabbit Anti-TBR1 Abcam Cat#ab31940; RRID: AB_2200219 Rabbit Anti-H2Bub1 Cell Signaling Technology Cat# 5546T; RRID: AB_10693452 Mouse Anti-FLAG Sigma Cat# F1804; RRID: AB_262044 Rabbit Anti-HA Abmart Cat#M20003; RRID: AB_2864345 Rabbit Anti-b-Catenin Cell Signaling Technology Cat# 8480s; RRID: AB_11127855 Rabbit Anti-p-b-Catenin Cell Signaling Technology Cat#9561s; RRID: AB_331729 Rabbit Anti-p-GSK3b (ser9) Cell Signaling Technology Cat#5558; RRID: AB_10013750 Rabbit Anti-b-ACTIN Proteintech Cat# 20536-1-AP; RRID: AB_10700003 Chemicals, peptides, and recombinant proteins Papain Worthington Cat# 38P18865 Alexa Fluor 555 Invitrogen Cat# a30677 IB4 Vector Labs Cat# B-1205 TOPFlash Beyotime Cat# D2501 5-bromo-2-deoxyuridine (BrdU) Sigma-Aldrich Cat# B5002-5G CILP2 Recombinant Human Protein Zeye Cat# IC2132402 Recombinant Murine Wnt-3a PeproTech Cat# 315-2010 IWP-2 Selleck Cat# S7085 Critical commercial assays In Situ Cell Death Detection Kit Roche Cat#12156792910 SYBR Green PCR Kit TIANGEN Cat#FP205 CILP2 ELISA Kit Boshen Cat#BS-E8014H1 Deposited data Raw and analyzed data of bulk RNA-seq This paper GEO: GSE192997 Experimental models: Cell lines HEK293 Cell Line ATCC ATCC CRL-1573; RRID: CVCL_0045 (Continued on next page) e1 Cell Reports 40, 111350, September 13, 2022

Techniques: Expressing, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Knockdown, Quantitative Proteomics

(A) IFN-β luciferase reporter assay of HEK293T cells transiently transfected with HA-tagged RIG-I, MDA5, MAVS, STING, or TBK1 (100 ng) together with either RNF20-Flag expression plasmid (100 ng) or an empty control vector, followed by infection with VSV-GFP (MOI = 1.0) for 12h . (B, C) HEK293T cells were transiently co-transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), and HA-tagged RIG-I or MDA5, together with increasing doses of RNF20-Flag expression plasmid (0, 50, 100, 200, 300, or 400 ng). (D) HEK293T cells were transiently co-transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), poly(I:C), and Flag-RIG-I (100 ng), together with RNF20-interfering plasmid. (E, F) HEK293T cells were co-transfected with Flag-RNF20 and either Myc-tagged RIG-I or Myc-tagged MDA5, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Flag antibody and analyzed by immunoblotting with the indicated antibodies. (G, H) Confocal microscopy analysis of HeLa cells infected with NDV for 24 h, followed by immunostaining with anti-RNF20, anti-RIG-I, or anti-MDA5 primary antibodies, and Alexa Fluor 488-conjugated goat anti-mouse IgG (green) or Cy3-conjugated goat anti-rat IgG (red) secondary antibodies. (I, J) Schematic representation of RIG-I and MDA5 structures and their truncated mutants. (K) HEK293T cells were co-transfected with Myc-tagged MDA5 or its mutants together with Flag-RNF20, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Myc antibody and analyzed by immunoblotting with the indicated antibodies. (L, M) HEK293T cells were co-transfected with Myc-tagged MDA5 or RIG-I (wild-type or dCARD mutants) together with Flag-RNF20, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Myc antibody and analyzed by immunoblotting with the indicated antibodies. Data are presented as mean ± SD; *** p < 0.001; Scale bars = 10 µm. The results were repeated in three independent experiments with three replicas each. (Note: UI denotes uninfected virus; pcDNA3.1: empty vector control; shNC: non-targeting shRNA control).

Journal: PLOS Pathogens

Article Title: RNF20 dynamically regulates RIG-I and MDA5 transcription and degradation via nucleocytoplasmic translocation to balance antiviral signaling

doi: 10.1371/journal.ppat.1013890

Figure Lengend Snippet: (A) IFN-β luciferase reporter assay of HEK293T cells transiently transfected with HA-tagged RIG-I, MDA5, MAVS, STING, or TBK1 (100 ng) together with either RNF20-Flag expression plasmid (100 ng) or an empty control vector, followed by infection with VSV-GFP (MOI = 1.0) for 12h . (B, C) HEK293T cells were transiently co-transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), and HA-tagged RIG-I or MDA5, together with increasing doses of RNF20-Flag expression plasmid (0, 50, 100, 200, 300, or 400 ng). (D) HEK293T cells were transiently co-transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), poly(I:C), and Flag-RIG-I (100 ng), together with RNF20-interfering plasmid. (E, F) HEK293T cells were co-transfected with Flag-RNF20 and either Myc-tagged RIG-I or Myc-tagged MDA5, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Flag antibody and analyzed by immunoblotting with the indicated antibodies. (G, H) Confocal microscopy analysis of HeLa cells infected with NDV for 24 h, followed by immunostaining with anti-RNF20, anti-RIG-I, or anti-MDA5 primary antibodies, and Alexa Fluor 488-conjugated goat anti-mouse IgG (green) or Cy3-conjugated goat anti-rat IgG (red) secondary antibodies. (I, J) Schematic representation of RIG-I and MDA5 structures and their truncated mutants. (K) HEK293T cells were co-transfected with Myc-tagged MDA5 or its mutants together with Flag-RNF20, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Myc antibody and analyzed by immunoblotting with the indicated antibodies. (L, M) HEK293T cells were co-transfected with Myc-tagged MDA5 or RIG-I (wild-type or dCARD mutants) together with Flag-RNF20, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Myc antibody and analyzed by immunoblotting with the indicated antibodies. Data are presented as mean ± SD; *** p < 0.001; Scale bars = 10 µm. The results were repeated in three independent experiments with three replicas each. (Note: UI denotes uninfected virus; pcDNA3.1: empty vector control; shNC: non-targeting shRNA control).

Article Snippet: The antibodies IgG were purchased from the indicated manufacturer’s, including HRP-conjugated anti-mouse or rabbit IgG (Cell Signaling Technologies, 7076 and 7074), Alexa Fluor 488-conjugated Goat anti-rabbit IgG (Sangon Biotech, D110061), Cy3-conjugated Goat anti-mouse IgG (Sangon Biotech, D110088), Anti-Flag tag mouse monoclonal antibody (Sangon Biotech, D191041), Anti-HA tag mouse monoclonal antibody (Abmart, 26D11), Anti-Myc tag mouse monoclonal antibody (Abmart, 19C2), Anti-GFP tag mouse monoclonal antibody (ABclonal,AE012), anti-β-tubulin (Abmart, 2H4), anti-Lamin B1 (Beyotime, AF1408), anti-RNF20 (proteintech, 21625–1-AP), anti-RNF20 (Santa Cruz biotechnology, sc-517358), anti-RIG-I (Cell Signaling Technologies, 3743), anti-MDA5 (Cell Signaling Technologies, 5321), anti-MAVS (Cell Signaling Technologies, 3993), anti-TBK1 (Cell Signaling Technologies, 3504), anti- p -TBK1 (Cell Signaling Technologies, 5483), anti-IRF3 (Cell Signaling Technologies, 11904), anti- p -IRF3 (Cell Signaling Technologies, 4947), anti-STING (Cell Signaling Technologies, 13647S), anti-STAT1 (Sangon Biotech, D120084).

Techniques: Luciferase, Reporter Assay, Transfection, Expressing, Plasmid Preparation, Control, Infection, Immunoprecipitation, Western Blot, Confocal Microscopy, Immunostaining, Virus, shRNA

(A-C) Confocal microscopy of HeLa cells that were uninfected (UI) or infected with H1N1, NDV, or VSV-GFP for the indicated time points (0, 3, 6, 9, 12, and 24h ) . Cells were stained with an anti-RNF20 primary antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (green) or Cy3-conjugated goat anti-rat IgG (red). Scale bars = 10 μm. (D) Immunoblot analysis of RNF20 in cytoplasmic and nuclear fractions of A549 cells infected with VSV-GFP for the indicated time points. (E) Immunoblot analysis of RNF20 in cytoplasmic and nuclear fractions of A549 cells that were UI or infected with VSV-GFP for 24h and then treated with DMSO and KPT330. (F, G) qPCR analysis of RNF20 and IFN-β mRNA levels in A549 cells that were transiently transfected with RNF20 expression or control plasmid, infected with VSV-GFP for 24h and treated with DMSO or KPT330. (H) Co-immunoprecipitation analysis of CRM1-Flag and RNF20-HA in HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with the indicated antibodies. (I, J) Confocal microscopy of HeLa cells transfected for 24 h with RNF20-dNLS-EGFP or RNF20-dNES-EGFP expression plasmids, followed by infection with NDV for 24 h or left uninfected. Scale bars = 10 μm. (K, L) Luciferase reporter assay of HEK293T cells transiently transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), and RNF20 (wild-type or mutant) expression plasmids or control vector. (M, N ) qPCR analysis of IFN-β, OAS1, IL-1β, IL-6, and IL-8 mRNA levels in A549 cells that were transiently transfected with RNF20 (WT) or RNF20-dNLS-expressing plasmid or control plasmid and infected with VSV-GFP for 24h. n = 3; Data were represented by the mean ± SD with corresponding significance. * p < 0.05, ** p < 0.01, *** p < 0.001. (Note: UI denotes uninfected virus; pcDNA3.1: empty vector control; shNC: non-targeting shRNA control).

Journal: PLOS Pathogens

Article Title: RNF20 dynamically regulates RIG-I and MDA5 transcription and degradation via nucleocytoplasmic translocation to balance antiviral signaling

doi: 10.1371/journal.ppat.1013890

Figure Lengend Snippet: (A-C) Confocal microscopy of HeLa cells that were uninfected (UI) or infected with H1N1, NDV, or VSV-GFP for the indicated time points (0, 3, 6, 9, 12, and 24h ) . Cells were stained with an anti-RNF20 primary antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (green) or Cy3-conjugated goat anti-rat IgG (red). Scale bars = 10 μm. (D) Immunoblot analysis of RNF20 in cytoplasmic and nuclear fractions of A549 cells infected with VSV-GFP for the indicated time points. (E) Immunoblot analysis of RNF20 in cytoplasmic and nuclear fractions of A549 cells that were UI or infected with VSV-GFP for 24h and then treated with DMSO and KPT330. (F, G) qPCR analysis of RNF20 and IFN-β mRNA levels in A549 cells that were transiently transfected with RNF20 expression or control plasmid, infected with VSV-GFP for 24h and treated with DMSO or KPT330. (H) Co-immunoprecipitation analysis of CRM1-Flag and RNF20-HA in HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with the indicated antibodies. (I, J) Confocal microscopy of HeLa cells transfected for 24 h with RNF20-dNLS-EGFP or RNF20-dNES-EGFP expression plasmids, followed by infection with NDV for 24 h or left uninfected. Scale bars = 10 μm. (K, L) Luciferase reporter assay of HEK293T cells transiently transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), and RNF20 (wild-type or mutant) expression plasmids or control vector. (M, N ) qPCR analysis of IFN-β, OAS1, IL-1β, IL-6, and IL-8 mRNA levels in A549 cells that were transiently transfected with RNF20 (WT) or RNF20-dNLS-expressing plasmid or control plasmid and infected with VSV-GFP for 24h. n = 3; Data were represented by the mean ± SD with corresponding significance. * p < 0.05, ** p < 0.01, *** p < 0.001. (Note: UI denotes uninfected virus; pcDNA3.1: empty vector control; shNC: non-targeting shRNA control).

Article Snippet: The antibodies IgG were purchased from the indicated manufacturer’s, including HRP-conjugated anti-mouse or rabbit IgG (Cell Signaling Technologies, 7076 and 7074), Alexa Fluor 488-conjugated Goat anti-rabbit IgG (Sangon Biotech, D110061), Cy3-conjugated Goat anti-mouse IgG (Sangon Biotech, D110088), Anti-Flag tag mouse monoclonal antibody (Sangon Biotech, D191041), Anti-HA tag mouse monoclonal antibody (Abmart, 26D11), Anti-Myc tag mouse monoclonal antibody (Abmart, 19C2), Anti-GFP tag mouse monoclonal antibody (ABclonal,AE012), anti-β-tubulin (Abmart, 2H4), anti-Lamin B1 (Beyotime, AF1408), anti-RNF20 (proteintech, 21625–1-AP), anti-RNF20 (Santa Cruz biotechnology, sc-517358), anti-RIG-I (Cell Signaling Technologies, 3743), anti-MDA5 (Cell Signaling Technologies, 5321), anti-MAVS (Cell Signaling Technologies, 3993), anti-TBK1 (Cell Signaling Technologies, 3504), anti- p -TBK1 (Cell Signaling Technologies, 5483), anti-IRF3 (Cell Signaling Technologies, 11904), anti- p -IRF3 (Cell Signaling Technologies, 4947), anti-STING (Cell Signaling Technologies, 13647S), anti-STAT1 (Sangon Biotech, D120084).

Techniques: Confocal Microscopy, Infection, Staining, Western Blot, Transfection, Expressing, Control, Plasmid Preparation, Immunoprecipitation, Luciferase, Reporter Assay, Mutagenesis, Virus, shRNA