rabbit polyclonal anti sars cov 2 orf8 Search Results


sars-cov-2 orf8 antibody - azide and bsa free  (Bio-Techne corporation)
93
Bio-Techne corporation sars-cov-2 orf8 antibody - azide and bsa free
Sars Cov 2 Orf8 Antibody Azide And Bsa Free, supplied by Bio-Techne corporation, 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/rabbit+polyclonal+anti+sars+cov+2+orf8/bio-techne+corporation___nbp3-07972?v=Bio-Techne+corporation
Average 93 stars, based on 1 article reviews
sars-cov-2 orf8 antibody - azide and bsa free - by Bioz Stars, 2026-07
93/100 stars
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rat monoclonal anti sars cov 2 orf6  (Novus Biologicals)
94
Novus Biologicals rat monoclonal anti sars cov 2 orf6
a Schematic illustrating the SARS-COV-2 genomic landscape and the deletions/substitutions in ΔE G /ΔE G 68, main structural and accessory proteins indicated. Four overlapping fragments covering the whole SARS-CoV-2 genome were amplified by PCR (Fragments A-D, see also Supplementary Fig. ). b Complementation efficiency of Vero-E2T cells, analyzed by FFU (focus forming units) quantification after infection with ΔE G 3* (ΔE G with an additional stop codon in ORF3a) at different multiplicities of infection (MOI) or medium-only control (ctrl) three and six days post-infection ( n = 2 individual cultures), for corresponding genome copies, see Supplementary Fig. . c Passaging of 1:10 and 1:100 (after p2) dilutions of cell-free supernatant (Input = Passage 0) of wild-type SARS-CoV-2 (Muc-1, B.1), ΔE G 3* and ΔE G 68 on non-complementing Vero E6 cells (initial infection MOI = 1). Data from one representative experiment are shown; analysis was performed in duplicates. d Transmission electron microscopy analysis of recombinant wild-type SARS-CoV-2 (rCoV2) or vaccine candidates ΔE G and ΔE G 68 showing the presence of the characteristic spike protein (indicated with arrows). e Immunoblot analysis of viral protein production in Vero E6-TMPRSS2 cells infected for 24 h with rCoV2, E**fs, ΔE G 3*, ΔE G 68 or medium only (ctrl), probed with anti-NSP2, anti-N, anti-S, anti-ORF3a (full-length [fl] and truncated [tr] forms indicated with arrows), <t>anti-ORF6,</t> anti-ORF7a, anti-ORF8, and anti-beta-actin (β-ACT) antibodies. f Detection of N and S (magenta), F-actin (green), nuclei (blue) and ORF6 or ORF8 in Vero E6-TMPRSS2 cells infected with rCoV2, E**fs, ΔE G 3* or ΔE G 68. Scale bar is 100 nm in ( d ), 50 µm and 20 µm in ( f ) (overview and ROI images, respectively).
Rat Monoclonal Anti Sars Cov 2 Orf6, supplied by Novus Biologicals, 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/rabbit+polyclonal+anti+sars+cov+2+orf8/pmc11522273-411-53-67?v=Novus+Biologicals
Average 94 stars, based on 1 article reviews
rat monoclonal anti sars cov 2 orf6 - by Bioz Stars, 2026-07
94/100 stars
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rabbit polyclonal anti-sars-cov-2 orf8 antibody cat. no. gtx135591  (GeneTex)
90
GeneTex rabbit polyclonal anti-sars-cov-2 orf8 antibody cat. no. gtx135591
( A ) Overview of the COVID-19 pregnancy cohort (total n = 29). Pregnant women included in the study had negative (Control, n = 6), or positive SARS-CoV-2 diagnoses (COVID-19 n = 23) during the first ( n = 2), second ( n = 5), or third ( n = 16) trimesters of pregnancy. Participants had biospecimens collected at the time of the delivery. Biospecimens collected included (i) biofluid specimens: maternal plasma, newborn blood, cord plasma, and amniotic fluid; and (ii) placental tissues: umbilical cord, chorion, and amnion. The biospecimens underwent ultrasensitive SARS-CoV-2 detection using digital droplet PCR (ddPCR), SARS-CoV-2 <t>ORF8</t> ELISA, proteomics profile for 97 inflammatory biomarkers, and global transcriptomics profile (~22,000 host genes) by RNAseq. ( B ) Box and whiskers plot representing average and minimum and maximum values of copies/mL of SARS-CoV-2 N1 and N2 proteins detected maternal plasma (controls, n = 5 for N1; n = 6 for N2; COVID-19, n = 23 for N1/N2), chorion (controls, n = 5 for N1, n = 6 for N2; COVID-19, n = 21), amnion (controls, n = 2 for N1; n = 4 for N2; COVID-19, n = 21), amniotic fluid (controls, n = 3; COVID-19, n = 9), cord plasma (controls, n = 5 for N1, n = 3 for N2; COVID-19, n = 20), newborn plasma (controls, n = 0; COVID-19, n = 12) from controls and COVID-19-affected pregnancies. ( C ) Bar charts representing the percentage of samples positive (purple bar) and negative (blue bar) from SARS-CoV-2 in the biospecimens analyzed. Pie chart representing the total SARS-CoV-2 positivity considering at least one fetal-skewed specimen. Cut-off value defined as counts/mL 3 ≥ 2. ( D ) Stacked bar charts (mean with SD) representing the levels of viral RNA of samples positive for SARS-CoV-2 in the fetal compartment, according to the trimester of pregnancy that maternal SARS-CoV-2 occurred (First trimester, n = 2; Second trimester, n = 5 and Third trimester, n = 16). .
Rabbit Polyclonal Anti Sars Cov 2 Orf8 Antibody Cat. No. Gtx135591, supplied by GeneTex, 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/rabbit+polyclonal+anti+sars+cov+2+orf8/pmc11574245-439-17-25?v=GeneTex
Average 90 stars, based on 1 article reviews
rabbit polyclonal anti-sars-cov-2 orf8 antibody cat. no. gtx135591 - by Bioz Stars, 2026-07
90/100 stars
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rabbit anti-sars-cov-2 orf8 a20235  (ABclonal Biotechnology)
90
ABclonal Biotechnology rabbit anti-sars-cov-2 orf8 a20235
SARS-CoV-2 <t>ORF8</t> interacts p62 to form condensates (A) Co-precipitation analysis of endogenous p62 with ORF8-Strep in HEK293T. (B) Purified MBP-ORF8 was incubated with purified GST or GST-p62, and analysis of the interaction between ORF8 and p62 by in vitro GST pull-down was performed. (C and D) Cells were co-transfected with ORF8-Strep and mCherry-p62 for 24 h, then stained with anti-Strep antibody and imaged using confocal microscopy. Scale bar, 10 μm. The number of ORF8 puncta in each cell was counted from 50 cells of three independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (E) p62 WT and KO Vero-E6 cells were transfected with ORF8-Strep for 24 h, then cells were stained with anti-Strep and anti-p62 antibodies, and imaged using confocal microscopy. Scale bar, 10 μm. The number of ORF8 puncta in each cell was counted from 50 cells of three independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (F) HeLa cells were co-transfected with GFP-ORF8 and mCherry-p62 for 24 h, then cells were mock treated or treated with 1,6-hexanediol for 1 min and imaged using confocal microscopy. Scale bar, 10 μm. The number of ORF8/p62 puncta in each cell was counted from 50 cells of three independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (G) HeLa cells with exogenous expression of GFP-ORF8 and mCherry-p62 undergo liquid-like behavior; fluorescence recovery after photobleaching (FRAP) images are shown before and at indicated time points after bleaching. Time 0 indicates the time of photobleaching. Scale bar, 10 μm. Quantification of fluorescence intensity recovery of GFP-ORF8 and mCherry-p62 in the bleached droplet. (H) HeLa cells with exogenous expression of GFP-ORF8 and mCherry-p62 undergo fusion, images are shown at indicated time points after imaging. (I) mCherry-p62 were purified from Escherichia coli BL21 and analyzed via Coomassie blue. (J) In vitro phase separation assay of GFP and mCherry-p62/His6-UBx8 or GFP-ORF8 and mCherry-p62/His6-UBx8. Fluorescence images of 10 μM each protein in phase separation assay buffer without PEG8000. Representative fluorescence images of three independent experiments are shown. Scale bar, 10 μm. (K) GFP-ORF8 truncations were expressed as indicated with p62-FLAG. Cell lysates were subjected to immunoprecipitation with GFP antibody and analyzed using western blotting (WB). (L) Cells were transfected with GFP-ORF8 or mutant with mCherry-p62 for 24 h, and imaged using confocal microscopy. Scale bar, 10 μm. (M) p62-FLAG mutations were expressed as indicated with ORF8-Strep. Cell lysates were subjected to immunoprecipitation with FLAG antibody and analyzed using WB. (N) p62 KO Vero-E6 cells were transfected with p62-FLAG or mutant with ORF8-Strep for 24 h, then stained with anti-FLAG and anti-Strep antibodies and imaged using confocal microscopy. Scale bar, 10 μm. (O) SARS-CoV-2 infected Vero-E6 cells were stained with anti-ORF8 and anti-p62 antibodies and imaged using confocal microscopy. Scale bar, 10 μm. (P) Co-precipitation analysis of p62-HA with SARS-CoV-2 ORF8-FLAG or SARS-CoV ORF8-FLAG or MERS-CoV ORF8-FLAG in HEK293T. (Q) Cells were co-transfected with mCherry-p62 with SARS-CoV ORF8-FLAG or MERS-CoV ORF8-FLAG for 24 h, then stained with anti-FLAG antibody and imaged using confocal microscopy. Scale bar, 10 μm. See also <xref ref-type=Figures S1 and . " width="250" height="auto" />
Rabbit Anti Sars Cov 2 Orf8 A20235, supplied by ABclonal 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/rabbit+polyclonal+anti+sars+cov+2+orf8/pmc09998290-439-18-25?v=ABclonal+Biotechnology
Average 90 stars, based on 1 article reviews
rabbit anti-sars-cov-2 orf8 a20235 - by Bioz Stars, 2026-07
90/100 stars
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orf8  (ProSci Incorporated)
91
ProSci Incorporated orf8
SARS-CoV-2 <t>ORF8</t> interacts p62 to form condensates (A) Co-precipitation analysis of endogenous p62 with ORF8-Strep in HEK293T. (B) Purified MBP-ORF8 was incubated with purified GST or GST-p62, and analysis of the interaction between ORF8 and p62 by in vitro GST pull-down was performed. (C and D) Cells were co-transfected with ORF8-Strep and mCherry-p62 for 24 h, then stained with anti-Strep antibody and imaged using confocal microscopy. Scale bar, 10 μm. The number of ORF8 puncta in each cell was counted from 50 cells of three independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (E) p62 WT and KO Vero-E6 cells were transfected with ORF8-Strep for 24 h, then cells were stained with anti-Strep and anti-p62 antibodies, and imaged using confocal microscopy. Scale bar, 10 μm. The number of ORF8 puncta in each cell was counted from 50 cells of three independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (F) HeLa cells were co-transfected with GFP-ORF8 and mCherry-p62 for 24 h, then cells were mock treated or treated with 1,6-hexanediol for 1 min and imaged using confocal microscopy. Scale bar, 10 μm. The number of ORF8/p62 puncta in each cell was counted from 50 cells of three independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (G) HeLa cells with exogenous expression of GFP-ORF8 and mCherry-p62 undergo liquid-like behavior; fluorescence recovery after photobleaching (FRAP) images are shown before and at indicated time points after bleaching. Time 0 indicates the time of photobleaching. Scale bar, 10 μm. Quantification of fluorescence intensity recovery of GFP-ORF8 and mCherry-p62 in the bleached droplet. (H) HeLa cells with exogenous expression of GFP-ORF8 and mCherry-p62 undergo fusion, images are shown at indicated time points after imaging. (I) mCherry-p62 were purified from Escherichia coli BL21 and analyzed via Coomassie blue. (J) In vitro phase separation assay of GFP and mCherry-p62/His6-UBx8 or GFP-ORF8 and mCherry-p62/His6-UBx8. Fluorescence images of 10 μM each protein in phase separation assay buffer without PEG8000. Representative fluorescence images of three independent experiments are shown. Scale bar, 10 μm. (K) GFP-ORF8 truncations were expressed as indicated with p62-FLAG. Cell lysates were subjected to immunoprecipitation with GFP antibody and analyzed using western blotting (WB). (L) Cells were transfected with GFP-ORF8 or mutant with mCherry-p62 for 24 h, and imaged using confocal microscopy. Scale bar, 10 μm. (M) p62-FLAG mutations were expressed as indicated with ORF8-Strep. Cell lysates were subjected to immunoprecipitation with FLAG antibody and analyzed using WB. (N) p62 KO Vero-E6 cells were transfected with p62-FLAG or mutant with ORF8-Strep for 24 h, then stained with anti-FLAG and anti-Strep antibodies and imaged using confocal microscopy. Scale bar, 10 μm. (O) SARS-CoV-2 infected Vero-E6 cells were stained with anti-ORF8 and anti-p62 antibodies and imaged using confocal microscopy. Scale bar, 10 μm. (P) Co-precipitation analysis of p62-HA with SARS-CoV-2 ORF8-FLAG or SARS-CoV ORF8-FLAG or MERS-CoV ORF8-FLAG in HEK293T. (Q) Cells were co-transfected with mCherry-p62 with SARS-CoV ORF8-FLAG or MERS-CoV ORF8-FLAG for 24 h, then stained with anti-FLAG antibody and imaged using confocal microscopy. Scale bar, 10 μm. See also <xref ref-type=Figures S1 and . " width="250" height="auto" />
Orf8, supplied by ProSci Incorporated, 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/rabbit+polyclonal+anti+sars+cov+2+orf8/bio_rxiv__2021__01__27__428534-127-43-44?v=ProSci+Incorporated
Average 91 stars, based on 1 article reviews
orf8 - by Bioz Stars, 2026-07
91/100 stars
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anti-dykddddk (flag)-tagged mouse monoclonal antibody (mab)  (FUJIFILM)
90
FUJIFILM anti-dykddddk (flag)-tagged mouse monoclonal antibody (mab)
SARS-CoV-2 <t>ORF8</t> interacts p62 to form condensates (A) Co-precipitation analysis of endogenous p62 with ORF8-Strep in HEK293T. (B) Purified MBP-ORF8 was incubated with purified GST or GST-p62, and analysis of the interaction between ORF8 and p62 by in vitro GST pull-down was performed. (C and D) Cells were co-transfected with ORF8-Strep and mCherry-p62 for 24 h, then stained with anti-Strep antibody and imaged using confocal microscopy. Scale bar, 10 μm. The number of ORF8 puncta in each cell was counted from 50 cells of three independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (E) p62 WT and KO Vero-E6 cells were transfected with ORF8-Strep for 24 h, then cells were stained with anti-Strep and anti-p62 antibodies, and imaged using confocal microscopy. Scale bar, 10 μm. The number of ORF8 puncta in each cell was counted from 50 cells of three independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (F) HeLa cells were co-transfected with GFP-ORF8 and mCherry-p62 for 24 h, then cells were mock treated or treated with 1,6-hexanediol for 1 min and imaged using confocal microscopy. Scale bar, 10 μm. The number of ORF8/p62 puncta in each cell was counted from 50 cells of three independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (G) HeLa cells with exogenous expression of GFP-ORF8 and mCherry-p62 undergo liquid-like behavior; fluorescence recovery after photobleaching (FRAP) images are shown before and at indicated time points after bleaching. Time 0 indicates the time of photobleaching. Scale bar, 10 μm. Quantification of fluorescence intensity recovery of GFP-ORF8 and mCherry-p62 in the bleached droplet. (H) HeLa cells with exogenous expression of GFP-ORF8 and mCherry-p62 undergo fusion, images are shown at indicated time points after imaging. (I) mCherry-p62 were purified from Escherichia coli BL21 and analyzed via Coomassie blue. (J) In vitro phase separation assay of GFP and mCherry-p62/His6-UBx8 or GFP-ORF8 and mCherry-p62/His6-UBx8. Fluorescence images of 10 μM each protein in phase separation assay buffer without PEG8000. Representative fluorescence images of three independent experiments are shown. Scale bar, 10 μm. (K) GFP-ORF8 truncations were expressed as indicated with p62-FLAG. Cell lysates were subjected to immunoprecipitation with GFP antibody and analyzed using western blotting (WB). (L) Cells were transfected with GFP-ORF8 or mutant with mCherry-p62 for 24 h, and imaged using confocal microscopy. Scale bar, 10 μm. (M) p62-FLAG mutations were expressed as indicated with ORF8-Strep. Cell lysates were subjected to immunoprecipitation with FLAG antibody and analyzed using WB. (N) p62 KO Vero-E6 cells were transfected with p62-FLAG or mutant with ORF8-Strep for 24 h, then stained with anti-FLAG and anti-Strep antibodies and imaged using confocal microscopy. Scale bar, 10 μm. (O) SARS-CoV-2 infected Vero-E6 cells were stained with anti-ORF8 and anti-p62 antibodies and imaged using confocal microscopy. Scale bar, 10 μm. (P) Co-precipitation analysis of p62-HA with SARS-CoV-2 ORF8-FLAG or SARS-CoV ORF8-FLAG or MERS-CoV ORF8-FLAG in HEK293T. (Q) Cells were co-transfected with mCherry-p62 with SARS-CoV ORF8-FLAG or MERS-CoV ORF8-FLAG for 24 h, then stained with anti-FLAG antibody and imaged using confocal microscopy. Scale bar, 10 μm. See also <xref ref-type=Figures S1 and . " width="250" height="auto" />
Anti Dykddddk (Flag) Tagged Mouse Monoclonal Antibody (Mab), supplied by FUJIFILM, 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/rabbit+polyclonal+anti+sars+cov+2+orf8/pmc08832879-251-15-20?v=FUJIFILM
Average 90 stars, based on 1 article reviews
anti-dykddddk (flag)-tagged mouse monoclonal antibody (mab) - by Bioz Stars, 2026-07
90/100 stars
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anti-his ae003  (ABclonal Biotechnology)
90
ABclonal Biotechnology anti-his ae003

Anti His Ae003, supplied by ABclonal Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 1 article reviews
anti-his ae003 - by Bioz Stars, 2026-07
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recombinant sars cov 2 nsp10 protein  (ProSci Incorporated)
94
ProSci Incorporated recombinant sars cov 2 nsp10 protein
Determining the stability of SARS-CoV-2 proteins. Human lung epithelial BEAS-2B cells were exposed to cycloheximide (CHX) (100 μg/mL) at four time points (2, 4, 6, and 8 h) prior to immunoblotting. The bands were quantitated and normalized to β-actin, and the half-lives of the indicated proteins are shown graphically. (A) NSP1, E, and ORF8. (B) NSP3d, NSP4, NSP6, NSP7, NSP8, NSP9, NSP12, NSP13, NSP14, NSP16, ORF3a, ORF3b, ORF6, ORF7b, and ORF9b. (C) NSP2, NSP5, <t>NSP10,</t> NSP15, spike, M, and N. Data are from three independent biological replicates.
Recombinant Sars Cov 2 Nsp10 Protein, supplied by ProSci Incorporated, 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/rabbit+polyclonal+anti+sars+cov+2+orf8/pmc09238396-129-0-18?v=ProSci+Incorporated
Average 94 stars, based on 1 article reviews
recombinant sars cov 2 nsp10 protein - by Bioz Stars, 2026-07
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rabbit anti-flag ae092  (ABclonal Biotechnology)
90
ABclonal Biotechnology rabbit anti-flag ae092

Rabbit Anti Flag Ae092, supplied by ABclonal Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit anti-flag ae092 - by Bioz Stars, 2026-07
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anti-mcherry ae002  (ABclonal Biotechnology)
90
ABclonal Biotechnology anti-mcherry ae002

Anti Mcherry Ae002, supplied by ABclonal Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti-gst ae001  (ABclonal Biotechnology)
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ABclonal Biotechnology anti-gst ae001

Anti Gst Ae001, supplied by ABclonal Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti-mbp ae016  (ABclonal Biotechnology)
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ABclonal Biotechnology anti-mbp ae016

Anti Mbp Ae016, supplied by ABclonal Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


a Schematic illustrating the SARS-COV-2 genomic landscape and the deletions/substitutions in ΔE G /ΔE G 68, main structural and accessory proteins indicated. Four overlapping fragments covering the whole SARS-CoV-2 genome were amplified by PCR (Fragments A-D, see also Supplementary Fig. ). b Complementation efficiency of Vero-E2T cells, analyzed by FFU (focus forming units) quantification after infection with ΔE G 3* (ΔE G with an additional stop codon in ORF3a) at different multiplicities of infection (MOI) or medium-only control (ctrl) three and six days post-infection ( n = 2 individual cultures), for corresponding genome copies, see Supplementary Fig. . c Passaging of 1:10 and 1:100 (after p2) dilutions of cell-free supernatant (Input = Passage 0) of wild-type SARS-CoV-2 (Muc-1, B.1), ΔE G 3* and ΔE G 68 on non-complementing Vero E6 cells (initial infection MOI = 1). Data from one representative experiment are shown; analysis was performed in duplicates. d Transmission electron microscopy analysis of recombinant wild-type SARS-CoV-2 (rCoV2) or vaccine candidates ΔE G and ΔE G 68 showing the presence of the characteristic spike protein (indicated with arrows). e Immunoblot analysis of viral protein production in Vero E6-TMPRSS2 cells infected for 24 h with rCoV2, E**fs, ΔE G 3*, ΔE G 68 or medium only (ctrl), probed with anti-NSP2, anti-N, anti-S, anti-ORF3a (full-length [fl] and truncated [tr] forms indicated with arrows), anti-ORF6, anti-ORF7a, anti-ORF8, and anti-beta-actin (β-ACT) antibodies. f Detection of N and S (magenta), F-actin (green), nuclei (blue) and ORF6 or ORF8 in Vero E6-TMPRSS2 cells infected with rCoV2, E**fs, ΔE G 3* or ΔE G 68. Scale bar is 100 nm in ( d ), 50 µm and 20 µm in ( f ) (overview and ROI images, respectively).

Journal: NPJ Vaccines

Article Title: High protection and transmission-blocking immunity elicited by single-cycle SARS-CoV-2 vaccine in hamsters

doi: 10.1038/s41541-024-00992-z

Figure Lengend Snippet: a Schematic illustrating the SARS-COV-2 genomic landscape and the deletions/substitutions in ΔE G /ΔE G 68, main structural and accessory proteins indicated. Four overlapping fragments covering the whole SARS-CoV-2 genome were amplified by PCR (Fragments A-D, see also Supplementary Fig. ). b Complementation efficiency of Vero-E2T cells, analyzed by FFU (focus forming units) quantification after infection with ΔE G 3* (ΔE G with an additional stop codon in ORF3a) at different multiplicities of infection (MOI) or medium-only control (ctrl) three and six days post-infection ( n = 2 individual cultures), for corresponding genome copies, see Supplementary Fig. . c Passaging of 1:10 and 1:100 (after p2) dilutions of cell-free supernatant (Input = Passage 0) of wild-type SARS-CoV-2 (Muc-1, B.1), ΔE G 3* and ΔE G 68 on non-complementing Vero E6 cells (initial infection MOI = 1). Data from one representative experiment are shown; analysis was performed in duplicates. d Transmission electron microscopy analysis of recombinant wild-type SARS-CoV-2 (rCoV2) or vaccine candidates ΔE G and ΔE G 68 showing the presence of the characteristic spike protein (indicated with arrows). e Immunoblot analysis of viral protein production in Vero E6-TMPRSS2 cells infected for 24 h with rCoV2, E**fs, ΔE G 3*, ΔE G 68 or medium only (ctrl), probed with anti-NSP2, anti-N, anti-S, anti-ORF3a (full-length [fl] and truncated [tr] forms indicated with arrows), anti-ORF6, anti-ORF7a, anti-ORF8, and anti-beta-actin (β-ACT) antibodies. f Detection of N and S (magenta), F-actin (green), nuclei (blue) and ORF6 or ORF8 in Vero E6-TMPRSS2 cells infected with rCoV2, E**fs, ΔE G 3* or ΔE G 68. Scale bar is 100 nm in ( d ), 50 µm and 20 µm in ( f ) (overview and ROI images, respectively).

Article Snippet: The following antibodies were used in this study: mouse monoclonal anti-β-actin (Cell Signaling Technology; 3700; RRID: AB_2242334; LOT# 20), rabbit polyclonal anti-SARS-CoV-2 nsp2 (GeneTex; GTX135717; RRID: AB_2909866; LOT# B318853), rabbit polyclonal anti-SARS-CoV Nucleocapsid protein (Rockland; 200-401-A50; RRID:AB_828403), mouse monoclonal anti-SARS-CoV-2 Nucleocapsid protein (4F3C4, gift from S. Reiche ), sheep polyclonal anti-SARS-CoV-2 ORF3a , rat monoclonal anti-SARS-CoV-2 ORF6 (8B10, gift from Y. Miyamoto ), rabbit polyclonal anti-SARS-CoV-2 ORF8 (Novus Biologicals; NBP3-07972; LOT# 25966-2102), mouse monoclonal anti-SARS-CoV-2 Spike protein (4B5C1, gift from S. Reiche).

Techniques: Amplification, Infection, Control, Passaging, Transmission Assay, Electron Microscopy, Recombinant, Western Blot

a – c Modulation after transfection: Flow cytometry staining of THP-1 cells for HLA-A/B/C, CD80, CD275, and HLA-DR surface expression 48 h after transfection with expression plasmids for ORF6 ( a ), ORF8 ( b ), or Envelope ( c ) proteins, compared with control transfection. d – i Modulation after infection: d A549-ACE2-TMPRSS2 cells were infected with recombinant wild-type (rCoV2), E**fs, ΔE G 68, or XBB.1.5 SARS-CoV-2 virus (MOI = 0.1) for 24 h and stained for HLA-A/B/C, CD44 and CD275. e Median fluorescence intensity (MFI) of HLA-A/B/C and CD275. The same infection was conducted on HEK293T-ACE2 and their respective supernatant was then applied on THP-1 for 48 h before surface staining and analysis. f Histogram showing the expression of CD44, HLA-A/B/C, CD80, CD275, and HLA-DR on THP-1 after 48 h. g Median fluorescence intensity of CD44, HLA-A/B/C, CD80, and CD275 markers on THP-1 after 48 h incubation. h Comparison of wild-type or ΔE G 68 conditions for their expression of CD80 and HLA-A/B/C. The frequency of cells inside the gate in ( h ) is shown in ( i ). Median is shown for ( e ) and ( g ), mean and S.D. for ( i ). The gating strategy is shown in Supplementary Fig. .

Journal: NPJ Vaccines

Article Title: High protection and transmission-blocking immunity elicited by single-cycle SARS-CoV-2 vaccine in hamsters

doi: 10.1038/s41541-024-00992-z

Figure Lengend Snippet: a – c Modulation after transfection: Flow cytometry staining of THP-1 cells for HLA-A/B/C, CD80, CD275, and HLA-DR surface expression 48 h after transfection with expression plasmids for ORF6 ( a ), ORF8 ( b ), or Envelope ( c ) proteins, compared with control transfection. d – i Modulation after infection: d A549-ACE2-TMPRSS2 cells were infected with recombinant wild-type (rCoV2), E**fs, ΔE G 68, or XBB.1.5 SARS-CoV-2 virus (MOI = 0.1) for 24 h and stained for HLA-A/B/C, CD44 and CD275. e Median fluorescence intensity (MFI) of HLA-A/B/C and CD275. The same infection was conducted on HEK293T-ACE2 and their respective supernatant was then applied on THP-1 for 48 h before surface staining and analysis. f Histogram showing the expression of CD44, HLA-A/B/C, CD80, CD275, and HLA-DR on THP-1 after 48 h. g Median fluorescence intensity of CD44, HLA-A/B/C, CD80, and CD275 markers on THP-1 after 48 h incubation. h Comparison of wild-type or ΔE G 68 conditions for their expression of CD80 and HLA-A/B/C. The frequency of cells inside the gate in ( h ) is shown in ( i ). Median is shown for ( e ) and ( g ), mean and S.D. for ( i ). The gating strategy is shown in Supplementary Fig. .

Article Snippet: The following antibodies were used in this study: mouse monoclonal anti-β-actin (Cell Signaling Technology; 3700; RRID: AB_2242334; LOT# 20), rabbit polyclonal anti-SARS-CoV-2 nsp2 (GeneTex; GTX135717; RRID: AB_2909866; LOT# B318853), rabbit polyclonal anti-SARS-CoV Nucleocapsid protein (Rockland; 200-401-A50; RRID:AB_828403), mouse monoclonal anti-SARS-CoV-2 Nucleocapsid protein (4F3C4, gift from S. Reiche ), sheep polyclonal anti-SARS-CoV-2 ORF3a , rat monoclonal anti-SARS-CoV-2 ORF6 (8B10, gift from Y. Miyamoto ), rabbit polyclonal anti-SARS-CoV-2 ORF8 (Novus Biologicals; NBP3-07972; LOT# 25966-2102), mouse monoclonal anti-SARS-CoV-2 Spike protein (4B5C1, gift from S. Reiche).

Techniques: Transfection, Flow Cytometry, Staining, Expressing, Control, Infection, Recombinant, Virus, Fluorescence, Incubation, Comparison

( A ) Overview of the COVID-19 pregnancy cohort (total n = 29). Pregnant women included in the study had negative (Control, n = 6), or positive SARS-CoV-2 diagnoses (COVID-19 n = 23) during the first ( n = 2), second ( n = 5), or third ( n = 16) trimesters of pregnancy. Participants had biospecimens collected at the time of the delivery. Biospecimens collected included (i) biofluid specimens: maternal plasma, newborn blood, cord plasma, and amniotic fluid; and (ii) placental tissues: umbilical cord, chorion, and amnion. The biospecimens underwent ultrasensitive SARS-CoV-2 detection using digital droplet PCR (ddPCR), SARS-CoV-2 ORF8 ELISA, proteomics profile for 97 inflammatory biomarkers, and global transcriptomics profile (~22,000 host genes) by RNAseq. ( B ) Box and whiskers plot representing average and minimum and maximum values of copies/mL of SARS-CoV-2 N1 and N2 proteins detected maternal plasma (controls, n = 5 for N1; n = 6 for N2; COVID-19, n = 23 for N1/N2), chorion (controls, n = 5 for N1, n = 6 for N2; COVID-19, n = 21), amnion (controls, n = 2 for N1; n = 4 for N2; COVID-19, n = 21), amniotic fluid (controls, n = 3; COVID-19, n = 9), cord plasma (controls, n = 5 for N1, n = 3 for N2; COVID-19, n = 20), newborn plasma (controls, n = 0; COVID-19, n = 12) from controls and COVID-19-affected pregnancies. ( C ) Bar charts representing the percentage of samples positive (purple bar) and negative (blue bar) from SARS-CoV-2 in the biospecimens analyzed. Pie chart representing the total SARS-CoV-2 positivity considering at least one fetal-skewed specimen. Cut-off value defined as counts/mL 3 ≥ 2. ( D ) Stacked bar charts (mean with SD) representing the levels of viral RNA of samples positive for SARS-CoV-2 in the fetal compartment, according to the trimester of pregnancy that maternal SARS-CoV-2 occurred (First trimester, n = 2; Second trimester, n = 5 and Third trimester, n = 16). .

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Overview of the COVID-19 pregnancy cohort (total n = 29). Pregnant women included in the study had negative (Control, n = 6), or positive SARS-CoV-2 diagnoses (COVID-19 n = 23) during the first ( n = 2), second ( n = 5), or third ( n = 16) trimesters of pregnancy. Participants had biospecimens collected at the time of the delivery. Biospecimens collected included (i) biofluid specimens: maternal plasma, newborn blood, cord plasma, and amniotic fluid; and (ii) placental tissues: umbilical cord, chorion, and amnion. The biospecimens underwent ultrasensitive SARS-CoV-2 detection using digital droplet PCR (ddPCR), SARS-CoV-2 ORF8 ELISA, proteomics profile for 97 inflammatory biomarkers, and global transcriptomics profile (~22,000 host genes) by RNAseq. ( B ) Box and whiskers plot representing average and minimum and maximum values of copies/mL of SARS-CoV-2 N1 and N2 proteins detected maternal plasma (controls, n = 5 for N1; n = 6 for N2; COVID-19, n = 23 for N1/N2), chorion (controls, n = 5 for N1, n = 6 for N2; COVID-19, n = 21), amnion (controls, n = 2 for N1; n = 4 for N2; COVID-19, n = 21), amniotic fluid (controls, n = 3; COVID-19, n = 9), cord plasma (controls, n = 5 for N1, n = 3 for N2; COVID-19, n = 20), newborn plasma (controls, n = 0; COVID-19, n = 12) from controls and COVID-19-affected pregnancies. ( C ) Bar charts representing the percentage of samples positive (purple bar) and negative (blue bar) from SARS-CoV-2 in the biospecimens analyzed. Pie chart representing the total SARS-CoV-2 positivity considering at least one fetal-skewed specimen. Cut-off value defined as counts/mL 3 ≥ 2. ( D ) Stacked bar charts (mean with SD) representing the levels of viral RNA of samples positive for SARS-CoV-2 in the fetal compartment, according to the trimester of pregnancy that maternal SARS-CoV-2 occurred (First trimester, n = 2; Second trimester, n = 5 and Third trimester, n = 16). .

Article Snippet: Ten-micron sections were dewaxed, rehydrated and stained with hematoxylin and eosin (H&E) or via immunohistochemical staining with rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody (Cat. No. GTX135591, Genetex) and recombinant anti-C3 antibody (clone: EPR19394; Abcam) and counterstained with hematoxylin.

Techniques: Negative Control, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

( A ) Graphical representation of samples analyzed for ORF8 quantitative and qualitative ELISA: maternal plasma (Control, n = 4; COVID-19, n = 23), umbilical cord plasma (Control, n = 4; COVID-19, n = 20), and amniotic fluid (Control, n = 3; COVID-19, n = 8). ORF8 levels in samples from control and COVID-19-affected pregnancies. Data are presented in violin plots showing the median (middle line) and describe numerical data distributions using density curves. Values are represented in ng/mL with cut off 1.7 ng/mL in maternal plasma, cut off of 13.4 ng/mL in umbilical cord plasma and cut off of 36.1 ng/mL in amniotic fluid. ( B ) Pie charts illustrating qualitative analysis of ORF8 ELISA. ( C ) Stacked bars representing the circulating ORF8 according to trimester of SARS-CoV-2 infection (First trimester, n = 2; Second trimester, n = 4; Third trimester, n = 10). ( D ) Boxplots representing levels of anti-Spike S1 IgM with cut off of 2720 ng/mL and anti-Nucleocapsid IgG with cut off of 6420 ng/mL in maternal plasma ( n = 22) and newborn cord plasma ( n = 15) from COVID-19-affected pregnancies. Data are presented in boxplot, middle line represents means, the bound of box represent interquartile range, and whiskers represent maximum and minimum values. ( E ) Comparative analysis of DEGs (−2 < FC > 2, p < 0.05) in COVID-19 amnion ORF8 negatives and positives. ( F ) Bar plot representing gene ontology of upregulated biological pathways in COVID-19 amnion ORF8 positive group. ( G , H ) Violin plots representing the fold change values for individual genes related with ( G ) complement (ORF8 (−), n = 5–6; ORF8 (+), n = 9–10) and ( H ) inflammation in amnion (ORF8 (−), n = 5–7; ORF8 (+), n = 8–11) transcriptomics. ( I ) Comparative analysis of differentially expressed proteins (DEPs) ( p < 0.05) in COVID-19 amniotic fluid ORF8 negatives ( n = 4) and positives ( n = 8). Bubble plot of DEPs upregulated exclusively in COVID-19 amniotic fluid ORF8 positive group. ( J ) Comparative analysis of DEGs in COVID-19 umbilical cord ORF8 negatives and positives. ( K ) Bar plot of gene ontology biological pathways associated with DEGs upregulated exclusively in COVID-19 umbilical cord ORF8 positive. ( L ) Violin plots representing the fold change values for individual genes related to complement in umbilical cord transcriptomics. ( M ) Amnion serial sections were analyzed by immunohistochemistry for detection of SARS-CoV-2 ORF8 and C3b. From left to right, tissues stained with (i) Hematoxylin and Eosin (H&E) (hematoxylin—purple, eosin—pink); (ii) Hematoxylin (purple) and the secondary antibody anti-rabbit (Motulsky and Brown, ); (iii) Hematoxylin (purple) and anti-ORF8 produced (Motulsky and Brown, ); and (iv) Hematoxylin (purple) and anti-C3b (Motulsky and Brown, ). Images were taken at 4X magnification. Representative images from three control and three COVID-19 amnions. Fold change calculated by individual expression divided by average expression of control. Violin plots data are presented as means ± SEMs pg/mL, using Mann–Whitney U test ( p < 0.05). Comparative analysis relative to controls. .

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Graphical representation of samples analyzed for ORF8 quantitative and qualitative ELISA: maternal plasma (Control, n = 4; COVID-19, n = 23), umbilical cord plasma (Control, n = 4; COVID-19, n = 20), and amniotic fluid (Control, n = 3; COVID-19, n = 8). ORF8 levels in samples from control and COVID-19-affected pregnancies. Data are presented in violin plots showing the median (middle line) and describe numerical data distributions using density curves. Values are represented in ng/mL with cut off 1.7 ng/mL in maternal plasma, cut off of 13.4 ng/mL in umbilical cord plasma and cut off of 36.1 ng/mL in amniotic fluid. ( B ) Pie charts illustrating qualitative analysis of ORF8 ELISA. ( C ) Stacked bars representing the circulating ORF8 according to trimester of SARS-CoV-2 infection (First trimester, n = 2; Second trimester, n = 4; Third trimester, n = 10). ( D ) Boxplots representing levels of anti-Spike S1 IgM with cut off of 2720 ng/mL and anti-Nucleocapsid IgG with cut off of 6420 ng/mL in maternal plasma ( n = 22) and newborn cord plasma ( n = 15) from COVID-19-affected pregnancies. Data are presented in boxplot, middle line represents means, the bound of box represent interquartile range, and whiskers represent maximum and minimum values. ( E ) Comparative analysis of DEGs (−2 < FC > 2, p < 0.05) in COVID-19 amnion ORF8 negatives and positives. ( F ) Bar plot representing gene ontology of upregulated biological pathways in COVID-19 amnion ORF8 positive group. ( G , H ) Violin plots representing the fold change values for individual genes related with ( G ) complement (ORF8 (−), n = 5–6; ORF8 (+), n = 9–10) and ( H ) inflammation in amnion (ORF8 (−), n = 5–7; ORF8 (+), n = 8–11) transcriptomics. ( I ) Comparative analysis of differentially expressed proteins (DEPs) ( p < 0.05) in COVID-19 amniotic fluid ORF8 negatives ( n = 4) and positives ( n = 8). Bubble plot of DEPs upregulated exclusively in COVID-19 amniotic fluid ORF8 positive group. ( J ) Comparative analysis of DEGs in COVID-19 umbilical cord ORF8 negatives and positives. ( K ) Bar plot of gene ontology biological pathways associated with DEGs upregulated exclusively in COVID-19 umbilical cord ORF8 positive. ( L ) Violin plots representing the fold change values for individual genes related to complement in umbilical cord transcriptomics. ( M ) Amnion serial sections were analyzed by immunohistochemistry for detection of SARS-CoV-2 ORF8 and C3b. From left to right, tissues stained with (i) Hematoxylin and Eosin (H&E) (hematoxylin—purple, eosin—pink); (ii) Hematoxylin (purple) and the secondary antibody anti-rabbit (Motulsky and Brown, ); (iii) Hematoxylin (purple) and anti-ORF8 produced (Motulsky and Brown, ); and (iv) Hematoxylin (purple) and anti-C3b (Motulsky and Brown, ). Images were taken at 4X magnification. Representative images from three control and three COVID-19 amnions. Fold change calculated by individual expression divided by average expression of control. Violin plots data are presented as means ± SEMs pg/mL, using Mann–Whitney U test ( p < 0.05). Comparative analysis relative to controls. .

Article Snippet: Ten-micron sections were dewaxed, rehydrated and stained with hematoxylin and eosin (H&E) or via immunohistochemical staining with rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody (Cat. No. GTX135591, Genetex) and recombinant anti-C3 antibody (clone: EPR19394; Abcam) and counterstained with hematoxylin.

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Control, Infection, Immunohistochemistry, Staining, Produced, Expressing, MANN-WHITNEY

( A ) Chorion serial sections were analyzed by immunohistochemistry for detection of SARS-CoV-2 ORF8 and C3b. From left to right, tissues stained with (i) Hematoxylin (purple) and the secondary antibody anti-rabbit (Motulsky and Brown, ); (ii) Hematoxylin (purple) and anti-ORF8 produced (Motulsky and Brown, ); and (iii) Hematoxylin (purple) and anti-C3b (Motulsky and Brown, ). Images were taken at 20X magnification. Representative images from two control and two COVID-19 chorion specimens.

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Chorion serial sections were analyzed by immunohistochemistry for detection of SARS-CoV-2 ORF8 and C3b. From left to right, tissues stained with (i) Hematoxylin (purple) and the secondary antibody anti-rabbit (Motulsky and Brown, ); (ii) Hematoxylin (purple) and anti-ORF8 produced (Motulsky and Brown, ); and (iii) Hematoxylin (purple) and anti-C3b (Motulsky and Brown, ). Images were taken at 20X magnification. Representative images from two control and two COVID-19 chorion specimens.

Article Snippet: Ten-micron sections were dewaxed, rehydrated and stained with hematoxylin and eosin (H&E) or via immunohistochemical staining with rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody (Cat. No. GTX135591, Genetex) and recombinant anti-C3 antibody (clone: EPR19394; Abcam) and counterstained with hematoxylin.

Techniques: Immunohistochemistry, Staining, Produced, Control

( A ) Chorion and ( B ) amnion serial sections were stained with SARS-CoV-2 ORF8 (red) and C3b (green); or with Krt8/18 (red) and ORF8 (green). Images were taken at 40× magnification. Images are representative of one control and one COVID-19+. Scale bars: 40 μm. .

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Chorion and ( B ) amnion serial sections were stained with SARS-CoV-2 ORF8 (red) and C3b (green); or with Krt8/18 (red) and ORF8 (green). Images were taken at 40× magnification. Images are representative of one control and one COVID-19+. Scale bars: 40 μm. .

Article Snippet: Ten-micron sections were dewaxed, rehydrated and stained with hematoxylin and eosin (H&E) or via immunohistochemical staining with rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody (Cat. No. GTX135591, Genetex) and recombinant anti-C3 antibody (clone: EPR19394; Abcam) and counterstained with hematoxylin.

Techniques: Staining, Control

( A ) Chorion and ( B ) amnion serial sections were stained with SARS-CoV-2 ORF8 (red) and C3b (green). Mander’s and Pearson’s correlations coefficient between SARS-CoV-2 ORF8 and C3b. White circles represent the colocalization areas analyzed. Images were taken at 40x magnification. Images are representative of one COVID-19+ pregnancy. Scale bars: 40 μm.

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Chorion and ( B ) amnion serial sections were stained with SARS-CoV-2 ORF8 (red) and C3b (green). Mander’s and Pearson’s correlations coefficient between SARS-CoV-2 ORF8 and C3b. White circles represent the colocalization areas analyzed. Images were taken at 40x magnification. Images are representative of one COVID-19+ pregnancy. Scale bars: 40 μm.

Article Snippet: Ten-micron sections were dewaxed, rehydrated and stained with hematoxylin and eosin (H&E) or via immunohistochemical staining with rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody (Cat. No. GTX135591, Genetex) and recombinant anti-C3 antibody (clone: EPR19394; Abcam) and counterstained with hematoxylin.

Techniques: Staining

( A ) Graphical representation detailing the placental cell culture models for in vitro SARS-CoV-2 ORF8 treatment. ( B ) Boxplots representing the fold change values of gene expression for individual genes, obtained from standardization of in vitro SARS-CoV-2 ORF8 treatment with 10 ng/mL, 20 ng/mL, or 100 ng/mL for 8 h from eight independent experiments ( n = 8) and 16 h from eleven independent experiments ( n = 11) on immortalized trophoblasts (HTR8/SVneo cells). Fold change calculated by DDCt method, relative to mock. Data are presented in boxplot where the middle line represent means, the bound of box represent interquartile range, and whiskers represent maximum and minimum values in ng/mL, using one-way ANOVA Kruskal–Wallis with uncorrected Dunn’s test. ( C – E ) Relative expression of genes associated with complement activation in the placental cell culture models treated with 20 ng/mL of SARS-CoV-2 ORF8 for 8 h. The models included ( C ) HTR8/SVneo cells (mock, n = 6–8; and ORF8, n = 7–8) ( D ) primary human villous trophoblasts (HVT) (mock, n = 4–5; and ORF8, n = 4–5), and ( E ) iPSC-derived trophoblasts (mock, n = 5–6; and ORF8, n = 6). Relative expression was calculated by DCt method using GAPDH as the normalizing gene. Mann–Whitney U test was used for analysis. ( F ) Boxplots representing the percentage of C3b + HTR8/SVneo cells treated with SARS-CoV-2 ORF8 for 30 min (mock, n = 5; and ORF8, n = 6), 8 h (mock, n = 4; and ORF8, n = 4) or 16 h (mock, n = 4; and ORF8, n = 4). Data are presented in boxplot, middle line represents means, the bound of box represent interquartile range, and whiskers represent maximum and minimum values. ( G ) Violin plots representing the levels of complement proteins in the supernatant of HTR8/SVneo cells treated with SARS-CoV-2 ORF8 for 8 h (mock, n = 5–6; and ORF8, n = 6) or 16 h (mock, n = 4–5; and ORF8, n = 6). Mann–Whitney U test was used for analysis ( p < 0.05). Immortalized pluripotent stem cells (iPSC). .

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Graphical representation detailing the placental cell culture models for in vitro SARS-CoV-2 ORF8 treatment. ( B ) Boxplots representing the fold change values of gene expression for individual genes, obtained from standardization of in vitro SARS-CoV-2 ORF8 treatment with 10 ng/mL, 20 ng/mL, or 100 ng/mL for 8 h from eight independent experiments ( n = 8) and 16 h from eleven independent experiments ( n = 11) on immortalized trophoblasts (HTR8/SVneo cells). Fold change calculated by DDCt method, relative to mock. Data are presented in boxplot where the middle line represent means, the bound of box represent interquartile range, and whiskers represent maximum and minimum values in ng/mL, using one-way ANOVA Kruskal–Wallis with uncorrected Dunn’s test. ( C – E ) Relative expression of genes associated with complement activation in the placental cell culture models treated with 20 ng/mL of SARS-CoV-2 ORF8 for 8 h. The models included ( C ) HTR8/SVneo cells (mock, n = 6–8; and ORF8, n = 7–8) ( D ) primary human villous trophoblasts (HVT) (mock, n = 4–5; and ORF8, n = 4–5), and ( E ) iPSC-derived trophoblasts (mock, n = 5–6; and ORF8, n = 6). Relative expression was calculated by DCt method using GAPDH as the normalizing gene. Mann–Whitney U test was used for analysis. ( F ) Boxplots representing the percentage of C3b + HTR8/SVneo cells treated with SARS-CoV-2 ORF8 for 30 min (mock, n = 5; and ORF8, n = 6), 8 h (mock, n = 4; and ORF8, n = 4) or 16 h (mock, n = 4; and ORF8, n = 4). Data are presented in boxplot, middle line represents means, the bound of box represent interquartile range, and whiskers represent maximum and minimum values. ( G ) Violin plots representing the levels of complement proteins in the supernatant of HTR8/SVneo cells treated with SARS-CoV-2 ORF8 for 8 h (mock, n = 5–6; and ORF8, n = 6) or 16 h (mock, n = 4–5; and ORF8, n = 6). Mann–Whitney U test was used for analysis ( p < 0.05). Immortalized pluripotent stem cells (iPSC). .

Article Snippet: Ten-micron sections were dewaxed, rehydrated and stained with hematoxylin and eosin (H&E) or via immunohistochemical staining with rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody (Cat. No. GTX135591, Genetex) and recombinant anti-C3 antibody (clone: EPR19394; Abcam) and counterstained with hematoxylin.

Techniques: Cell Culture, In Vitro, Gene Expression, Expressing, Activation Assay, Derivative Assay, MANN-WHITNEY

( A ) Line chart representing absorbance values obtained in the SARS-CoV-2 ORF8 - C1q in vitro binding assay using different molarities of C1q (5, 10, 20, and 50 nM) and SARS-CoV-2 ORF8 (0, 20, 40, 60, 80, and 100 nM). Absorbance calculated by discounting the blank (0 nM C1q). ( B ) Co-immunoprecipitation of SARS-CoV-2 ORF8 with C1qA, C1qB and C1qC. Graphical representation of the plasmid constructs containing the expression cassettes for the subcomponents C1qA, C1qB, and C1qC contained an N-terminal signal peptide (SP), and a C-terminal FLAG tag. Western blot images for co-immunoprecipitation using an anti-ORF8 antibody. ( C ) Co-immunoprecipitation of C1qA full-length (FL), globular (G), disordered domain (D) with SARS-CoV-2 ORF8. Graphical representation of the plasmid constructs containing expression cassettes for the C1qA-FL, C1qA-G, and C1qA-D, with N-terminal SP and a C-terminal 3FLAG tag, cloned in pIRES vectors. Western blot images for co-immunoprecipitation using an anti-ORF8 antibody. Co-immunoprecipitated product (IP). Whole-cell lysate (WCL). .

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Line chart representing absorbance values obtained in the SARS-CoV-2 ORF8 - C1q in vitro binding assay using different molarities of C1q (5, 10, 20, and 50 nM) and SARS-CoV-2 ORF8 (0, 20, 40, 60, 80, and 100 nM). Absorbance calculated by discounting the blank (0 nM C1q). ( B ) Co-immunoprecipitation of SARS-CoV-2 ORF8 with C1qA, C1qB and C1qC. Graphical representation of the plasmid constructs containing the expression cassettes for the subcomponents C1qA, C1qB, and C1qC contained an N-terminal signal peptide (SP), and a C-terminal FLAG tag. Western blot images for co-immunoprecipitation using an anti-ORF8 antibody. ( C ) Co-immunoprecipitation of C1qA full-length (FL), globular (G), disordered domain (D) with SARS-CoV-2 ORF8. Graphical representation of the plasmid constructs containing expression cassettes for the C1qA-FL, C1qA-G, and C1qA-D, with N-terminal SP and a C-terminal 3FLAG tag, cloned in pIRES vectors. Western blot images for co-immunoprecipitation using an anti-ORF8 antibody. Co-immunoprecipitated product (IP). Whole-cell lysate (WCL). .

Article Snippet: Ten-micron sections were dewaxed, rehydrated and stained with hematoxylin and eosin (H&E) or via immunohistochemical staining with rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody (Cat. No. GTX135591, Genetex) and recombinant anti-C3 antibody (clone: EPR19394; Abcam) and counterstained with hematoxylin.

Techniques: In Vitro, Binding Assay, Immunoprecipitation, Plasmid Preparation, Construct, Expressing, FLAG-tag, Western Blot, Clone Assay

( A ) Co-immunoprecipitation of SARS-CoV-2 ORF8 with C1qA, C1qB, and C1qC. Graphical representation of the plasmid constructs containing the expression cassettes for the subcomponents C1qA, C1qB, and C1qC contained an N-terminal signal peptide (SP), and a C-terminal FLAG tag. Western blot images for co-immunoprecipitation using an anti-ORF8 antibody. ( B ) Co-immunoprecipitation of C1qA full-length (FL), globular (G), disordered domain (D) with SARS-CoV-2 ORF8. Graphical representation of the plasmid constructs containing expression cassettes for the C1qA-FL, C1qA-G, and C1qA-D, with N-terminal SP and a C-terminal 3FLAG tag, cloned in pIRES vectors. Western blot images for co-immunoprecipitation using an anti-Flag antibody. Co-immunoprecipitated product (IP). Whole-cell lysate (WCL).

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Co-immunoprecipitation of SARS-CoV-2 ORF8 with C1qA, C1qB, and C1qC. Graphical representation of the plasmid constructs containing the expression cassettes for the subcomponents C1qA, C1qB, and C1qC contained an N-terminal signal peptide (SP), and a C-terminal FLAG tag. Western blot images for co-immunoprecipitation using an anti-ORF8 antibody. ( B ) Co-immunoprecipitation of C1qA full-length (FL), globular (G), disordered domain (D) with SARS-CoV-2 ORF8. Graphical representation of the plasmid constructs containing expression cassettes for the C1qA-FL, C1qA-G, and C1qA-D, with N-terminal SP and a C-terminal 3FLAG tag, cloned in pIRES vectors. Western blot images for co-immunoprecipitation using an anti-Flag antibody. Co-immunoprecipitated product (IP). Whole-cell lysate (WCL).

Article Snippet: Ten-micron sections were dewaxed, rehydrated and stained with hematoxylin and eosin (H&E) or via immunohistochemical staining with rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody (Cat. No. GTX135591, Genetex) and recombinant anti-C3 antibody (clone: EPR19394; Abcam) and counterstained with hematoxylin.

Techniques: Immunoprecipitation, Plasmid Preparation, Construct, Expressing, FLAG-tag, Western Blot, Clone Assay

( A ) Line chart of absorbance values obtained in the SARS-CoV-2 ORF8 peptides - C1q binding assay using different molarities of C1q (10, 20, and 50 nM) and 1 mM of 28 different peptides spanning SARS-CoV-2 ORF8 region. Absorbance calculated by discounting the blank (0 nM C1q). ( B ) The ORF8 peptides presenting dose-dependent binding with C1q (#4 red, #10 purple, #19 blue, and #25 yellow) in molecular binding model between SARS-CoV-2 ORF8 dimers and C1qA-Globular (model 1). ( C ) Bar charts representing docking and confidence scores for in silico docking models SARS-CoV-2 ORF8 and C1qA-G. The ORF8 peptides #4, #10, #19, and #25 represented on amino acid (aa) sequence alignment of docking models. ( D ) Representative docking poses of the globular domain of C1q (PDB ID: 1PK6) with SARS-CoV-2 ORF8 protein (PDB ID:7JTL) in 2D interaction diagrams. The ORF8 and C1q residues with significant interactions from each protein are highlighted, labeled, and properly annotated. The hydrogen bonds, pi-pi, and salt bridges formed between ORF8 and C1q are also shown by colored dash or arrow lines. The polar (turquoise), hydrophobic (green), positively charged (purple), and negatively charged (orange) residues are represented in colored spheres. Shown below each of the interaction diagrams is a table of interacting or contact residues between the SARS-CoV-2 ORF8 protein and the globular C1q domain, including their aa positions, aa distance measured in Angstrom (Å), and the corresponding specific binding interactions. ( E ) Boxplots representing the relative expression of genes analyzed in HTR8/SVneo trophoblast treated with ORF8 peptides #4, #10, #19, and #25 using 0.1 nM and 1 nM ( n = 5–6) or mock ( n = 7–10). ( F ) Boxplots representing the relative expression of genes analyzed in primary human villous trophoblast (HVT) treated with 0.1 nM and 1 nM of ORF8 peptide #10 or Mock. Relative expression was calculated by DCt method using GAPDH as the normalizing gene. ( G ) Boxplots representing the percentage of live C3b+ cells in mock ( n = 4) and ORF8 peptides #4, #10, #19, and #25 0.1 nM and 1 nM treated ( n = 4–6) HTR8/SVneo trophoblasts. Data are presented in boxplot, middle line represents means, the bound of box represents interquartile range, and whiskers represent maximum and minimum values. ( H ) Graphical representation of main results. Comparisons using one-way ANOVA Kruskal–Wallis with Fisher test ( p < 0.05). .

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Line chart of absorbance values obtained in the SARS-CoV-2 ORF8 peptides - C1q binding assay using different molarities of C1q (10, 20, and 50 nM) and 1 mM of 28 different peptides spanning SARS-CoV-2 ORF8 region. Absorbance calculated by discounting the blank (0 nM C1q). ( B ) The ORF8 peptides presenting dose-dependent binding with C1q (#4 red, #10 purple, #19 blue, and #25 yellow) in molecular binding model between SARS-CoV-2 ORF8 dimers and C1qA-Globular (model 1). ( C ) Bar charts representing docking and confidence scores for in silico docking models SARS-CoV-2 ORF8 and C1qA-G. The ORF8 peptides #4, #10, #19, and #25 represented on amino acid (aa) sequence alignment of docking models. ( D ) Representative docking poses of the globular domain of C1q (PDB ID: 1PK6) with SARS-CoV-2 ORF8 protein (PDB ID:7JTL) in 2D interaction diagrams. The ORF8 and C1q residues with significant interactions from each protein are highlighted, labeled, and properly annotated. The hydrogen bonds, pi-pi, and salt bridges formed between ORF8 and C1q are also shown by colored dash or arrow lines. The polar (turquoise), hydrophobic (green), positively charged (purple), and negatively charged (orange) residues are represented in colored spheres. Shown below each of the interaction diagrams is a table of interacting or contact residues between the SARS-CoV-2 ORF8 protein and the globular C1q domain, including their aa positions, aa distance measured in Angstrom (Å), and the corresponding specific binding interactions. ( E ) Boxplots representing the relative expression of genes analyzed in HTR8/SVneo trophoblast treated with ORF8 peptides #4, #10, #19, and #25 using 0.1 nM and 1 nM ( n = 5–6) or mock ( n = 7–10). ( F ) Boxplots representing the relative expression of genes analyzed in primary human villous trophoblast (HVT) treated with 0.1 nM and 1 nM of ORF8 peptide #10 or Mock. Relative expression was calculated by DCt method using GAPDH as the normalizing gene. ( G ) Boxplots representing the percentage of live C3b+ cells in mock ( n = 4) and ORF8 peptides #4, #10, #19, and #25 0.1 nM and 1 nM treated ( n = 4–6) HTR8/SVneo trophoblasts. Data are presented in boxplot, middle line represents means, the bound of box represents interquartile range, and whiskers represent maximum and minimum values. ( H ) Graphical representation of main results. Comparisons using one-way ANOVA Kruskal–Wallis with Fisher test ( p < 0.05). .

Article Snippet: Ten-micron sections were dewaxed, rehydrated and stained with hematoxylin and eosin (H&E) or via immunohistochemical staining with rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody (Cat. No. GTX135591, Genetex) and recombinant anti-C3 antibody (clone: EPR19394; Abcam) and counterstained with hematoxylin.

Techniques: Binding Assay, In Silico, Sequencing, Labeling, Expressing

Correlation analyses between the number of days post SARS-CoV-2 infection to delivery and ( A ) SARS-CoV-2 N1 RNA copies, SARS-CoV-2 N2 RNA copies, and ( B ) circulating ORF8 levels in all biospecimens analyzed, including maternal plasma, chorion, amnion, cord plasma and newborn plasma. Simples linear regression and Spearman’s rank correlation test was used for all the correlations analysis. Scatter plots with Pearson correlation coefficients (r) and dotted lines represent 95% confidence intervals.

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: Correlation analyses between the number of days post SARS-CoV-2 infection to delivery and ( A ) SARS-CoV-2 N1 RNA copies, SARS-CoV-2 N2 RNA copies, and ( B ) circulating ORF8 levels in all biospecimens analyzed, including maternal plasma, chorion, amnion, cord plasma and newborn plasma. Simples linear regression and Spearman’s rank correlation test was used for all the correlations analysis. Scatter plots with Pearson correlation coefficients (r) and dotted lines represent 95% confidence intervals.

Article Snippet: Ten-micron sections were dewaxed, rehydrated and stained with hematoxylin and eosin (H&E) or via immunohistochemical staining with rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody (Cat. No. GTX135591, Genetex) and recombinant anti-C3 antibody (clone: EPR19394; Abcam) and counterstained with hematoxylin.

Techniques: Infection, Clinical Proteomics

( A ) Sequence alignment of SARS-CoV-2 ORF8 and human immunoglobulin variable lambda chain (IGLV) genes. Representative amino acid residues of SARS-CoV-2 ORF8-specific region located at the loop flanking β 4- β 5 strands were compared with a portion of the framework 3 (FR3) region of the variable light chain of human IGLV5-39*01, IGLV5-39*02, and IGLV1-47*01 germline gene sequences. Identical residues were highlighted in red box and marked by an asterisk (*), while semi-conserved positions due to minor structural differences are in blue boxes and are indicated by a period (.). The β strands are shown by green arrows above the ORF8 sequence while the spanning loops were represented by black lines (Flower et al, ). The FR3 region (shown in the yellow box) was defined according to the IMGT delimitations. ( B ) Comparison of the overlapping sequences of SARS-CoV-2 ORF8 and human IGLV genes highlighted in the dashed boxes using peptide properties: buried index, hydrophobicity, polarity, and turn and helix propensity. The legend for each property is shown as a gradient color scheme on the right. ( C – E ) Structures of ORF8 ( C ), IGLV5-39*01 ( D ), and superimposed images after pairwise structural alignment as viewed from the top (left) and side (right). PBD entries: 7JTL (SARS-CoV-2, blue) and 2CD0 (human IGLV5-39, green). Overlapping sequences between the two structures are highlighted in red. ( F ) Pairwise structural alignment of the human IgG-Fc region (cyan, PDB ID:1FC1) and ORF8 (in blue) showing close structural similarity at the CH2 domain (193 atoms aligned; RMSD: 5.9 Å). The CH2 and CH3 domains are highlighted, while residues in red sphere show the neonatal Fc receptor (FcRn) interaction site. ( G ) Superimposition of CH2 domain of the human IgG-Fc and ORF8 from different rotational perspectives. CH2 residues with the closest folding to ORF8 (blue) is shown in flesh while CH3 are in cyan. The glycosylation site at N297 at the CH2 interface is shown in cyan stick models.

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Sequence alignment of SARS-CoV-2 ORF8 and human immunoglobulin variable lambda chain (IGLV) genes. Representative amino acid residues of SARS-CoV-2 ORF8-specific region located at the loop flanking β 4- β 5 strands were compared with a portion of the framework 3 (FR3) region of the variable light chain of human IGLV5-39*01, IGLV5-39*02, and IGLV1-47*01 germline gene sequences. Identical residues were highlighted in red box and marked by an asterisk (*), while semi-conserved positions due to minor structural differences are in blue boxes and are indicated by a period (.). The β strands are shown by green arrows above the ORF8 sequence while the spanning loops were represented by black lines (Flower et al, ). The FR3 region (shown in the yellow box) was defined according to the IMGT delimitations. ( B ) Comparison of the overlapping sequences of SARS-CoV-2 ORF8 and human IGLV genes highlighted in the dashed boxes using peptide properties: buried index, hydrophobicity, polarity, and turn and helix propensity. The legend for each property is shown as a gradient color scheme on the right. ( C – E ) Structures of ORF8 ( C ), IGLV5-39*01 ( D ), and superimposed images after pairwise structural alignment as viewed from the top (left) and side (right). PBD entries: 7JTL (SARS-CoV-2, blue) and 2CD0 (human IGLV5-39, green). Overlapping sequences between the two structures are highlighted in red. ( F ) Pairwise structural alignment of the human IgG-Fc region (cyan, PDB ID:1FC1) and ORF8 (in blue) showing close structural similarity at the CH2 domain (193 atoms aligned; RMSD: 5.9 Å). The CH2 and CH3 domains are highlighted, while residues in red sphere show the neonatal Fc receptor (FcRn) interaction site. ( G ) Superimposition of CH2 domain of the human IgG-Fc and ORF8 from different rotational perspectives. CH2 residues with the closest folding to ORF8 (blue) is shown in flesh while CH3 are in cyan. The glycosylation site at N297 at the CH2 interface is shown in cyan stick models.

Article Snippet: Ten-micron sections were dewaxed, rehydrated and stained with hematoxylin and eosin (H&E) or via immunohistochemical staining with rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody (Cat. No. GTX135591, Genetex) and recombinant anti-C3 antibody (clone: EPR19394; Abcam) and counterstained with hematoxylin.

Techniques: Sequencing, Comparison, Glycoproteomics

Reagents and tools table

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Ten-micron sections were dewaxed, rehydrated and stained with hematoxylin and eosin (H&E) or via immunohistochemical staining with rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody (Cat. No. GTX135591, Genetex) and recombinant anti-C3 antibody (clone: EPR19394; Abcam) and counterstained with hematoxylin.

Techniques: Recombinant, Variant Assay, Generated, Plasmid Preparation, FLAG-tag, Sequencing, Digital PCR, cDNA Synthesis, SYBR Green Assay, Blocking Assay, Software, Cell Culture, Enzyme-linked Immunosorbent Assay, Transfection, Real-time Polymerase Chain Reaction, Spectrophotometry, Imaging

SARS-CoV-2 ORF8 interacts p62 to form condensates (A) Co-precipitation analysis of endogenous p62 with ORF8-Strep in HEK293T. (B) Purified MBP-ORF8 was incubated with purified GST or GST-p62, and analysis of the interaction between ORF8 and p62 by in vitro GST pull-down was performed. (C and D) Cells were co-transfected with ORF8-Strep and mCherry-p62 for 24 h, then stained with anti-Strep antibody and imaged using confocal microscopy. Scale bar, 10 μm. The number of ORF8 puncta in each cell was counted from 50 cells of three independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (E) p62 WT and KO Vero-E6 cells were transfected with ORF8-Strep for 24 h, then cells were stained with anti-Strep and anti-p62 antibodies, and imaged using confocal microscopy. Scale bar, 10 μm. The number of ORF8 puncta in each cell was counted from 50 cells of three independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (F) HeLa cells were co-transfected with GFP-ORF8 and mCherry-p62 for 24 h, then cells were mock treated or treated with 1,6-hexanediol for 1 min and imaged using confocal microscopy. Scale bar, 10 μm. The number of ORF8/p62 puncta in each cell was counted from 50 cells of three independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (G) HeLa cells with exogenous expression of GFP-ORF8 and mCherry-p62 undergo liquid-like behavior; fluorescence recovery after photobleaching (FRAP) images are shown before and at indicated time points after bleaching. Time 0 indicates the time of photobleaching. Scale bar, 10 μm. Quantification of fluorescence intensity recovery of GFP-ORF8 and mCherry-p62 in the bleached droplet. (H) HeLa cells with exogenous expression of GFP-ORF8 and mCherry-p62 undergo fusion, images are shown at indicated time points after imaging. (I) mCherry-p62 were purified from Escherichia coli BL21 and analyzed via Coomassie blue. (J) In vitro phase separation assay of GFP and mCherry-p62/His6-UBx8 or GFP-ORF8 and mCherry-p62/His6-UBx8. Fluorescence images of 10 μM each protein in phase separation assay buffer without PEG8000. Representative fluorescence images of three independent experiments are shown. Scale bar, 10 μm. (K) GFP-ORF8 truncations were expressed as indicated with p62-FLAG. Cell lysates were subjected to immunoprecipitation with GFP antibody and analyzed using western blotting (WB). (L) Cells were transfected with GFP-ORF8 or mutant with mCherry-p62 for 24 h, and imaged using confocal microscopy. Scale bar, 10 μm. (M) p62-FLAG mutations were expressed as indicated with ORF8-Strep. Cell lysates were subjected to immunoprecipitation with FLAG antibody and analyzed using WB. (N) p62 KO Vero-E6 cells were transfected with p62-FLAG or mutant with ORF8-Strep for 24 h, then stained with anti-FLAG and anti-Strep antibodies and imaged using confocal microscopy. Scale bar, 10 μm. (O) SARS-CoV-2 infected Vero-E6 cells were stained with anti-ORF8 and anti-p62 antibodies and imaged using confocal microscopy. Scale bar, 10 μm. (P) Co-precipitation analysis of p62-HA with SARS-CoV-2 ORF8-FLAG or SARS-CoV ORF8-FLAG or MERS-CoV ORF8-FLAG in HEK293T. (Q) Cells were co-transfected with mCherry-p62 with SARS-CoV ORF8-FLAG or MERS-CoV ORF8-FLAG for 24 h, then stained with anti-FLAG antibody and imaged using confocal microscopy. Scale bar, 10 μm. See also <xref ref-type=Figures S1 and . " width="100%" height="100%">

Journal: Cell Reports

Article Title: Coronavirus subverts ER-phagy by hijacking FAM134B and ATL3 into p62 condensates to facilitate viral replication

doi: 10.1016/j.celrep.2023.112286

Figure Lengend Snippet: SARS-CoV-2 ORF8 interacts p62 to form condensates (A) Co-precipitation analysis of endogenous p62 with ORF8-Strep in HEK293T. (B) Purified MBP-ORF8 was incubated with purified GST or GST-p62, and analysis of the interaction between ORF8 and p62 by in vitro GST pull-down was performed. (C and D) Cells were co-transfected with ORF8-Strep and mCherry-p62 for 24 h, then stained with anti-Strep antibody and imaged using confocal microscopy. Scale bar, 10 μm. The number of ORF8 puncta in each cell was counted from 50 cells of three independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (E) p62 WT and KO Vero-E6 cells were transfected with ORF8-Strep for 24 h, then cells were stained with anti-Strep and anti-p62 antibodies, and imaged using confocal microscopy. Scale bar, 10 μm. The number of ORF8 puncta in each cell was counted from 50 cells of three independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (F) HeLa cells were co-transfected with GFP-ORF8 and mCherry-p62 for 24 h, then cells were mock treated or treated with 1,6-hexanediol for 1 min and imaged using confocal microscopy. Scale bar, 10 μm. The number of ORF8/p62 puncta in each cell was counted from 50 cells of three independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (G) HeLa cells with exogenous expression of GFP-ORF8 and mCherry-p62 undergo liquid-like behavior; fluorescence recovery after photobleaching (FRAP) images are shown before and at indicated time points after bleaching. Time 0 indicates the time of photobleaching. Scale bar, 10 μm. Quantification of fluorescence intensity recovery of GFP-ORF8 and mCherry-p62 in the bleached droplet. (H) HeLa cells with exogenous expression of GFP-ORF8 and mCherry-p62 undergo fusion, images are shown at indicated time points after imaging. (I) mCherry-p62 were purified from Escherichia coli BL21 and analyzed via Coomassie blue. (J) In vitro phase separation assay of GFP and mCherry-p62/His6-UBx8 or GFP-ORF8 and mCherry-p62/His6-UBx8. Fluorescence images of 10 μM each protein in phase separation assay buffer without PEG8000. Representative fluorescence images of three independent experiments are shown. Scale bar, 10 μm. (K) GFP-ORF8 truncations were expressed as indicated with p62-FLAG. Cell lysates were subjected to immunoprecipitation with GFP antibody and analyzed using western blotting (WB). (L) Cells were transfected with GFP-ORF8 or mutant with mCherry-p62 for 24 h, and imaged using confocal microscopy. Scale bar, 10 μm. (M) p62-FLAG mutations were expressed as indicated with ORF8-Strep. Cell lysates were subjected to immunoprecipitation with FLAG antibody and analyzed using WB. (N) p62 KO Vero-E6 cells were transfected with p62-FLAG or mutant with ORF8-Strep for 24 h, then stained with anti-FLAG and anti-Strep antibodies and imaged using confocal microscopy. Scale bar, 10 μm. (O) SARS-CoV-2 infected Vero-E6 cells were stained with anti-ORF8 and anti-p62 antibodies and imaged using confocal microscopy. Scale bar, 10 μm. (P) Co-precipitation analysis of p62-HA with SARS-CoV-2 ORF8-FLAG or SARS-CoV ORF8-FLAG or MERS-CoV ORF8-FLAG in HEK293T. (Q) Cells were co-transfected with mCherry-p62 with SARS-CoV ORF8-FLAG or MERS-CoV ORF8-FLAG for 24 h, then stained with anti-FLAG antibody and imaged using confocal microscopy. Scale bar, 10 μm. See also Figures S1 and .

Article Snippet: Mouse anti-GFP (AE012), anti-mCherry (AE002), anti-GST (AE001), anti-MBP (AE016), anti-His (AE003), rabbit anti-Flag (AE092), rabbit anti-SARS-CoV-2 ORF3a (A20234), rabbit anti-SARS-CoV-2 ORF8 (A20235) were obtained from ABclonal.

Techniques: Purification, Incubation, In Vitro, Transfection, Staining, Confocal Microscopy, Two Tailed Test, Expressing, Fluorescence, Imaging, Immunoprecipitation, Western Blot, Mutagenesis, Infection

ORF8/p62 condensates subvert ER-phagy (A) Co-precipitation analysis of GFP-FAM134B with ORF8-Strep in HEK293T. (B) Purified MBP or MBP-ORF8 was incubated with purified GST or GST-FAM134B, and analyzed the interaction between ORF8 and FAM134B by GST pull-down. (C) Immunofluorescence of cells expressing FAM134B-HA or FAM134B-HA and GFP-ORF8 or FAM134B-HA, p62-FLAG and GFP-ORF8 with anti-HA and anti-FLAG antibodies. Scale bar, 10 μm. The number of FAM134B puncta (>1 μm) in each cell was counted from 50 cells of three independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (D) In vitro phase separation assay of GFP-FAM134B alone or GFP-FAM134B and MBP-ORF8 or GFP-FAM134B and mCherry-p62/His-UBx8 or GFP-FAM134B, MBP-ORF8 and mCherry-p62/His-UBx8. Fluorescence images of 10 μM each protein in phase separation assay buffer without PEG8000. Representative images of three independent experiments are shown. Scale bar, 10 μm. (E) GFP-ORF8 truncations were expressed as indicated with FAM134B-HA. Cell lysates were subjected to immunoprecipitation with GFP antibody and analyzed using WB. (F) HeLa cells were transfected with GFP-ORF8 truncations with FAM134B-HA for 24 h, then stained with anti-HA antibody and imaged using confocal microscopy. Scale bar, 10 μm. (G and H) U2OS cells expressing mCherry-GFP-RAMP4 were transfected with p62-FLAG with or without ORF8-Strep or mutants for 24 h, then cells were starved in EBSS for 12 h and imaged using confocal microscopy (G). Scale bar, 10 μm. The number of red puncta in each cell (H) was counted from 50 cells of three independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (I and J) U2OS cells expressing mCherry-Sec61B were transfected with indicated plasmids for 24 h and then starved in EBSS for 12 h. Cell lysates were analyzed using WB. (K) U2OS cells expressing mCherry-Sec61B were transfected with indicated plasmids for 24 h. Cell lysates were analyzed using WB. (L) Vero-E6 cells were transfected with control or ORF8-KD plasmid for 36 h and analyzed the knockdown efficiency using WB. (M and N) Vero-E6 WT cells were transfected with control or ORF8-KD plasmid, p62 KO Vero-E6 cells were transfected with p62 EHGG-AAAA -FLAG for 12 h and then infected with SARS-CoV-2 for 24 h, then cells were stained with anti-FAM134B and anti-p62 or anti-FLAG antibodies and imaged using confocal microscopy. Scale bar, 10 μm. The number of colocalization between FAM134B and p62 droplets in each cell was counted from 20 cells of two independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (O) Vero-E6 cells were transfected with ORF8-KD plasmid with or without si-ORF3a or si-Atg7 for 24 h and then infected with SARS-CoV-2 for another 24 h. Cell lysates were analyzed using WB. See also <xref ref-type=Figure S3 . " width="100%" height="100%">

Journal: Cell Reports

Article Title: Coronavirus subverts ER-phagy by hijacking FAM134B and ATL3 into p62 condensates to facilitate viral replication

doi: 10.1016/j.celrep.2023.112286

Figure Lengend Snippet: ORF8/p62 condensates subvert ER-phagy (A) Co-precipitation analysis of GFP-FAM134B with ORF8-Strep in HEK293T. (B) Purified MBP or MBP-ORF8 was incubated with purified GST or GST-FAM134B, and analyzed the interaction between ORF8 and FAM134B by GST pull-down. (C) Immunofluorescence of cells expressing FAM134B-HA or FAM134B-HA and GFP-ORF8 or FAM134B-HA, p62-FLAG and GFP-ORF8 with anti-HA and anti-FLAG antibodies. Scale bar, 10 μm. The number of FAM134B puncta (>1 μm) in each cell was counted from 50 cells of three independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (D) In vitro phase separation assay of GFP-FAM134B alone or GFP-FAM134B and MBP-ORF8 or GFP-FAM134B and mCherry-p62/His-UBx8 or GFP-FAM134B, MBP-ORF8 and mCherry-p62/His-UBx8. Fluorescence images of 10 μM each protein in phase separation assay buffer without PEG8000. Representative images of three independent experiments are shown. Scale bar, 10 μm. (E) GFP-ORF8 truncations were expressed as indicated with FAM134B-HA. Cell lysates were subjected to immunoprecipitation with GFP antibody and analyzed using WB. (F) HeLa cells were transfected with GFP-ORF8 truncations with FAM134B-HA for 24 h, then stained with anti-HA antibody and imaged using confocal microscopy. Scale bar, 10 μm. (G and H) U2OS cells expressing mCherry-GFP-RAMP4 were transfected with p62-FLAG with or without ORF8-Strep or mutants for 24 h, then cells were starved in EBSS for 12 h and imaged using confocal microscopy (G). Scale bar, 10 μm. The number of red puncta in each cell (H) was counted from 50 cells of three independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (I and J) U2OS cells expressing mCherry-Sec61B were transfected with indicated plasmids for 24 h and then starved in EBSS for 12 h. Cell lysates were analyzed using WB. (K) U2OS cells expressing mCherry-Sec61B were transfected with indicated plasmids for 24 h. Cell lysates were analyzed using WB. (L) Vero-E6 cells were transfected with control or ORF8-KD plasmid for 36 h and analyzed the knockdown efficiency using WB. (M and N) Vero-E6 WT cells were transfected with control or ORF8-KD plasmid, p62 KO Vero-E6 cells were transfected with p62 EHGG-AAAA -FLAG for 12 h and then infected with SARS-CoV-2 for 24 h, then cells were stained with anti-FAM134B and anti-p62 or anti-FLAG antibodies and imaged using confocal microscopy. Scale bar, 10 μm. The number of colocalization between FAM134B and p62 droplets in each cell was counted from 20 cells of two independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (O) Vero-E6 cells were transfected with ORF8-KD plasmid with or without si-ORF3a or si-Atg7 for 24 h and then infected with SARS-CoV-2 for another 24 h. Cell lysates were analyzed using WB. See also Figure S3 .

Article Snippet: Mouse anti-GFP (AE012), anti-mCherry (AE002), anti-GST (AE001), anti-MBP (AE016), anti-His (AE003), rabbit anti-Flag (AE092), rabbit anti-SARS-CoV-2 ORF3a (A20234), rabbit anti-SARS-CoV-2 ORF8 (A20235) were obtained from ABclonal.

Techniques: Purification, Incubation, Immunofluorescence, Expressing, Two Tailed Test, In Vitro, Fluorescence, Immunoprecipitation, Transfection, Staining, Confocal Microscopy, Control, Plasmid Preparation, Knockdown, Infection

ORF8/p62 condensates facilitate DMV formation (A) Vero-E6 cells were transfected with control or ORF8-KD plasmid for 12 h and infected with SARS-CoV-2 for 24 h. Cells were stained with dsRNA antibody (red). Nuclear DNA was stained with DAPI (blue). Cells were imaged using confocal microscopy. Scale bar, 10 μm. The number of dsRNA puncta in each cell was counted from 50 cells of two independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (B and C) Transmission electron microscopy images of p62 WT and KO Vero-E6 cells transfected with p62 EHGG-AAAA with control or ORF8-KD plasmid for 12 h and then infected with or without SARS-CoV-2 for 24 h. The DMV structures (red pentagram), ER-DMVs interaction (blue arrowhead), autophagosomes (green arrowhead), ER in autophagosomes (red arrowhead) are shown. (D and E) The number of DMV (D) and ER-phagy (E) in each cell was counted from 25 cells of two independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (F) Vero-E6 cells were transfected with control or ORF8-KD plasmid for 12 h and then infected with or without SARS-CoV-2 for 24 h, then cells were stained with anti-FAM134B and anti-LC3 antibodies and imaged using confocal microscopy. Scale bar, 10 μm. (G) The number of co-localizations between FAM134B and LC3 in each cell from (F) was counted from 25 cells of two independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (H) p62 KO Vero-E6 cells transfected with WT or p62 EHGG-AAAA for 12 h and then infected with SARS-CoV-2 for 24 h. Cells were stained with dsRNA antibody (red). Nuclear DNA was stained with DAPI (blue). Cells were imaged using confocal microscopy. Scale bar, 10 μm. (I) The number of dsRNA puncta in each cell from (H) was counted from 50 cells of two independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (J) Vero-E6 cells were transfected with sh-FAM134B or FAM134B-HA or si-Atg7 for 12 h, cells were infected with SARS-CoV-2 for 24 h, and media were collected and analyzed using plaque assay. Error bars, mean ± SD of three independent experiments. Two-tailed unpaired Student’s t test, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (K) p62 KO Vero-E6 cells were transfected with p62-FLAG or p62 EHGG-AAAA -FLAG for 12 h and infected with SARS-CoV-2 for 24 h, and media were collected and analyzed using plaque assay. Error bars, mean ± SD of three independent experiments Two-tailed unpaired Student’s t test, ∗∗ p < 0.01. (L) HeLa cells were transfected with FAM134B-HA, Nsp3/4-Myc for 24 h, then stained with anti-HA and anti-Myc, and imaged using confocal microscopy. Scale bar, 10 μm. (M) HeLa cells were transfected with Nsp3/4-Myc for 24 h and starved for another 12 h with or without 2 h CQ treatment. Cell lysates were analyzed using WB. See also <xref ref-type=Figure S4 . " width="100%" height="100%">

Journal: Cell Reports

Article Title: Coronavirus subverts ER-phagy by hijacking FAM134B and ATL3 into p62 condensates to facilitate viral replication

doi: 10.1016/j.celrep.2023.112286

Figure Lengend Snippet: ORF8/p62 condensates facilitate DMV formation (A) Vero-E6 cells were transfected with control or ORF8-KD plasmid for 12 h and infected with SARS-CoV-2 for 24 h. Cells were stained with dsRNA antibody (red). Nuclear DNA was stained with DAPI (blue). Cells were imaged using confocal microscopy. Scale bar, 10 μm. The number of dsRNA puncta in each cell was counted from 50 cells of two independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (B and C) Transmission electron microscopy images of p62 WT and KO Vero-E6 cells transfected with p62 EHGG-AAAA with control or ORF8-KD plasmid for 12 h and then infected with or without SARS-CoV-2 for 24 h. The DMV structures (red pentagram), ER-DMVs interaction (blue arrowhead), autophagosomes (green arrowhead), ER in autophagosomes (red arrowhead) are shown. (D and E) The number of DMV (D) and ER-phagy (E) in each cell was counted from 25 cells of two independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (F) Vero-E6 cells were transfected with control or ORF8-KD plasmid for 12 h and then infected with or without SARS-CoV-2 for 24 h, then cells were stained with anti-FAM134B and anti-LC3 antibodies and imaged using confocal microscopy. Scale bar, 10 μm. (G) The number of co-localizations between FAM134B and LC3 in each cell from (F) was counted from 25 cells of two independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (H) p62 KO Vero-E6 cells transfected with WT or p62 EHGG-AAAA for 12 h and then infected with SARS-CoV-2 for 24 h. Cells were stained with dsRNA antibody (red). Nuclear DNA was stained with DAPI (blue). Cells were imaged using confocal microscopy. Scale bar, 10 μm. (I) The number of dsRNA puncta in each cell from (H) was counted from 50 cells of two independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (J) Vero-E6 cells were transfected with sh-FAM134B or FAM134B-HA or si-Atg7 for 12 h, cells were infected with SARS-CoV-2 for 24 h, and media were collected and analyzed using plaque assay. Error bars, mean ± SD of three independent experiments. Two-tailed unpaired Student’s t test, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (K) p62 KO Vero-E6 cells were transfected with p62-FLAG or p62 EHGG-AAAA -FLAG for 12 h and infected with SARS-CoV-2 for 24 h, and media were collected and analyzed using plaque assay. Error bars, mean ± SD of three independent experiments Two-tailed unpaired Student’s t test, ∗∗ p < 0.01. (L) HeLa cells were transfected with FAM134B-HA, Nsp3/4-Myc for 24 h, then stained with anti-HA and anti-Myc, and imaged using confocal microscopy. Scale bar, 10 μm. (M) HeLa cells were transfected with Nsp3/4-Myc for 24 h and starved for another 12 h with or without 2 h CQ treatment. Cell lysates were analyzed using WB. See also Figure S4 .

Article Snippet: Mouse anti-GFP (AE012), anti-mCherry (AE002), anti-GST (AE001), anti-MBP (AE016), anti-His (AE003), rabbit anti-Flag (AE092), rabbit anti-SARS-CoV-2 ORF3a (A20234), rabbit anti-SARS-CoV-2 ORF8 (A20235) were obtained from ABclonal.

Techniques: Transfection, Control, Plasmid Preparation, Infection, Staining, Confocal Microscopy, Two Tailed Test, Transmission Assay, Electron Microscopy, Plaque Assay

ORF8 homo-dimerization is important for ER-phagy inhibition (A) Co-precipitation analysis of GFP-ORF8 with ORF8-Strep in HEK293T. (B) Purified MBP-ORF8 was incubated with purified GST or GST-ORF8, and analysis of the self-interaction by GST pull-down. (C) Analysis of the homo-dimerization of ORF8 by cross-linking with DSS. Cells were transfected with ORF8-FLAG for 24 h and treated with DSS as indicated for 30 min. Cell lysates were analyzed using WB. (D) Analysis of the self-interaction of ORF8 with p62 overexpression by coIP assay. (E) Analysis of the self-interaction of ORF8 under starvation treatment by coIP assay. (F) Analysis of the homo-dimerization of ORF8 by cross-linking with DSS under starvation treatment. Cells stable expressing ORF8-FLAG were starved for 12 h. Cell lysates were analyzed using WB. (G) Schematic illustration of ORF8 point mutations. (H and I) Cross-linking with DSS assay (H) and coIP (I) analyzed ORF8 homo-dimerization. Cells were transfected with indicated plasmids for 24 h. (J) Interaction between Strep-tagged WT ORF8 and mutants with GFP-ORF8-HA observed in a coIP assay. (K) Analysis of the homodimerization of ORF8 deletion mutants by cross-linking with DSS and analyzed using WB. (L) Prediction of ORF8 aggregation domain. (M and N) Analysis of the homodimerization of ORF8 deletion mutants by coIP (M) and DSS assay (N). (O) Interaction between GFP-ORF8 and mutants with p62-FLAG observed in a coIP assay. (P and Q) Interaction between GFP-ORF8 and mutants with FAM134B-HA (P) or ATL3-HA (Q) observed in a coIP assay. (R) Analysis of the colocalization of GFP-ORF8 and deletion mutant with FAM134B-HA or ATL3-HA with anti-HA or anti-FLAG antibodies using confocal microscopy. Scale bar, 10 μm. The number of colocalization of ORF8/p62 bodies with FAM134B or ATL3 in each cell was counted from 25 cells of three independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (S) U2OS cells expressing mCherry-Sec61B were transfected with indicated plasmids for 24 h and then starved in EBSS for 12 h. Lysates were analyzed using WB. (T) HEK293T cells were transfected with indicated plasmids for 24 h. Lysates were analyzed using WB. (U) Proposed model for the role of ORF8/p62 condensates in viral replication through ER-phagy inhibition.

Journal: Cell Reports

Article Title: Coronavirus subverts ER-phagy by hijacking FAM134B and ATL3 into p62 condensates to facilitate viral replication

doi: 10.1016/j.celrep.2023.112286

Figure Lengend Snippet: ORF8 homo-dimerization is important for ER-phagy inhibition (A) Co-precipitation analysis of GFP-ORF8 with ORF8-Strep in HEK293T. (B) Purified MBP-ORF8 was incubated with purified GST or GST-ORF8, and analysis of the self-interaction by GST pull-down. (C) Analysis of the homo-dimerization of ORF8 by cross-linking with DSS. Cells were transfected with ORF8-FLAG for 24 h and treated with DSS as indicated for 30 min. Cell lysates were analyzed using WB. (D) Analysis of the self-interaction of ORF8 with p62 overexpression by coIP assay. (E) Analysis of the self-interaction of ORF8 under starvation treatment by coIP assay. (F) Analysis of the homo-dimerization of ORF8 by cross-linking with DSS under starvation treatment. Cells stable expressing ORF8-FLAG were starved for 12 h. Cell lysates were analyzed using WB. (G) Schematic illustration of ORF8 point mutations. (H and I) Cross-linking with DSS assay (H) and coIP (I) analyzed ORF8 homo-dimerization. Cells were transfected with indicated plasmids for 24 h. (J) Interaction between Strep-tagged WT ORF8 and mutants with GFP-ORF8-HA observed in a coIP assay. (K) Analysis of the homodimerization of ORF8 deletion mutants by cross-linking with DSS and analyzed using WB. (L) Prediction of ORF8 aggregation domain. (M and N) Analysis of the homodimerization of ORF8 deletion mutants by coIP (M) and DSS assay (N). (O) Interaction between GFP-ORF8 and mutants with p62-FLAG observed in a coIP assay. (P and Q) Interaction between GFP-ORF8 and mutants with FAM134B-HA (P) or ATL3-HA (Q) observed in a coIP assay. (R) Analysis of the colocalization of GFP-ORF8 and deletion mutant with FAM134B-HA or ATL3-HA with anti-HA or anti-FLAG antibodies using confocal microscopy. Scale bar, 10 μm. The number of colocalization of ORF8/p62 bodies with FAM134B or ATL3 in each cell was counted from 25 cells of three independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (S) U2OS cells expressing mCherry-Sec61B were transfected with indicated plasmids for 24 h and then starved in EBSS for 12 h. Lysates were analyzed using WB. (T) HEK293T cells were transfected with indicated plasmids for 24 h. Lysates were analyzed using WB. (U) Proposed model for the role of ORF8/p62 condensates in viral replication through ER-phagy inhibition.

Article Snippet: Mouse anti-GFP (AE012), anti-mCherry (AE002), anti-GST (AE001), anti-MBP (AE016), anti-His (AE003), rabbit anti-Flag (AE092), rabbit anti-SARS-CoV-2 ORF3a (A20234), rabbit anti-SARS-CoV-2 ORF8 (A20235) were obtained from ABclonal.

Techniques: Inhibition, Purification, Incubation, Transfection, Over Expression, Co-Immunoprecipitation Assay, Expressing, Mutagenesis, Confocal Microscopy, Two Tailed Test

Journal: Cell Reports

Article Title: Coronavirus subverts ER-phagy by hijacking FAM134B and ATL3 into p62 condensates to facilitate viral replication

doi: 10.1016/j.celrep.2023.112286

Figure Lengend Snippet:

Article Snippet: Mouse anti-GFP (AE012), anti-mCherry (AE002), anti-GST (AE001), anti-MBP (AE016), anti-His (AE003), rabbit anti-Flag (AE092), rabbit anti-SARS-CoV-2 ORF3a (A20234), rabbit anti-SARS-CoV-2 ORF8 (A20235) were obtained from ABclonal.

Techniques: Virus, Control, Recombinant, Transfection, Saline, Modification, Protease Inhibitor, Magnetic Beads, Bicinchoninic Acid Protein Assay, shRNA, Software

Journal: Cell Reports

Article Title: Coronavirus subverts ER-phagy by hijacking FAM134B and ATL3 into p62 condensates to facilitate viral replication

doi: 10.1016/j.celrep.2023.112286

Figure Lengend Snippet:

Article Snippet: Mouse anti-GFP (AE012), anti-mCherry (AE002), anti-GST (AE001), anti-MBP (AE016), anti-His (AE003), rabbit anti-Flag (AE092), rabbit anti-SARS-CoV-2 ORF3a (A20234), rabbit anti-SARS-CoV-2 ORF8 (A20235) were obtained from ABclonal.

Techniques: Virus, Control, Recombinant, Transfection, Saline, Modification, Protease Inhibitor, Magnetic Beads, Bicinchoninic Acid Protein Assay, shRNA, Software

Determining the stability of SARS-CoV-2 proteins. Human lung epithelial BEAS-2B cells were exposed to cycloheximide (CHX) (100 μg/mL) at four time points (2, 4, 6, and 8 h) prior to immunoblotting. The bands were quantitated and normalized to β-actin, and the half-lives of the indicated proteins are shown graphically. (A) NSP1, E, and ORF8. (B) NSP3d, NSP4, NSP6, NSP7, NSP8, NSP9, NSP12, NSP13, NSP14, NSP16, ORF3a, ORF3b, ORF6, ORF7b, and ORF9b. (C) NSP2, NSP5, NSP10, NSP15, spike, M, and N. Data are from three independent biological replicates.

Journal: mSystems

Article Title: Stability of SARS-CoV-2-Encoded Proteins and Their Antibody Levels Correlate with Interleukin 6 in COVID-19 Patients

doi: 10.1128/msystems.00058-22

Figure Lengend Snippet: Determining the stability of SARS-CoV-2 proteins. Human lung epithelial BEAS-2B cells were exposed to cycloheximide (CHX) (100 μg/mL) at four time points (2, 4, 6, and 8 h) prior to immunoblotting. The bands were quantitated and normalized to β-actin, and the half-lives of the indicated proteins are shown graphically. (A) NSP1, E, and ORF8. (B) NSP3d, NSP4, NSP6, NSP7, NSP8, NSP9, NSP12, NSP13, NSP14, NSP16, ORF3a, ORF3b, ORF6, ORF7b, and ORF9b. (C) NSP2, NSP5, NSP10, NSP15, spike, M, and N. Data are from three independent biological replicates.

Article Snippet: Recombinant SARS-CoV-2 NSP10 protein (catalog number 10-408) and recombinant SARS-CoV-2 ORF8 protein (catalog number 10-436) were purchased from ProSci (Poway, CA).

Techniques: Western Blot

SARS-CoV-2 protein-specific antibodies in COVID-19 patients. (A) Plasma samples from 19 COVID-19 patients and 5 healthy controls were collected. The recombinant SARS-CoV-2 proteins, including NSP2, NSP5, NSP10, NSP15, spike, M, E, and N, were used to determine the corresponding IgG antibody titers in COVID-19 patients using an indirect ELISA. (B) Correlation of OD values (450 nm) and half-lives of proteins in COVID-19 patients. The half-lives of stable NSP2, NSP5, NSP10, NSP15, spike, N, M, and E proteins and their corresponding antibody levels were analyzed. IgG titers are indicated with OD values (at 450 nm).

Journal: mSystems

Article Title: Stability of SARS-CoV-2-Encoded Proteins and Their Antibody Levels Correlate with Interleukin 6 in COVID-19 Patients

doi: 10.1128/msystems.00058-22

Figure Lengend Snippet: SARS-CoV-2 protein-specific antibodies in COVID-19 patients. (A) Plasma samples from 19 COVID-19 patients and 5 healthy controls were collected. The recombinant SARS-CoV-2 proteins, including NSP2, NSP5, NSP10, NSP15, spike, M, E, and N, were used to determine the corresponding IgG antibody titers in COVID-19 patients using an indirect ELISA. (B) Correlation of OD values (450 nm) and half-lives of proteins in COVID-19 patients. The half-lives of stable NSP2, NSP5, NSP10, NSP15, spike, N, M, and E proteins and their corresponding antibody levels were analyzed. IgG titers are indicated with OD values (at 450 nm).

Article Snippet: Recombinant SARS-CoV-2 NSP10 protein (catalog number 10-408) and recombinant SARS-CoV-2 ORF8 protein (catalog number 10-436) were purchased from ProSci (Poway, CA).

Techniques: Clinical Proteomics, Recombinant, Indirect ELISA

Correlation of plasma IL-6 concentrations in COVID-19 patients and SARS-CoV-2 protein-specific antibodies. (A) Correlation between IL-6 and NSP10. (B to J) Correlation between IL-6 and spike, N, M, NSP2, NSP5, NSP15, E, NSP1, and ORF8, respectively.

Journal: mSystems

Article Title: Stability of SARS-CoV-2-Encoded Proteins and Their Antibody Levels Correlate with Interleukin 6 in COVID-19 Patients

doi: 10.1128/msystems.00058-22

Figure Lengend Snippet: Correlation of plasma IL-6 concentrations in COVID-19 patients and SARS-CoV-2 protein-specific antibodies. (A) Correlation between IL-6 and NSP10. (B to J) Correlation between IL-6 and spike, N, M, NSP2, NSP5, NSP15, E, NSP1, and ORF8, respectively.

Article Snippet: Recombinant SARS-CoV-2 NSP10 protein (catalog number 10-408) and recombinant SARS-CoV-2 ORF8 protein (catalog number 10-436) were purchased from ProSci (Poway, CA).

Techniques: Clinical Proteomics

Journal: Cell Reports

Article Title: Coronavirus subverts ER-phagy by hijacking FAM134B and ATL3 into p62 condensates to facilitate viral replication

doi: 10.1016/j.celrep.2023.112286

Figure Lengend Snippet:

Article Snippet: Mouse anti-GFP (AE012), anti-mCherry (AE002), anti-GST (AE001), anti-MBP (AE016), anti-His (AE003), rabbit anti-Flag (AE092), rabbit anti-SARS-CoV-2 ORF3a (A20234), rabbit anti-SARS-CoV-2 ORF8 (A20235) were obtained from ABclonal.

Techniques: Virus, Control, Recombinant, Transfection, Saline, Modification, Protease Inhibitor, Magnetic Beads, Bicinchoninic Acid Protein Assay, shRNA, Software

Journal: Cell Reports

Article Title: Coronavirus subverts ER-phagy by hijacking FAM134B and ATL3 into p62 condensates to facilitate viral replication

doi: 10.1016/j.celrep.2023.112286

Figure Lengend Snippet:

Article Snippet: Mouse anti-GFP (AE012), anti-mCherry (AE002), anti-GST (AE001), anti-MBP (AE016), anti-His (AE003), rabbit anti-Flag (AE092), rabbit anti-SARS-CoV-2 ORF3a (A20234), rabbit anti-SARS-CoV-2 ORF8 (A20235) were obtained from ABclonal.

Techniques: Virus, Control, Recombinant, Transfection, Saline, Modification, Protease Inhibitor, Magnetic Beads, Bicinchoninic Acid Protein Assay, shRNA, Software

Journal: Cell Reports

Article Title: Coronavirus subverts ER-phagy by hijacking FAM134B and ATL3 into p62 condensates to facilitate viral replication

doi: 10.1016/j.celrep.2023.112286

Figure Lengend Snippet:

Article Snippet: Mouse anti-GFP (AE012), anti-mCherry (AE002), anti-GST (AE001), anti-MBP (AE016), anti-His (AE003), rabbit anti-Flag (AE092), rabbit anti-SARS-CoV-2 ORF3a (A20234), rabbit anti-SARS-CoV-2 ORF8 (A20235) were obtained from ABclonal.

Techniques: Virus, Control, Recombinant, Transfection, Saline, Modification, Protease Inhibitor, Magnetic Beads, Bicinchoninic Acid Protein Assay, shRNA, Software

Journal: Cell Reports

Article Title: Coronavirus subverts ER-phagy by hijacking FAM134B and ATL3 into p62 condensates to facilitate viral replication

doi: 10.1016/j.celrep.2023.112286

Figure Lengend Snippet:

Article Snippet: Mouse anti-GFP (AE012), anti-mCherry (AE002), anti-GST (AE001), anti-MBP (AE016), anti-His (AE003), rabbit anti-Flag (AE092), rabbit anti-SARS-CoV-2 ORF3a (A20234), rabbit anti-SARS-CoV-2 ORF8 (A20235) were obtained from ABclonal.

Techniques: Virus, Control, Recombinant, Transfection, Saline, Modification, Protease Inhibitor, Magnetic Beads, Bicinchoninic Acid Protein Assay, shRNA, Software