antibody rabbit polyclonal anti-c-gal8  (Thermo Fisher)

Journal: Cell Reports

Article Title: Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CL pro substrate degradome

doi: 10.1016/j.celrep.2021.109892

Figure Lengend Snippet: 3CL pro disrupts galectin-8 binding to Spike in antiviral-autophagy (A) Immunoblot of human galectin-8 (Gal8) in BEAS-2B cells in response to IFN- α , IFN-β, or vehicle. One way ANOVA and Dunnett's posthoc test (mean ± SD, n = 3 each, *** p ≤ 0.001, * p ≤ 0.05). (B) MALDI-TOF-MS of intact versus 3CL pro -cleaved synthetic Gal8 P4–P4’ peptide. (C) SDS-PAGE and Edman sequencing of Gal8 incubated with 3CL pro +/− inhibitor GC376, or 3CL pro C145A (1:5 mol/mol, E:S). (D) Structural model of Gal8 docked onto 3CL pro . 3CL pro cleavage site identified by the neo-N-terminal peptide (red) in 9/9 independent TAILS analyses. (E) Gal8 immunoblots of lysates from primary HAECs incubated with 3CL pro or 3CL pro -C145A (1:200 w/w, E:S) for 18 h, 37°C (N = 5). (F) Gal8 immunoblot of infected Calu-3 cells at 24 (n = 4) and 48 (n = 4) hpi (MOI 1.0, mock n = 3). β-actin and β-tubulin loading controls. (G) ELISA of SARS-CoV-2 Spike S1 protein binding intact Gal8 or 3CL pro -cleaved (ΔGal8) (mean ± SD, n = 2, N = 2, ∗∗∗∗ p ≤ 0.0001, ∗∗ p ≤ 0.01, ns p > 0.05, two-way ANOVA with Šídák’s multiple comparison test). (H) Immunoprecipitation (IP) by α-FLAG agarose-beads of HeLa cell lysates co-transfected with GFP-NDP52, WT-Gal8-FLAG or C-Gal8 (159-317)-FLAG. (I) Intact or cleaved Gal8 binding immobilized NDP52 detected with anti-C-Gal8 antibody. Student's t test, (mean ± SD, n = 3, N = 2, ns p > 0.05). (J) NDP52 binding Spike S1-associated intact or cleaved ΔGal8 (mean ± SD, n = 2, N = 2, ∗∗∗∗ p ≤ 0.0001, ∗∗∗ p ≤ 0.001, ∗ p ≤ 0.05, ns p > 0.05, two-way ANOVA with Šídák’s multiple comparison test). (K) Confocal microscopy of NDP52 and FLAG immunofluorescence after osmotic shock of HEK293 cells transfected with FLAG-tagged (WT) Gal8, N-Gal8 (1-158), or C-Gal8 (159-317). Scale bar, 20 μm. Quantification of NDP52 and Gal8-positive puncta, mean ± SD, n = 30, N = 5, ∗∗∗∗ p ≤ 0.0001, ns p > 0.05, one-way ANOVA with Tukey’s multiple comparison test. (L) Human lung tissue sections from normal subjects (N = 3) and post-mortem COVID-19 patients (N = 4) stained with hematoxylin and eosin (H&E), DAPI (blue) and immunofluorescence on the same sections for Gal8 (green), NDP52 (red), Spike (magenta) and merged image (orange). Scale bar, 50 μm. (M) Numbers of immunopositive cells for Gal8, NDP52 and Gal8 colocalized with NDP52 in healthy versus COVID-19 lung tissue. Total cell count distribution shown as a scatter dot plot, bar = median. See also Figure S6 .

Article Snippet: rabbit polyclonal anti-C-Gal8 antibody (1:500) , Thermo Fisher Scientific , Cat# PA5-19729; RRID: AB_10984508.

Techniques: Binding Assay, Western Blot, SDS Page, Sequencing, Incubation, Infection, Enzyme-linked Immunosorbent Assay, Protein Binding, Comparison, Immunoprecipitation, Transfection, Confocal Microscopy, Immunofluorescence, Staining, Cell Counting

Journal: Cell Reports

Article Title: Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CL pro substrate degradome

doi: 10.1016/j.celrep.2021.109892

Figure Lengend Snippet:

Article Snippet: rabbit polyclonal anti-C-Gal8 antibody (1:500) , Thermo Fisher Scientific , Cat# PA5-19729; RRID: AB_10984508.

Techniques: Virus, Recombinant, Sequencing, Fluorescence, Modification, Protease Inhibitor, Polymer, Staining, Blocking Assay, Binding Assay, Activity Assay, Western Blot, Synthesized, Software, Control, Targeted Proteomics, Microscopy, Spectrophotometry, Mass Spectrometry

Journal: Cell Reports

Article Title: Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CL pro substrate degradome

doi: 10.1016/j.celrep.2021.109892

Figure Lengend Snippet: 3CL pro disrupts galectin-8 binding to Spike in antiviral-autophagy (A) Immunoblot of human galectin-8 (Gal8) in BEAS-2B cells in response to IFN- α , IFN-β, or vehicle. One way ANOVA and Dunnett's posthoc test (mean ± SD, n = 3 each, *** p ≤ 0.001, * p ≤ 0.05). (B) MALDI-TOF-MS of intact versus 3CL pro -cleaved synthetic Gal8 P4–P4’ peptide. (C) SDS-PAGE and Edman sequencing of Gal8 incubated with 3CL pro +/− inhibitor GC376, or 3CL pro C145A (1:5 mol/mol, E:S). (D) Structural model of Gal8 docked onto 3CL pro . 3CL pro cleavage site identified by the neo-N-terminal peptide (red) in 9/9 independent TAILS analyses. (E) Gal8 immunoblots of lysates from primary HAECs incubated with 3CL pro or 3CL pro -C145A (1:200 w/w, E:S) for 18 h, 37°C (N = 5). (F) Gal8 immunoblot of infected Calu-3 cells at 24 (n = 4) and 48 (n = 4) hpi (MOI 1.0, mock n = 3). β-actin and β-tubulin loading controls. (G) ELISA of SARS-CoV-2 Spike S1 protein binding intact Gal8 or 3CL pro -cleaved (ΔGal8) (mean ± SD, n = 2, N = 2, ∗∗∗∗ p ≤ 0.0001, ∗∗ p ≤ 0.01, ns p > 0.05, two-way ANOVA with Šídák’s multiple comparison test). (H) Immunoprecipitation (IP) by α-FLAG agarose-beads of HeLa cell lysates co-transfected with GFP-NDP52, WT-Gal8-FLAG or C-Gal8 (159-317)-FLAG. (I) Intact or cleaved Gal8 binding immobilized NDP52 detected with anti-C-Gal8 antibody. Student's t test, (mean ± SD, n = 3, N = 2, ns p > 0.05). (J) NDP52 binding Spike S1-associated intact or cleaved ΔGal8 (mean ± SD, n = 2, N = 2, ∗∗∗∗ p ≤ 0.0001, ∗∗∗ p ≤ 0.001, ∗ p ≤ 0.05, ns p > 0.05, two-way ANOVA with Šídák’s multiple comparison test). (K) Confocal microscopy of NDP52 and FLAG immunofluorescence after osmotic shock of HEK293 cells transfected with FLAG-tagged (WT) Gal8, N-Gal8 (1-158), or C-Gal8 (159-317). Scale bar, 20 μm. Quantification of NDP52 and Gal8-positive puncta, mean ± SD, n = 30, N = 5, ∗∗∗∗ p ≤ 0.0001, ns p > 0.05, one-way ANOVA with Tukey’s multiple comparison test. (L) Human lung tissue sections from normal subjects (N = 3) and post-mortem COVID-19 patients (N = 4) stained with hematoxylin and eosin (H&E), DAPI (blue) and immunofluorescence on the same sections for Gal8 (green), NDP52 (red), Spike (magenta) and merged image (orange). Scale bar, 50 μm. (M) Numbers of immunopositive cells for Gal8, NDP52 and Gal8 colocalized with NDP52 in healthy versus COVID-19 lung tissue. Total cell count distribution shown as a scatter dot plot, bar = median. See also Figure S6 .

Article Snippet: To detect the bound protein, mouse monoclonal anti-His-tag antibody (1:1,000, Cedarlane Labs, CLH101AP) (for Spike S1 glycoprotein) or goat polyclonal anti-Gal8 antibody (1:200, R&D Systems, AF1305, RRID: AB_2137229 ) and rabbit polyclonal anti-C-Gal8 antibody (1:500, Thermo Fisher Scientific, PA5-19729, RRID: AB_10984508 ) (for galectin-8) were added as appropriate and incubated for 1 h at RT.

Techniques: Binding Assay, Western Blot, SDS Page, Sequencing, Incubation, Infection, Enzyme-linked Immunosorbent Assay, Protein Binding, Immunoprecipitation, Transfection, Confocal Microscopy, Immunofluorescence, Staining, Cell Counting

Journal: Cell Reports

Article Title: Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CL pro substrate degradome

doi: 10.1016/j.celrep.2021.109892

Figure Lengend Snippet:

Article Snippet: To detect the bound protein, mouse monoclonal anti-His-tag antibody (1:1,000, Cedarlane Labs, CLH101AP) (for Spike S1 glycoprotein) or goat polyclonal anti-Gal8 antibody (1:200, R&D Systems, AF1305, RRID: AB_2137229 ) and rabbit polyclonal anti-C-Gal8 antibody (1:500, Thermo Fisher Scientific, PA5-19729, RRID: AB_10984508 ) (for galectin-8) were added as appropriate and incubated for 1 h at RT.

Techniques: Recombinant, Sequencing, Fluorescence, Modification, Protease Inhibitor, Staining, Blocking Assay, Binding Assay, Activity Assay, Western Blot, Synthesized, Software, Microscopy, Spectrophotometry, Mass Spectrometry