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: Figure 5. 3CLpro disrupts galectin-8 binding to Spike in antiviral-autophagy (A) Immunoblot of human galectin-8 (Gal8) in BEAS-2B cells in response to IFN-a, IFN-b, 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 3CLpro-cleaved synthetic Gal8 P4–P4’ peptide. (C) SDS-PAGE and Edman sequencing of Gal8 incubated with 3CLpro+/ inhibitor GC376, or 3CLpro C145A (1:5 mol/mol, E:S). (D) Structural model of Gal8 docked onto 3CLpro. 3CLpro 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 3CLpro or 3CLpro-C145A (1:200 w/w, E:S) for 18 h, 37C (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). b-actin and b-tubulin loading controls. (G) ELISA of SARS-CoV-2 Spike S1 protein binding intact Gal8 or 3CLpro-cleaved (DGal8) (mean ± SD, n = 2, N = 2, ****p % 0.0001, **p % 0.01, ns p > 0.05, two- way ANOVA with Sı´da´ k’s multiple comparison test). (legend continued on next page)
Article Snippet: The primary antibodies and dilutions used were: mousemonoclonal anti-SARS-CoV-2 nucleocapsid antibody (1:1,000, Invitrogen, MA5-29981, RRID: AB_2785780); rabbit anti-SARS-CoV-1 3CLpro antibody (1:2000, Rockland, 200-401-A51, RRID: AB_828457); rabbit polyclonal anti-RPAP1 antibody (1:1,000, Proteintech, 15138-1-AP, RRID: AB_2301137); mouse monoclonal anti-PTBP1 antibody (1:500, Biolegend, 630101, 3H7, RRID: AB_2171285); rabbit polyclonal anti-MAP4K5 antibody (1:1,000, Cusabio, CSBPA013440DSR2HU, RRID: AB_2892084); rabbit polyclonal anti-CREB1 antibody (1:1,000, Abclonal, A11989, RRID: AB_2758916); rabbit polyclonal anti-YAP1 antibody (1:1,000, Abclonal, A11430, RRID: AB_2758556); rabbit polyclonal anti-FYCO1 antibody (1:1,000, Cusabio, CSB-PA866262LA01HU, RRID: AB_2892085); rabbit polyclonal anti-FAF1 antibody (1:1,000, Abclonal, A2921, RRID: AB_2764739); goat polyclonal anti-Gal8 antibody (1:400, R&D Systems, AF1305, RRID: AB_2137229); rabbit polyclonal anti-KPNA3 (IMA4) antibody (1:1,000, Abclonal, A8347, RRID: AB_2770124); rabbit polyclonal anti-NUP107 antibody (1:1,000, Abclonal, A13110, RRID: AB_2759959); mouse monoclonal anti-IRS2 antibody (1:300, R&D Systems, MAB6347, 676415, RRID: AB_10992928); mouse monoclonal anti-FLAG M2 antibody (1:10,000, Sigma, F3165, RRID: AB_259529); mouse monoclonal antib-tubulin antibody (1:2000, AbLab, 21-0018-00, clone BT7R); mouse monoclonal anti-b-actin antibody (1:1,000, Abcam, ab8226, RRID: AB_306371); rabbit monoclonal anti-b-actin antibody (1:200, Abcam, ab115777, RRID: AB_10899528).
Techniques: Binding Assay, Western Blot, SDS Page, Sequencing, Incubation, Infection, Enzyme-linked Immunosorbent Assay, Protein Binding, Comparison