Journal: Viruses

Article Title: Ivermectin Inhibits HBV Entry into the Nucleus by Suppressing KPNA2

doi: 10.3390/v14112468

Figure Lengend Snippet: PCR primer and probe sequences.

Article Snippet: KPNA3 , Bethyl Laboratories , A301-626A , Rabbit , 1:500.

Techniques: Sequencing

Journal: Viruses

Article Title: Ivermectin Inhibits HBV Entry into the Nucleus by Suppressing KPNA2

doi: 10.3390/v14112468

Figure Lengend Snippet: Primary antibodies for Western blotting.

Article Snippet: KPNA3 , Bethyl Laboratories , A301-626A , Rabbit , 1:500.

Techniques: Western Blot

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: Characterization of 3CL pro cleavage specificity (A and B) MALDI-TOF-MS spectra of synthetic peptides spanning P4–P4’ of protein cleavage sites after incubation with 3CL pro (1:20 molar ratio, E:S). Product generation (red) and substrate consumption (black) were calculated as the peak area normalized to the total peak area in the spectrum. Apparent (app) k cat / K M values for 1 μM 3CL pro to convert 50% of substrate in 5, 15, 30, 60, 120 or 240 min are listed alongside bins of 4 peptides that share similar kinetic values arranged on a row-by-row basis. P4–P4’ sequence alignment using the Shapley color scale. Green protein names had cut sites identified by Edman sequencing of recombinant substrate digests. Boxed peptides, no cleavage. (C–I) Structures of the highest-ranked of 50,000 models of the active site of 3CL pro protomer 1 (PDB: 6XHM ) docked with P4–P4’ peptides from six 3CL pro substrates exhibiting a range of app k cat / K M values (circled in B). (C) 3CL pro dimer. Protomer 1, gray surface or green ribbons with catalytic Cys 145 shown. Protomer 2, orange surface with Ser 1 shown. Docking models with P4–P4’ peptide of: (C and D) RPAP1 (I_sc = −31.7), (E) IMA4 (I_sc = −30.6), (F) PTBP1 (I_sc = −31.7), (G) RBM15 (I_sc = −39.65), (H) MAP4K5 (I_sc = −28.1), and (I) CREB1 (I_sc = −30.3). Blue and red sticks, P and P’ amino acid residues, respectively. Yellow dashed sticks, hydrogen-bonds. See also Figure S2 .

Article Snippet: The primary antibodies and dilutions used were: mouse monoclonal anti-SARS-CoV-2 nucleocapsid antibody (1:1,000, Invitrogen, MA5-29981, RRID: AB_2785780 ); rabbit anti-SARS-CoV-1 3CL pro 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, CSB-PA013440DSR2HU, 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 anti-β-tubulin antibody (1:2000, AbLab, 21-0018-00, clone BT7R); mouse monoclonal anti-β-actin antibody (1:1,000, Abcam, ab8226, RRID: AB_306371 ); rabbit monoclonal anti-β-actin antibody (1:200, Abcam, ab115777, RRID: AB_10899528 ).

Techniques: Incubation, Sequencing, Recombinant

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: The primary antibodies and dilutions used were: mouse monoclonal anti-SARS-CoV-2 nucleocapsid antibody (1:1,000, Invitrogen, MA5-29981, RRID: AB_2785780 ); rabbit anti-SARS-CoV-1 3CL pro 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, CSB-PA013440DSR2HU, 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 anti-β-tubulin antibody (1:2000, AbLab, 21-0018-00, clone BT7R); mouse monoclonal anti-β-actin antibody (1:1,000, Abcam, ab8226, RRID: AB_306371 ); rabbit monoclonal anti-β-actin antibody (1:200, Abcam, ab115777, RRID: AB_10899528 ).

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

Journal: Journal of Medical Genetics

Article Title: CIC de novo loss of function variants contribute to cerebral folate deficiency by downregulating FOLR1 expression

doi: 10.1136/jmedgenet-2020-106987

Figure Lengend Snippet: Functional analysis of the CIC variant. (A) Protein amino acid locus of variant p.R353X, which is predicted to create premature stop codons. Lollipop plot shows all four CIC mutants identified in our CFD cohort. (B) Localisation of GFP tagged CIC variants in stably transfected HeLa cells. HeLa cells were transduced with lentiviral particles coding for GFP, GFP-CIC wildtype or GFP-CIC p.R353X. Subcellular localisation was analysed by microscopy for GFP and endogenous CIC. Nuclei were stained with DAPI, Bars, 20 µm. (C) Analysis of CIC p.R353X variant effect on interaction between CIC and KPNA3. Mutant CIC abolished interaction between CIC and KPNA3. (D) Analysis of CIC p.R353X variant effect on interaction between CIC and ATXN1. CFD, cerebral folate deficiency.

Article Snippet: KPNA3 antibody was purchased from Bethyl Laboratories (Montgomery, Texas, USA).

Techniques: Functional Assay, Variant Assay, Stable Transfection, Transfection, Transduction, Microscopy, Staining, Mutagenesis