rabbit polyclonal anti-a3g  (Santa Cruz Biotechnology)

Journal: Nature Communications

Article Title: Crystal structure of the catalytic domain of HIV-1 restriction factor APOBEC3G in complex with ssDNA

doi: 10.1038/s41467-018-04872-8

Figure Lengend Snippet: Generation of a hyperactive A3G-CTD variant. a Amino acid residues of wild-type A3G-CTD and the catalytically hyper-active variant, namely CTD2, are aligned. 2K3A substitutions (L234K, C243A, F310K, C321A, and C356A) are colored green, and the additional five substitutions (P200A, N236A, P247K, Q318K, and Q322A) are colored orange. b Real-time NMR deamination assay. The product (5ʹ-AATCCdeoxy-UAAA) concentration as a function of reaction time is plotted for CTD2 (red dots) and wild-type A3G-CTD (black dots) deamination at pH 7.5 with 200 nM protein and 200 µM 5ʹ-AATCCCAAA substrate. For CTD2, the first reaction reached completion within 5 h, and the second deamination (5ʹ-AATCCdeoxy-UAAA to 5ʹ-AATCdeoxy-Udeoxy-UAAA) started, thus decreasing the concentration of the initial product. c EMSA for binding of CTD2* to the 9nt ssDNA (5′-AATCCCAAA-6-FAM). CTD2*-DNA indicates position of the CTD2*–ssDNA complex, and free DNA indicates the position of protein-free ssDNA. Fluorescent-unprobed 9nt polyA (5′-AAAAAAAAA) or fluorescent-unprobed 9nt ssDNA (5′-AATCCCAAA) were added with incremental amounts in lanes 3 (25 nM), 4 (250 nM), and 5 (2500 nM) or 7 (25 nM), 8 (250 nM), and 9 (2500 nM), respectively. Uncropped gel is shown in Supplementary Fig. . d Each dot represents microscale thermophoresis (MST) measurement of a mixture containing fluorescent-labeled CTD2* (50 nM) and the 9nt ssDNA 5ʹ-AATCCCAAA at various concentrations including 0.12 µM, 0.24 µM, 0.48 µM, 0.97 µM, 1.95 µM, 3.90 µM, 7.81 µM, 15.62 µM, 31.25 µM, 62.5 µM, 125 µM, 250 µM, 500 µM, 1 mM, 2 mM, and 4 mM. Three independent MST experiments were performed, and bars of data points represent standard error of n = 3 measurements

Article Snippet: Proteins were detected with primary antibodies as follows: FLAG-A3G or FLAG-NTD-CTD2 (rabbit anti-FLAG polyclonal antibody, 1:5000 dilution, Sigma #F7425); Vif-HA (mouse anti-HA monoclonal antibody, 1:5000 dilution, Sigma #H3663); α-tubulin (mouse anti-α-tubulin antibody, 1:10,000 dilution, Sigma #T9026); Antibody against HIV-1 p24 (monoclonal, 1:10,000 dilution) was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 p24 Gag Monoclonal (#24-3) from Dr. Michael Malim (catalog #6458) .

Techniques: Variant Assay, Concentration Assay, Binding Assay, Microscale Thermophoresis, Labeling

Journal: Nature Communications

Article Title: Crystal structure of the catalytic domain of HIV-1 restriction factor APOBEC3G in complex with ssDNA

doi: 10.1038/s41467-018-04872-8

Figure Lengend Snippet: Antiviral restriction activity of FLAG-NTD-CTD2. a Representative western blot showing 293T cells co-transfected with increasing amounts of wild-type FLAG-A3G or FLAG- NTD-CTD2 (21, 42, 84, 170 ng; black triangles), HDV-EGFP, and VSV-G in the presence or absence of Vif-HA. Percent A3G expression is shown for each respective lane. Single-cycle infectivity of Vif+ ( b ) and Vif− ( c ) HDV-EGFP virus prepared in the presence of increasing amounts of FLAG-A3G (black bars) or FLAG-NTD-CTD2 (light gray bars) assayed in TZM-bl target cells. Data reflects the average relative light units (RLU) normalized to the no A3G control. Error bars represent the standard deviation for three independent experiments

Article Snippet: Proteins were detected with primary antibodies as follows: FLAG-A3G or FLAG-NTD-CTD2 (rabbit anti-FLAG polyclonal antibody, 1:5000 dilution, Sigma #F7425); Vif-HA (mouse anti-HA monoclonal antibody, 1:5000 dilution, Sigma #H3663); α-tubulin (mouse anti-α-tubulin antibody, 1:10,000 dilution, Sigma #T9026); Antibody against HIV-1 p24 (monoclonal, 1:10,000 dilution) was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 p24 Gag Monoclonal (#24-3) from Dr. Michael Malim (catalog #6458) .

Techniques: Activity Assay, Western Blot, Transfection, Expressing, Infection, Standard Deviation

Journal: Nature Communications

Article Title: Crystal structure of the catalytic domain of HIV-1 restriction factor APOBEC3G in complex with ssDNA

doi: 10.1038/s41467-018-04872-8

Figure Lengend Snippet: Structure of CTD2* in complex with ssDNA. a The asymmetric unit contains one protein (yellow) and one ssDNA (blue) molecule. A 2Fo–Fc electron density map contoured at 1 σ is shown in cyan around the ssDNA. Zn 2+ ion is colored purple. N and C indicate the N- and C-terminal ends of the protein, respectively. b An enlarged view shows interactions between the 5ʹ-TCCCA target sequence and the protein. Protein is colored yellow, 5ʹ-TCCCA is blue, and amino acid sidechains interacting with DNA are shown as sticks. c – f Enlarged views show interactions between T −3 , C −2 , C −1 , C 0 , or A +1 and protein. C, N, and O atoms are colored yellow, blue, and red, respectively, for amino acid residues of the protein. Atoms in nucleotides are colored blue, navy blue, red, and orange for C, N, O, and P, respectively. Water molecules are shown as red spheres, and Zn 2+ is shown as a purple sphere. Dotted lines indicate hydrogen bonds. In ( c ), the double arrow-headed line points to the neighboring backbone phosphorous atoms of C −1 and C −2 . In ( d ), sidechains of F289 and Q318K are not shown. g Summary of the interactions between CTD2* and nucleotides in the 5ʹ-TCCCA target sequence

Article Snippet: Proteins were detected with primary antibodies as follows: FLAG-A3G or FLAG-NTD-CTD2 (rabbit anti-FLAG polyclonal antibody, 1:5000 dilution, Sigma #F7425); Vif-HA (mouse anti-HA monoclonal antibody, 1:5000 dilution, Sigma #H3663); α-tubulin (mouse anti-α-tubulin antibody, 1:10,000 dilution, Sigma #T9026); Antibody against HIV-1 p24 (monoclonal, 1:10,000 dilution) was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 p24 Gag Monoclonal (#24-3) from Dr. Michael Malim (catalog #6458) .

Techniques: Sequencing

Journal: Nature Communications

Article Title: Crystal structure of the catalytic domain of HIV-1 restriction factor APOBEC3G in complex with ssDNA

doi: 10.1038/s41467-018-04872-8

Figure Lengend Snippet: Comparison of CTD2* and A3A recognition of the nucleotide at −1 position. a Interaction of cytidine at −1 position (C −1 , blue) with residues of CTD2* (yellow) in the CTD2*–ssDNA complex (this study). b Interaction of thymidine at −1 position (T −1 , cyan) with residues of A3A (gray) in the A3A–ssDNA complex (PDB ID: 5KEG). c Superimposition of the CTD2*–ssDNA and A3A–ssDNA complexes showing nucleotides at the −1 position and their interacting residues from both structures

Article Snippet: Proteins were detected with primary antibodies as follows: FLAG-A3G or FLAG-NTD-CTD2 (rabbit anti-FLAG polyclonal antibody, 1:5000 dilution, Sigma #F7425); Vif-HA (mouse anti-HA monoclonal antibody, 1:5000 dilution, Sigma #H3663); α-tubulin (mouse anti-α-tubulin antibody, 1:10,000 dilution, Sigma #T9026); Antibody against HIV-1 p24 (monoclonal, 1:10,000 dilution) was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 p24 Gag Monoclonal (#24-3) from Dr. Michael Malim (catalog #6458) .

Techniques:

Journal: Nature Communications

Article Title: Crystal structure of the catalytic domain of HIV-1 restriction factor APOBEC3G in complex with ssDNA

doi: 10.1038/s41467-018-04872-8

Figure Lengend Snippet: Comparison of structures of CTD2* with apo-CTD-2K3A. Proteins are in the same orientation in all three figures. a Superimposed yellow and gray cartoons show structural features of CTD2* (this study) and CTD-2K3A (PDB ID# 3IR2), respectively. W211, R213, H216, Y315, D316, and D317 sidechains of CTD2* are shown in sticks. Zn 2+ ion is shown as a purple sphere. ssDNA is not shown. b An enlarged view of ( a ) that shows the repositioning of the critical residues W211, R213, and H216 of loop1, and Y315, D316, and D317 of loop7. Double headed arrows point to positions of Cα atoms of W211 and D317. c Surface representation of ssDNA-bound CTD2* (this study). Locations of the loop1 and loop7 residues are labeled except D317 because D317 is not seen on the surface. The 5ʹ-TCCCA target sequence is shown as sticks, and C, N, O, and P atoms are colored blue, dark blue, red, and orange, respectively

Article Snippet: Proteins were detected with primary antibodies as follows: FLAG-A3G or FLAG-NTD-CTD2 (rabbit anti-FLAG polyclonal antibody, 1:5000 dilution, Sigma #F7425); Vif-HA (mouse anti-HA monoclonal antibody, 1:5000 dilution, Sigma #H3663); α-tubulin (mouse anti-α-tubulin antibody, 1:10,000 dilution, Sigma #T9026); Antibody against HIV-1 p24 (monoclonal, 1:10,000 dilution) was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 p24 Gag Monoclonal (#24-3) from Dr. Michael Malim (catalog #6458) .

Techniques: Labeling, Sequencing