Journal: Nature Communications
Article Title: Human ANP32A/B are SUMOylated and utilized by avian influenza virus NS2 protein to overcome species-specific restriction
doi: 10.1038/s41467-024-55034-y
Figure Lengend Snippet: a Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated constructs on H9N2 vPol activity. Values above K0 were statistically analyzed using one-way ANOVA followed by a Dunnett’s multiple comparisons test against huANP32A-K0 (error bars represent the mean ± SD of n = 4 independent biological replicates). b Schematic representation of the huANP32A-K0 mutants generated. Western blots demonstrate comparable expression levels for all indicated constructs. c , d Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated huANP32A-K0 constructs on the H9N2 ( c ) and H7N9 (PB2-627E) ( d ) vPol activity in the presence of NS2. e The replication kinetics of the avian H9N2 virus were evaluated in control MDCK-TKO cells or those stably expressing huANP32A-K0-Flag or its mutant (MOI = 0.01), with viral titers determined at the indicated time points. f Western blot analysis of MDCK-TKO cells stably reconstituted with the indicated huANP32A-K0-Flag constructs or empty vector. g Ni 2+ -NTA bead affinity pull-down assay showing that the K68 and K153 sites of huANP32A can be modified by SUMO1. h Co-IP experiments showing that the K68R/K153R mutations in huANP32A suppress its interaction with H9N2-NS2. i , j Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated huANP32A constructs on the H9N2 ( i ) and H7N9 (PB2-627E) ( j ) vPol activity. Statistical analyses were performed relative to huANP32A. k The replication kinetics of the avian H9N2 virus were evaluated in control MDCK-TKO cells or those stably expressing huANP32A-Flag or huANP32A-K68R/K153R-Flag (MOI = 0.01), with viral titers determined at the indicated time points. l Western blot analysis of control MDCK-TKO cells or those stably expressing indicated huANP32A-Flag constructs. In ( c to e ) and ( i to k ), error bars represent the mean ± SD of n = 3 independent biological replicates; NS, not significant; Statistical significance was determined by two-tailed unpaired t-test ( c , d , i and j ) or two-way ANOVA ( k ). In ( g , h ), experiments were independently repeated three times with consistent results. Source data are provided as a Source Data file.
Article Snippet: Western blot analysis was conducted following established protocols using the following antibodies: rabbit anti-Flag (Sigma, F7425), mouse anti-Flag (Sigma, F1804), rabbit anti-HA (Sigma, H6908), rabbit anti-ACTB (Abclonal, AC026), mouse anti-ACTB (Abclonal, AC004), rabbit anti-Myc (Abclonal, AE070), rabbit anti-SUMO1 (Abclonal, A19121), rabbit anti-SUMO2/3 (Abclonal, A5066), rabbit anti-SENP1(Abcam, ab108981), mouse anti-His (Proteintech, 66005-1-Ig), rabbit anti-V5 (Proteintech, 14440-1-AP), rabbit anti-SENP1 (Proteintech, 25349-1-AP), rabbit anti-PIAS2 (Proteintech, 16074-1-AP), rabbit anti-GST (Proteintech, 10000-0-AP), rabbit anti-ANP32A (Proteintech,15810-1-AP), rabbit anti-ANP32B (Proteintech, 10843-1-AP), mouse anti-ANP32A (Proteintech, 67687-1-Ig), mouse anti-ANP32B (Proteintech, 66160-1-Ig), rabbit anti-influenza A virus NS2 (GeneTex, GTX125953), rabbit anti-influenza B virus NP (GeneTex, GTX128538), rabbit anti-influenza A virus PB2 (GeneTex, GTX125926), rabbit anti-influenza A virus PA (GeneTex, GTX118991), mouse anti-influenza A virus PA (prepared in our laboratory, 1:5000 for WB), mouse anti-influenza A virus NP (prepared in our laboratory, 1:5000 for WB), Biotin-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00004-2), DyLight 800-labeled Anti-Mouse IgG (H + L) Antibody (KPL, 5230-0415), DyLight 680-labeled Anti-Rabbit IgG (H + L) Antibody (KPL, 5230-0402) and DyLight™ 680-labeled streptavidin (KPL, 5270-0025).
Techniques: Construct, Activity Assay, Generated, Western Blot, Expressing, Virus, Control, Stable Transfection, Mutagenesis, Plasmid Preparation, Pull Down Assay, Modification, Co-Immunoprecipitation Assay, Two Tailed Test