Journal: PLoS Pathogens

Article Title: PIAS1-mediated SUMOylation of influenza A virus PB2 restricts viral replication and virulence

doi: 10.1371/journal.ppat.1010446

Figure Lengend Snippet: (A, B) Interaction of V5-WSNNP and Myc-PIAS1 in HEK293T cells by using a co-IP assay. HEK293T cells were individually transfected or co-transfected with plasmids expressing V5-WSNNP and Myc-PIAS1. Cell lysates were immunoprecipitated with a mouse anti-V5 mAb (A) or a mouse anti-Myc mAb (B), and subjected to western blotting with a rabbit anti-V5 pAb and a rabbit anti-Myc pAb for the detection of WSNNP and PIAS1, respectively. (C) Interaction of GST-WSNNP and His-PIAS1 by using a GST pull-down assay. His-tagged PIAS1 was expressed in E . coli BL21 (DE3) and purified by using Ni Sepharose Excel resin, and the GST or GST-NP protein was expressed in HEK293T cells and purified by using Glutathione Sepharose 4 Fast Flow. An equal amount of purified PIAS1 was mixed with the Glutathione Sepharose 4 Fast Flow samples that bind GST or GST-NP. After rocking and washing, the mixed samples were separated by SDS-PAGE and stained with Coomassie blue. (D) Interaction of IAV NP and PIAS1 in virus-infected cells. HEK293T cells were transfected for 24 h to express Myc-PIAS1, and were then infected with WSN (H1N1) virus (MOI = 5). At 30 h p.i., cell lysates were immunoprecipitated with a mouse anti-NP mAb, followed by western blotting with a rabbit anti-NP pAb and a rabbit anti-Myc pAb. (E) Co-localization of IAV NP and PIAS1 in A549 cells infected with WSN (H1N1) virus. A549 cells were infected with WSN (H1N1) virus (MOI = 5). At 2, 4, 6, and 8 h p.i., the infected cells were fixed and stained with a mouse anti-NP mAb and a rabbit anti-PIAS1 pAb, followed by incubation with Alexa Fluor 633 goat anti-mouse IgG (H+L) (red) and Alexa Fluor 488 donkey anti-rabbit IgG (H+L) (green). The nuclei were stained with DAPI. (F-H) Co-IP assay to examine the interactions between Myc-PIAS1 and PB2, PB1, and PA of WSN (H1N1) virus in HEK293T cells. HEK293T cells were individually transfected or co-transfected with plasmids expressing WSNPB2, WSNPB1, WSNPA, and Myc-PIAS1. Cell lysates were immunoprecipitated with a mouse anti-Myc mAb and were subjected to western blotting with a rabbit anti-PB2 pAb (F), a rabbit anti-PB1 pAb (G), a rabbit anti-PA pAb (H), and a rabbit anti-Myc pAb (F-H) for the detection of PB2, PB1, PA, and PIAS1, respectively.

Article Snippet: Whole cell lysates were prepared with SDS-PAGE loading buffer (Solarbio Science & Technology, Beijing, China) at 0, 12, and 24 h p.i.

Techniques: Co-Immunoprecipitation Assay, Transfection, Expressing, Immunoprecipitation, Western Blot, Pull Down Assay, Purification, SDS Page, Staining, Infection, Incubation