Journal: PLOS Pathogens

Article Title: The African swine fever virus p22 inhibits the JAK-STAT signaling pathway by promoting the TAX1BP1-mediated degradation of the type I interferon receptor

doi: 10.1371/journal.ppat.1013319

Figure Lengend Snippet: (A) HEK293T cells were cotransfected with the plasmids expressing HA-NBR1, -CALCOCO2, -TAX1BP1, -p62, -OPTN, -NIX, or -TOLLIP, and Flag-p22, and then lysed for co-IP using the anti-Flag MAb, followed by western blotting analysis using the indicated antibodies. (B) HEK293T cells were cotransfected with the plasmids expressing Flag-p22 and HA-TAX1BP1, and then lysed for Co-IP using anti-Flag or anti-HA MAb, followed by western blotting analysis using the indicated antibodies. (C) HEK293T cells were transfected with pHA-TAX1BP1, harvested and lysed at 24 hpt, and the supernatants were incubated with the prokaryotically expressed GST-p22 protein bound to the GST beads. The formed complexes were analyzed using western blotting and the indicated antibodies. (D) IBRS-2 cells were cotransfected with the plasmids expressing HA-TAX1BP1 and Flag-p22, and then fixed with 4% paraformaldehyde. TAX1BP1 and p22 were immunoblotted using anti-HA and anti-Flag antibodies, respectively. The nuclei were stained with DAPI and subjected to confocal microscopy. The colocalization of p22 and TAX1BP1 was analyzed using the Coloc2 tool in ImageJ, and it is shown as Pearson’s R value (R). Scale bar = 10 μm. (E) PAMs were infected with ASFV-WT or ASFV-ΔKP177R (MOI = 3). At 24 hpi, the cells were lysed for co-IP using anti-p22 MAb, followed by western blotting analysis using the indicated antibodies. (F) HEK293T cells were transfected with different amounts of the HA-TAX1BP1-expressing plasmid with pFlag-KP177R, followed by western blotting analysis using the indicated antibodies at 24 hpt. (G) The TAX1BP1-knockout or WT HEK293T cells were transfected with different amounts of the Flag-p22-expressing plasmid or pRK (Vec), followed by western blotting analysis using the indicated antibodies at 24 hpt. The densitometric analysis of the protein expression levels was performed using the ImageJ software.

Article Snippet: Rabbit anti-HA tag PAb (51064–2-AP), mouse anti-HA tag MAb (66006–2-Ig), mouse anti-Flag tag MAb (66008–4-Ig), rabbit anti-Myc tag PAb (16286–1-AP), mouse anti-GST tag MAb (66001–2-Ig), rabbit anti-TAX1BP1 PAb (14424–1-AP), HRP-conjugated goat anti-rabbit IgG(H + L) (SA00001–2), and HRP-conjugated goat anti-mouse IgG(H + L) (SA00001–1) were purchased from Proteintech (Chicago, USA).

Techniques: Expressing, Co-Immunoprecipitation Assay, Western Blot, Transfection, Incubation, Staining, Confocal Microscopy, Infection, Plasmid Preparation, Knock-Out, Software