Journal: The Journal of Biological Chemistry

Article Title: Influenza A virus-induced host caspase and viral PA-X antagonize the antiviral host factor, histone deacetylase 4

doi: 10.1074/jbc.RA119.010650

Figure Lengend Snippet: The IAV-induced phosphorylation of STAT1 and the expression of ISGs: IFITM3, ISG15, and viperin are reduced in HDAC4-depleted cells. A–E, A549 cells were transfected with 1 nm of either control siRNA (CT) or HDAC4 (HD4) siRNA for 72 h. Cells were then infected with PR8 at a m.o.i. of 1.0. A, after 0, 6, 12, and 24 h of infection, cells were harvested and the total cell lysates were prepared. Then, the pSTAT1 (91/84 kDa), tSTAT1 (91/84 kDa), IFITM3 (15 kDa), ISG15 (15 kDa), viperin (42 kDa), PDI, and viral NP polypeptides were detected by Western blotting. Note: the HDAC4, IFITM3, viperin, and ISG15 blots were reused to probe for corresponding PDI, the pSTAT1 blot was reused to probe for tSTAT1 and viperin blot was reused to probe for NP. B–E, the levels of pSTAT1 and IFITM3, ISG15, and viperin in panel A were quantified and normalized with tSTAT1 and PDI, respectively, as described in the legend to Fig. 1B. Then, the normalized levels of pSTAT1 (B), IFITM3 (C), ISG15 (D), and viperin (E) at each time point in control siRNA-transfected cells were considered 100% to compare their levels in HDAC4 siRNA-transfected cells at the respective time points. Error bars represent the mean ± S.E. of three independent experiments. The asterisks represent p values mentioned in the text calculated by ANOVA, and indicate the significant differences in means. MW, molecular weight.

Article Snippet: Subsequently, membranes were probed with the rabbit anti-HDAC4 (1:1,000; D15C3, Cell Signaling), rabbit anti-β-actin (1:10,000; ab8227, Abcam), goat anti-NP (1:10,000; G150, kindly provided by Richard Webby, St. Jude Children's Research Hospital), mouse anti-NP (1:10,000; NR-19868, obtained through BEI Resources, NIAID, NIH), goat anti-HA (1:1,000; G57, kindly provided by Richard Webby), rabbit anti-PDI (1:10,000; P7496, Sigma-Aldrich), rabbit anti-caspase 3 (1:1,000; 8G10, Cell Signaling), mouse anti-pSTAT1 (1:1,000; pY701, BD Bioscience), mouse anti-STAT1 (1:1,000; 610185, BD Bioscience), rabbit anti-viperin (1:1,000; D5T2X, Cell Signaling), rabbit anti-IFITM3 (1:1000; ab15592, Abcam), or rabbit anti-ISG15 (1:1,000; F-9, Cell Signaling) followed by horseradish peroxidase-conjugated anti-rabbit, anti-mouse, or anti-goat IgG antibody (1:2,000 or 1:5,000, Life Technologies).

Techniques: Expressing, Transfection, Infection, Western Blot, Molecular Weight