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. 2019 Nov 22;294(52):20207–20221. doi: 10.1074/jbc.RA119.010650

Figure 3.

Figure 3.

IAV down-regulates the level of HDAC4 polypeptide in epithelial cells. MDCK (A and C) or A549 (B and D) cells were infected with IAV PR8 (A and B), CA09 (C), or WSN (D) strains at a m.o.i. of 0.5 or 5.0. After 24 h, the uninfected (UNI) and infected (0.5, 5.0) cells were harvested, total cell lysates were prepared, and the HDAC4, actin (42 kDa), and viral NP polypeptides were detected by Western blotting. E, MDCK cells were infected with live (INF) or UV-irradiated (INFUV) PR8 at an m.o.i. of 0.5. After 24 h, total cell lysates were prepared, and the HDAC4, actin, and NP polypeptides were detected by Western blotting. Note: the HDAC4 blots were reused to probe for actin. F, the level of HDAC4 polypeptide in panels A and B blots were quantified and normalized as described in the legend to Fig. 1B. Then, the normalized level of HDAC4 polypeptide in uninfected cells (UNI) was considered 100% to compare its level in cells infected (INF) with 0.5 and 5.0 m.o.i. G, MDCK cells were infected with PR8 at a m.o.i. of 0.5, and harvested after 2, 6, 12, and 24 h of infection. The total cell lysates were resolved on 7–12% gradient SDS-PAGE, and the HDAC4, PDI, and NP polypeptides were detected by Western blotting. Arrows indicate the HDAC4 polypeptide cleavage products. Note: the HDAC4 blot was reused to probe for PDI, and the MW lane was combined with the rest of the lanes after cropping out the inconsistent second lane of same blot. H, the level of full-length HDAC4 polypeptide in panel G was quantified and normalized as described in the legend to Fig. 1B. Then, the normalized level of HDAC4 polypeptide at 2 h infection was considered 100% to compare its levels at 6, 12, and 24 h infection. I, MDCK cells were infected with PR8 and harvested at the times indicated in panel G. Then, the full-length caspase 3 (Cas3-FL) and cleaved caspase 3 (Cas3-CL) polypeptides were detected in total cell lysates by Western blotting. Note: the MW lane was combined with rest of the lanes after cropping out the less relevant middle lanes of same blot. Error bars represent the mean ± S.E. of three independent experiments. The asterisks represent p values mentioned in the text calculated by unpaired t test (F) or ANOVA (H), and indicate the significant differences in means. MW, molecular weight.