FIGURE 2.

HDAC4 specifically regulates HIF1α proteins. A, Hek293 cells were transduced with lentivirus containing shRNA against scramble sequence (sh-Scr), HIF1α (sh-HIF1α), HDAC1 (sh-HDAC1), HDAC3 (sh-HDAC3), or HDAC4 (sh-HDAC4). The indicated cells were cultured under either normal or hypoxic conditions for 6 h before Western blot analysis. B, an equal amount of HA-HIF1α plasmid was co-overexpressed with either empty vector (-) or FLAG-HDAC4 (+). 48 h after transfection, whole cell lysates were used for IP with anti-HA antibodies. The level of HA-HIF1α protein and its acetylation were measured by Western blot analyses using anti-HA and anti-acetyl-lysine (Ace-K) antibodies, respectively. The FLAG-HDAC4 was measured by the anti-FLAG antibody. C, the HA-HIF1α plasmid or FLAG-HDAC4 or FLAG-HDAC1 plasmid was overexpressed in HEK293T cells. 48 h after transfection, the HA-HIF1α, FLAG-HDAC4, and FLAG-HDAC1 were IP-purified using anti-HA and anti-FLAG antibodies, respectively. The purified HA-HIF1α was mixed with purified HDAC4 or HDAC1 in deacetylase buffer for the indicated time in an in vitro deacetylation assay. The resulted proteins were analyzed by Western blot analysis using anti-HA or anti-ace-K antibodies.