Figure 2. HDAC6 Deacetylates MSH2.

(A) 293T cells were transfected with either HA-MSH2 alone or HA-MSH2 with F-HDAC6. IP-WB and direct WB were performed as indicated.

(B) The levels of acetylated MSH2 are enhanced in HDAC6 knockout (KO) MEFs. IP-WB and direct WB were performed as indicated. Please note that in WT and KO MEFs, equal amounts of immunoprecipitated MSH2, not equal amounts of cell lysates, were loaded on SDS-PAGE. mHD6 represents mouse HDAC6.

(C) C13 cells were stably transfected with control shRNA(pRS) and HDAC6shRNA (Origene) to generate control (C13-pRS) and HDAC6 knockdown cells (C13-HD6KD), respectively. IP-WB and direct WB were performed as indicated. Please note that in C13-pRS and C13-HD6KD cells, equal amounts of immunoprecipitated MSH2, not equal amounts of cell lysates, were loaded on SDS-PAGE.

(D) The HDAC6-selective inhibitor, tubacin, enhances MSH2 acetylation. MEFs were treated with a vehicle control (DMSO), 5 μM tubacin, or 5 mM NaB for 12 hr. IP-WB and direct WB were performed as indicated. For the fourth panel, the original lanes between 1 and 2 were deleted.

(E) SAHA increases the acetylation of MSH2. 293T cells were transfected with HA-MSH2. One sample was treated with a vehicle and the other with 2 μM SAHA overnight. The IP-WB analyses were performed as indicated.

(F) The SirT2 inhibitor, AGK2, does not increase MSH2 acetylation. HeLa cells were transfected with HA-MSH2, then treated with a vehicle control or 25 μM AGK2 (EMD Chemicals) 24 hr prior to harvest. IP-WB and direct WB were performed as indicated.

(G) Depletion of SirT2 in HeLa cells does not increase MSH2 acetylation. HeLa cells were transfected with scramble or SMARTpool siRNA containing four sets of siRNAs against human SirT2 (Dharmacon Inc.) for 3 days. IP-WB and direct WB were performed as indicated. See also Figure S2.