Figure 2.

The interactions among the subunits of the HDA complex HDA1, HDA2, and HDA3. (A) The HDA complex coimmunoprecipitates with any single subunit of the complex. In lanes 1, 4, and 7, the whole-cell extracts were prepared from YDS2 (containing the WT HDA1 gene). In lanes 2, 5, and 8, strain WJY201 (containing HDA2∷MYC gene) was used. In lanes 3, 6, and 9, strain WJY202 (containing HDA3∷MYC gene) was used. The antibodies used for immunoprecipitation are shown. Blots were probed with α-HDA1 (lanes 1–3), α-HDA2 (lanes 4–6), and α-HDA3 (lanes 7–9). (B) The α-myc antibody does not recognize HDA1, HDA2, or HDA3 lacking a myc tag. In lanes 1, 3, and 5, strain WJY200 (HDA1∷myc) was used. In lanes 2, 4, and 6, YDS2 (containing WT HDA1, HDA2, and HDA3 with no myc tag) was used. Blots were probed with α-HDA1 (lanes 1 and 2), α-HDA2 (lanes 3 and 4), and α-HDA3 (lanes 5 and 6). (C) The α-HDA1 antibody does not recognize HDA2 or HDA3. In lanes 1, 3, and 5, strain YDS2 was used. In lanes 2, 4, and 6, the hda1Δ strain WJY110 was used. Blots were probed with α-HDA1 (lanes 1 and 2), α-HDA2 (lanes 3 and 4), and α-HDA3 (lanes 5 and 6). (D) The interaction between HDA2 and HDA3 is independent of HDA1. In lanes 1–6, strains YDS2 (WT, lanes 1 and 4), WJY103 (hda2Δ, lanes 2 and 5) and WJY104 (hda3Δ, lanes 3 and 6) were used. In lanes 7 and 8, the strains WJY201 (WT, lane 7) and WJY211 (hda1Δ, lane 8) were used, where the natural HDA2 gene has been replaced by HDA2∷MYC. The resulting blots were probed with α-HDA3 (lanes 1–3, 7, and 8) and α-HDA2 (lanes 4–6). (E) HDA1-HDA1 interact. HDA1 was tagged with myc18 in WT (WJY200), hda2Δ (WJY203), hda3Δ (WJY204), and hda2Δhda3Δ (WJY205). The plasmid expressing GAL4AD-HDA1 (pWJ104) was transformed into these strains. α-Myc antibody was used to pull down HDA1-myc from cell extracts of these strains, and α-myc antibody (lanes 1–4) and α-GAL4 AD antibody (lanes 5–8) were used to detect the presence of HDA1-myc and GAL4-AD-HDA1. IP, immunoprecipitation.