Figure 4.

Interaction of CYLD with HDAC6. (A) Cytoplasmic and nuclear extracts of keratinocytes (Cyld^+/+ and Cyld^−/−) in the presence or absence of TPA (100 nM for 30 min), immunoblotted with antibodies against HDAC6, lamin-B, and tubulin. (B) Immunoprecipitation of CYLD and immunoblotting against HDAC6 and CYLD untreated, TPA- (100 nM for 30 min) and/or nocodazole (10 μM for 60 min)-treated Cyld^+/+ keratinocytes. The lysate (lower panel) shows equal amount of protein used for immunoprecipitation. (C) The confocal plane of Cyld^+/+ keratinocytes stimulated for 30 min with 100 nM TPA and stained for HDAC6 (red) and CYLD (green). (D) HeLa cells were transiently transfected with FLAG-tagged, wild-type or different deletion mutants of CYLD, incubated for 24 h, lysed, immunoprecipitated using α-FLAG M2 agarose beads, and immunoblotted against endogenous HDAC6 or FLAG. The lysate (lower panel) shows equal amount of protein used for immunoprecipitation. (E) HeLa cells were transiently transfected with HA-tagged, full-length or different deletion mutants of HDAC6 and FLAG-tagged, full-length CYLD. After transfection (24 h) the cells were lysed, immunoprecipitated using α-FLAG M2 agarose beads, and immunoblotted against HA or FLAG. The lysate (lower panel) shows equal amount of protein used for immunoprecipitation. (F) His-tagged CYLD was purified from COS cells transiently transfected with His–CYLD (1.0 μg for 48 h) and incubated with recombinant N-terminal GST–HDAC6 for 30 min followed by precipitation of His-tagged protein complexes using Ni–NTA agarose beads. The bound proteins were recovered from the beads and analysed by immunoblotting against GST to detect GST–HDAC6 or His to detect His–CYLD. (G) Purified GST or GST–CYLD^1–212 protein was incubated with His–HDAC6 in vitro for 30 min followed by GST pull down using glutathione–agarose beads. The complex was recovered from the beads and analysed by immunoblotting against His to detect His–HDAC6 or GST to detect GST–CYLD^1–212.