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. 2009 Nov 5;29(1):131–144. doi: 10.1038/emboj.2009.317

Figure 5.

Figure 5

The N-terminal domain of CYLD is responsible for inhibition of HDAC6 activity. (A) MAP-enriched bovine tubulin (cytoskeleton) was polymerised into microtubules in the absence of taxol or glycerol by incubation for 30 min at 35°C. The polymerised microtubules were incubated with endogenous HDAC6 and/or EGFP–CYLD immunoprecipitates from melanoma cells in the absence or presence of TSA (0.5 μM) at 37°C for 2 h. Samples were placed on ice for 15 min and the supernatant was collected by centrifugation and analysed by immunoblotting. (B) COS cells were transiently transfected with different concentrations of FLAG–CYLD (0.5, 1.0 and 3.0 μg) or HA–HDAC6 (1.0 μg) for 24 h. Polymerised microtubules were incubated with CYLD and/or HDAC6 eluted from the corresponding immunoprecipitates at 37°C for 2 h. Samples were placed on ice for 15 min and the supernatant was collected by centrifugation and analysed by immunoblotting. (C) COS cells were transiently transfected with FLAG-tagged, wild-type or truncation mutants of CYLD or HA-tagged HDAC6 constructs (1.0 μg of each for 24 h). Polymerised microtubules were then incubated with CYLD and/or HDAC6 eluted from the corresponding immunoprecipitates at 37°C for 2 h. Samples were placed on ice for 15 min and the supernatant was collected by centrifugation and analysed by immunoblotting. (D) Immunoprecipitation of endogenous HDAC6 from Cyld+/+ and Cyld−/− keratinocytes in the absence or presence of TPA (100 nM for 30 min) and immunoblotting against tubulin and HDAC6 in the absence or presence of TPA (100 nM for 30 min). The lysate (lower panel) shows equal amount of protein used for immunoprecipitation. (E) Immunoblot analysis of HDAC6, acetylated α-tubulin, and total tubulin in EGFP- or EGFP–CYLD-expressing melanoma cells before or after 24 h of transient transfection with HDAC6 siRNAs (0.2 μM). (F) Immunoblot analysis of HDAC6, acetylated α-tubulin, and total tubulin in untreated or TPA (100 nM for 30 min)-treated Cyld−/− keratinocytes before or after transient transfection with HDAC6 siRNAs.