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. 2017 May 24;8(29):47890–47901. doi: 10.18632/oncotarget.18141

Figure 4. Deletion of the degron motif in HDAC6 confers resistance to SPOP-mediated degradation.

Figure 4

(A) Sequence comparison of HDAC6 with the putative SPOP binding motif (degron) with other known SPOP substrates, and a schematic illustration of HDAC6. NLS: Nuclear localization signal, NES: Nuclear export signal, CD: Catalytic domain, SET14: Cytoplasmic retention domain, UBP: Ubiquitin binding domain. (B) IB analysis of WCLs derived from 293T cells transfected with indicated constructs. (C) IB analysis of WCL and GST pull-down products derived from 293T cells transfected with indicated constructs. Cells were treated with 10 μM MG132 for 12 hours before harvesting. (D-E) IB analysis of WCL derived from 293T cells transfected with indicated Flag-HDAC6 plasmids and treated for indicated times with 100 μg/ml CHX before harvesting (D). Quantification of the band intensities of (D) using the ImageJ software (E). HDAC6 immunoblot bands were normalized to Vinculin, then normalized to the t = 0 time point. (F) IB of WCL and His pull-down from HCT116 cells transfected with the indicated constructs. Cells were treated with 30 μM MG132 for 6 hours and lysed with denature buffer.