Figure 5

Meq mediates the degradation of chHDAC1 and 2. (A) DF-1 cells were transfected with pcDNA-FLAG-Meq or pcDNA empty vector (Ev) and 48 h later, cells were harvested for protein and RNA extraction. Western blot (WB) analysis was processed with the indicated antibodies (left) and quantified with Image J and presented as fold change compared to Ev (middle). qRT-PCR was processed with primers targeting chHDAC1 and chHDAC2 and presented as fold change compared to Ev (right). pcDNA-FLAG-chHDAC1 (B) or pcDNA-FLAG-chHDAC2 (C) were co-transfected with different amounts of pcDNA-HA-Meq into 293T cells for 48 h. Whole cell lysates were subjected to WB with the indicated antibodies (left). WB results were quantified with Image J, normalized to HSP90, and presented as fold change compared to the least amount of Meq transfection (right). pcDNA-FLAG-Meq or pcDNA Ev were co-transfected with pcDNA-HA-chHDAC1 (D) or pcDNA-HA-chHDAC2 (E) into 293T cells and 24 h later, cells were treated with cycloheximide (CHX, 1 mg/ml) for the indicated length of time. WB were performed with HA, FLAG, and HSP90 antibodies (upper). HA-chHDAC1 or HA-chHDAC2 protein levels were quantified with Image J, normalized to HSP90, and presented as fold change compared to non-treated cells (bottom). All experiments were repeated two times. Error bars indicate standard deviation (SD). (F) pcDNA-FLAG-chp53, pcDNA-FLAG-chCREB, or pcDNA-FLAG-chc-Jun were co-transfected with different amounts of pcDNA-HA-Meq into 293T cells and 48 h later, whole cell lysates were subjected to WB with the indicated antibodies. The statistical differences were analyzed by Student t test. *p < 0.05, **p < 0.01, ***p < 0.001.