Figure 7

The 32 kDa and 47 kDa T1-LIR species have half-lives of less than two hours. (a) The half-life of the 32 kDa (**) and 47 kDa (arrow) transfection-dependent T1-LIR species was calculated by transfection of HEK293T cells with pCDNA-V5/hThap1 and treatment with 50 µg/ml cycloheximide (CHX) 24 h post-transfection. Cells were harvested at the indicated time points and total cellular extracts (35 µg) were immunoblotted with the Proteintech antibody (left panel), followed by the Neuromab antibody (right panel). Each panel is representative of two experiments. Note that the Neuromab antibody again recognizes a 27 kDa species (*), with similar half-life. (b) Densitometry of the transfection-dependent 32 kDa and 47 kDa species in the left panel, normalized to GAPDH, and level of transfection-dependent species prior to cycloheximide arbitrarily set to 1.0.