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. 2015 Mar 25;55(5):671–687. doi: 10.1002/mc.22312

Figure 6.

Figure 6

Cathepsin B reduces p27Kip1 stability. (A) The cDNA encoding HA‐tagged wild‐type p27Kip1 and R152A/△96–100 mutant were transiently transfected in 293T cells. Twenty‐four hours following transfection, cells were treated with cycloheximide (CHX, 30 μg/mL) and harvested after 0, 2, 6, 8, or 10 h. Cells were processed and lysed at the same time. Expression of HA‐27Kip1 proteins and β‐actin was analyzed by Western blotting. Representative Western blot analysis is shown in upper panel. The graph illustrates the densitometric analysis of data from three independent experiments. HA‐p27Kip1 expression at 0 h of cycloheximide was set at 100%. Relative HA‐p27Kip1 protein levels were calculated using β‐actin as reference. (B) The pcDNA3 vectors without or with the cDNA encoding HA‐tagged wild‐type p27Kip1 or HA‐tagged R152A/△96–100 mutant were transiently co‐transfected with 0, 150, 300, or 600 ng of the cDNA encoding cathepsin B in 293T cells. After 48 h, protein lysates were analyzed by Western blotting for the expression of HA‐27Kip1, cathepsin B, and β‐actin.