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. 2019 Feb 25;9:2724. doi: 10.1038/s41598-019-39368-y

Figure 3.

Figure 3

Depletion of FBXO38 or USP7 decreases KIF20B levels. AGS cells were transfected with two different siRNAs targeting FBXO38 (A) or USP7 (B) or with negative control siRNA (siControl) and whole cell lysates were analyzed by Western blotting using the indicated antibodies. Bands corresponding to FBXO38 are indicated with arrow heads. In AGS cells, an additional nonspecific band is also detected by this antibody running just below FBXO38. (C) KIF20B protein bands were quantified in three independent FBXO38 and USP7 silencing experiments in AGS cells and normalized to actin. The average values are shown relative the silencing control. ***P < 0.001. (D) AGS cells were transfected with an siRNA targeting KIF20B, and whole cell lysates were analyzed by Western blotting as in (A). (E) KIF20B mRNA levels were determined by RT-qPCR in AGS cells transfected with siRNA targeting FBXO38 or with negative control siRNA. The experiment was performed in triplicate and values normalized to actin. Average values and standard deviations are shown relative to the silencing control. (F) HCT116 cells were transfected with siRNA targeting FBXO38 or USP7 or with negative control siRNA (siControl) and whole cell lysates were analyzed by Western blotting as in (A). (G) Whole cell lysates of HCT116 USP7 KO or the WT parental cell line (USP7 WT) were analyzed by Western blotting as in (A). (H) AGS or HCT116 cells were treated with 5 µM of compound 4 (USP7 inhibitor) or an inactive enantiomer and harvested after 24 hours. Whole cell lysates were analyzed by Western blotting using the indicated antibodies.