Figure 8: PIN1 isomerase facilitates KLHL15-dependent degradation of CtIP.

(a) HEK293T cells were cotransfected with FLAG-KLHL15 and either GFP-CtIP-wt, -S276A/T315A (2A, defective in PIN1 interaction), or -K467A (KA, defective in Cdh1 interaction) expression constructs. Forty-eight hours post transfection, cells were analysed by immunoblotting (left). Relative CtIP protein levels were determined by quantification of CtIP band intensity (normalized to Actin) with the ImageJ software (right). Data are represented as mean values of densitometric quantification±s.e.m. (n≥3). Statistical analysis was carried out using unpaired, two-tailed t-tests. P values expressed as *(P<0.05) was considered significant. (b) HEK293T cells were transfected twice with the indicated siRNA oligos for two consecutive days. Forty-eight hours after the first siRNA transfection, cells were transfected with either empty vector (EV) or FLAG-KLHL15 expression constructs. 72 h after the first siRNA transfection, cells were lysed and whole-cell extracts were subjected to IP using anti-FLAG M2 affinity resin. Inputs and recovered protein complexes were analysed by immunoblotting. The asterisk indicates neddylated CUL3. (c) HEK293^Cas9/wt and HEK293^{Cas9/KLHL15Δ} cells were transfected with EV or HA-PIN1 expression construct. Forty-eight hours after transfection, cells were either mock-treated or treated with CPT (1 μM) for 1 h. Whole-cell lysates were analysed by immunoblotting using the indicated antibodies. SE and LE denote short and long exposures of the same membrane. Asterisks indicate hyperphosphorylated forms of CtIP and RPA2, respectively.