Fig. 5.

siRNA knockdown of nicastrin inhibits CTF15 accumulation. Panel A: Cytokine-stimulated HES cells were incubated with or without 1 µM L685,458 for 72 h and subjected to a cold lysis. Lysates were immunoprecipitated with CT1 antibody and the blots probed with antibody recognizing both mature and immature forms of nicastrin (Nct) as described in “Experimental Procedures Section.” Mature nicastrin was present only in immunoprecipitates containing accumulated CTF15 (upper panel). Panel B: HES cells were transfected with nicastrin or GAPDH siRNA as described in “Experimental Procedures Section.” Cytokine treatment was initiated following the second transfection in the absence (−) or presence (+) of 1 µM L685,458 as indicated. Equal concentrations of total cell protein collected after 56 h of treatment were probed with antibody to nicastrin or CT1 (upper and lower panels, respectively). Densitometric analysis of Panel B samples not treated with L685,458 is presented in Panel C (ADU, arbitrary densitometric units). Treatment with nicastrin siRNA stimulated CTF15 accumulation relative to cells receiving mock or GAPDH siRNA treatment. Data show the means ±s.d. of triplicate determinations in each case. *P< 0.001 versus mock or GAPDH siRNA treated.