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. 2009 Jan 2;284(1):685–695. doi: 10.1074/jbc.M806124200

FIGURE 2.

FIGURE 2.

ASK1, MKK4, and JNK3 bind arrestin3. A, COS-7 cells were transfected with FLAG-arrestin3 (FLAG-Arr3) with GFP-JNK3, HA-ASK1, HA-MKK4, or with indicated combinations of these kinases. Cell lysates were immunoprecipitated with rabbit anti-FLAG antibody, and then immunoblotted with mouse anti-HA, anti-GFP and anti-FLAG antibodies. B, COS-7 cells were transfected with HA-MKK4 with indicated FLAG-tagged arrestins. Cell lysates were immunoprecipitated and immunoblotted as in panel A. C, HEK-293A cells were transfected with HA-ASK-NLS alone or with indicated FLAG-tagged arrestins. Arrestins were visualized with M2 anti-FLAG, HA-ASK1 with rat anti-HA high affinity antibody, followed by Alexa 593 anti-mouse (Red) and Alexa 488 anti-rat (Green) secondary antibodies, respectively. The representative images show arrestins (red), HA-ASK1-NLS (green), and both channels merged. At least 20 cells expressing indicated proteins were scored for the subcellular distribution of HA-ASK1-NLS. Means ± S.D. (n = 3) of the fraction of cells with more ASK1 in the cytoplasm than in the nucleus are shown. ****, p < 0.0001.