Fig. 3.
Crk II knockdown decreased levels of IGF-1R and its downstream signaling in CWR22rv1 cancer cells. (A) WB detection (30 μg protein loaded in each well) of IGF-1R (antibody against IGF-1R β subunit) and the downstream signaling molecules of PI3K-Akt pathway. Bar graph shows the quantification of the staining intensities of each protein using the software of the FluorChem M imager (ProteinSimple); (B) Interaction of Crk II and IGF-1R by co-immunoprecipitation (i.p.). The CWR22rv1 cancer cell lysates were used to conduct pull down with either anti-Crk II antibody or anti-IGF-1Rβ antibody and isotype IgG (IgG) was used as a control; (C) Effects of IGF 1 stimulation on IGF-1R signaling in Crk II-shRNA knockdown cancer cells in comparison with the scramble control. WB detection was conducted similarly as in (A). Cancer cells were cultured in a low serum medium (1% FBS) over night and stimulated with IGF (10 ng/ml) for different times (15 and 30 min).