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Fig. 5. Effects of CNIH on TGF ${alpha}$ maturation. (A) CNIH increased the immature TGF ${alpha}$ form. CHO cells were transfected to express TGF ${alpha}$ in the presence of increasing CNIH-Myc levels. Upper panel: expression of TGF ${alpha}$ in cell lysates, showing an increase in immature TGF ${alpha}$ (form 1), relative to the mature forms 2 and 3. Lower panels: increased TGF ${alpha}$ levels co-precipitated with CNIH-Myc. (B) Effect of CNIH on endogenous TGF ${alpha}$ in HCA-7 cells. Cells were transfected to express CNIH-Myc (20 and 100 ng plasmid), and co-precipitation of endogenous TGF ${alpha}$ with CNIH was examined. (C) Effect of CNIH-HA on TGF ${alpha}$ localization in transfected CHO cells. In the absence of HA-CNIH, TGF ${alpha}$ had a diffuse distribution. CNIH expression conferred TGF ${alpha}$ accumulation in the ER, where CNIH is localized. Bar, 20 µm. (D) The Semi-quantitative image analysis of TGF ${alpha}$ immunostaining (given as relative TGF ${alpha}$ intensity units) shows three times more accumulation of intracellular TGF ${alpha}$ signal in comparison with cells not transfected with CNIH. Error bars represent s.e.m.; ^*t-test, P<0.05. (E) Comparison of full-length CNIH and CD1 deletion mutant; TM, transmembrane. Transfected cells expressed full-length or CD1 mutant CNIH-Myc and TGF ${alpha}$ . Cell lysates were processed for anti-Myc immunoprecipitation and immunoblotting with anti-TGF ${alpha}$ . Dividing vertical lines indicate parts from the same gel (B).

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