Fig. 5. Effects of CNIH on TGF maturation. (A) CNIH increased the immature TGF form. CHO cells were transfected to express TGF in the presence of increasing CNIH-Myc levels. Upper panel: expression of TGF in cell lysates, showing an increase in immature TGF (form 1), relative to the mature forms 2 and 3. Lower panels: increased TGF levels co-precipitated with CNIH-Myc. (B) Effect of CNIH on endogenous TGF in HCA-7 cells. Cells were transfected to express CNIH-Myc (20 and 100 ng plasmid), and co-precipitation of endogenous TGF with CNIH was examined. (C) Effect of CNIH-HA on TGF localization in transfected CHO cells. In the absence of HA-CNIH, TGF had a diffuse distribution. CNIH expression conferred TGF accumulation in the ER, where CNIH is localized. Bar, 20 µm. (D) The Semi-quantitative image analysis of TGF immunostaining (given as relative TGF intensity units) shows three times more accumulation of intracellular TGF 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. Cell lysates were processed for anti-Myc immunoprecipitation and immunoblotting with anti-TGF. Dividing vertical lines indicate parts from the same gel (B).