Figure 3
From: Congenital lactose intolerance is triggered by severe mutations on both alleles of the lactase gene
Potential interaction of LPH-Y1473X and LPH-D1796fs with wild type LPH in co-transfection experiments. COS-1 cells were transiently transfected either with cDNA encoding wild type LPH tagged with myc (LPH-myc) or cDNA clones encoding the mutants LPH-Y1473X and LPH-D1796fs. In another set of experiments, coexpression of LPH-myc with LPH-Y1473X or with LPH-D1796fs was performed in COS-1 cells. 48 h posttransfection the cells were metabolically labeled with [35S] methionine for 6 h continuously, lysed and the lysates were immunoprecipitated with either an antibody against myc to detect wild type LPH or a mixture of mAb anti-LPH antibodies to detect the mutants. Lactase activity was measured by determining the concentration of glucose generated by lactose hydrolysis by the Glucose oxidase-peroxidase mono-reagent method using photometric analysis. The total protein amount of each sample was determined by SDS-PAGE and autoradiography to calculate subsequently the relative specific activity. The quantification was performed by Quantity One® software. The results of LPH-Y1473X were taken from another gel with higher quality.