Total protein expression of WT and A531V CLC-1 channels.

Biochemical analyses of CLC-1 channels expressed in HEK293T cells. (A) Immunoblotting analyses of myc-tagged WT CLC-1 and the A531V mutant. The position of molecular weight markers (in kilodaltons, kDa) are at the left of the blots. Expressions of β-actin are displayed as controls for the loaded protein amounts. (B) Quantification of CLC-1 protein expression level. Protein density was standardized as the ratio of the myc-CLC-1 signal to the cognate β-actin signal. Values from the A531 mutant were then normalized to those for WT. Densitometric scans of immunoblots were obtained from 13 independent experiments. The mean normalized value of A531V is 0.57±0.02. Asterisks denote significant difference from WT (*, t-test: pC) Quantification of the percentage of transfected HEK293T cells emitting GFP fluorescence (GFP+/live cells). Flow cytometry was employed to determine the ratio for each of the three listed cDNA constructs. Data were pooled from 3 independent experiments.