Fig. 3. Immunoblot analysis of endogenous tight junction proteins in parental, GFP-transfected, and claudin-7-GFP-transfected cells (mixed clones). (A) Parental (P), GFP-transfected (G), and claudin-7-GFP-transfected (7) cells were lysed in RIPA buffer, and a total of 20 µg protein for each lane was loaded onto the SDS-polyacrylamide gel. Membranes were blotted against claudin-1, -2, -3, -4, -7 and ZO-1 antibodies. Actin staining was used as a loading control. (B) Densitometry analysis of protein expression levels. Following immunoblotting, X-ray films were scanned and band images were analyzed. The relative signal intensity of each band was obtained after background subtraction. The band intensity for parental cells was normalized to 1 and set as the reference. Data were collected from three independent clones with high expression levels of GFP and claudin-7-GFP. The average density values for three separate experiments were plotted for each designated claudin protein.