FIGURE 2:

Claudin-2 expression in MDCK II cells is decreased in the absence of Slp2-a. (A) Increased TER of Slp2-a KD cells. TER was measured in confluent control cells and Slp2-a KD cells. The TER value of the Slp2-a KD cells was significantly higher than the TER value of the control cells. Data are expressed as the means ± SEM of three determinations. *p < 0.01 (Student's unpaired t test). (B) Decreased expression of claudin-2 protein in Slp2-a KD #1 and #2 cells. Total cell lysates of confluent control cells and Slp2-a KD #1/#2 cells were subjected to 10% SDS–PAGE, followed by immunoblotting with the antibodies indicated on the right. The level of claudin-2 protein expression in Slp2-a KD #1/#2 cells was much lower than in the control cells, whereas the level of claudin-1 protein expression appeared to be the same. Actin was used as an internal control. The positions of the molecular mass markers (in kilodaltons) are shown on the left. (C) Decreased expression of claudin-2 mRNA in Slp2-a KD #1/#2 cells as revealed by RT-PCR analysis. Note that the level of claudin-2 mRNA expression in Slp2-a KD #1/#2 cells, but not the level of claudin-1 mRNA expression, was also dramatically decreased, the same as shown for claudin-2 protein expression in B. GAPDH was used as an internal control. The size of the molecular weight markers (kilobase pairs [kbp]) is shown on the left. (D) Correct targeting of claudin-2 to the tight junction in Slp2-a KD cells. Confluent control cells and Slp2-a KD cells were fixed, permeabilized, and stained with anti–ZO-1 antibody (red) and anti–claudin-1 antibody (left, green) or anti–claudin-2 antibody (right, green). Nuclei were stained with DAPI (blue). Confocal xz sections are shown. Although the intensity of claudin-2 staining was dramatically reduced in the Slp2-a KD cells, even in the absence of Slp2-a both claudin-1 and claudin-2 were correctly localized at the tight junction labeled with anti–ZO-1 antibody (yellow dots; bottom, arrowheads). Scale bars, 10 μm.