Figure 1.

Protein 4.1R isoform expression and localization during different confluent states of MDCK. A, immunofluorescence staining of 4.1R with an α-exon 13 Ab in sub-confluent and confluent MDCK cells. Bar, 10 μm. B, cell-cycle profiles of sub-confluent and confluent MDCK cells. C, RT-PCR 4.1R products amplified using an exon 21 antisense primer with an exon 1A, 1B, or 1C sense primer generated two major species with a 450-bp difference from all sets of primers. Molecular markers (kb) are provided at the left margin of the gels. D, major 4.1R isoforms expressed in MDCK cells. Schematic diagram of 4.1R and its domains. Constitutive exons are indicated as dark gray boxes, and alternatively-spliced cassettes are depicted as light gray boxes. Exon numbers are indicated. E, reciprocal expression of exons 16 and 17b in maturing MDCK cells. Schematic representation of regions between exons 13 and 18 of 4.1R and the primer sets used in PCR. RNA isolated from nonconfluent (non-), sub-confluent (sub-), and confluent (con-) MDCK cells were analyzed for exon 16 (E16) and 17b (E17b) expression by RT-PCR. E16 or E17b inclusion was calculated as the percent of total RNA products containing exon 16 or exon 17b, respectively. Averages and S.D. were obtained from three independent experiments (n = 3) and presented at the bottom of each lane. Molecular markers (bp) are provided at the right margin of the gels. F, 4.1R protein isoforms expression during MDCK maturation. Lysates from different confluency MDCK probed with anti-ex13, anti-HP, anti-ex16, and anti-ex17b Abs. β-Actin served as a loading control. Molecular mass markers (kDa) are provided at the left margin of the blots. G, exon 5 is required for the localization of 4.1R at the cell–cell contacts in confluent MDCK cells. MDCK 4.1R isoforms with exon compositions as indicated in D were fused with EGFP, transfected into MDCK, and examined for their subcellular localization as revealed by Zeiss microscopy. 135⁻⁵, 4.1R 135-kDa form without exon 5. Bar, 10 μm.