JCS03103 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Established cell lines display epithelial markers before and after adenoviral infection. Monolayers of uninfected MEpG cells (left panel) or those infected with either a control or Cre-expressing adenovirus to generate DG^+/+ cells (middle panel) or DG^−/− cells (right panel), respectively, were fixed in acetone-methanol and immunostained as described in Materials and Methods. Mouse mAb specific for E-cadherin (BD Transduction Labs) was used at 1:200. Rat mAb TROMA-1 specific for keratin 8 was used at 1:30 (obtained from Developmental Studies Hybridoma Bank under the auspices of the NICHD; maintained by University of Iowa, Dept. of Biol. Sciences, Iowa City, IA) (Kemler et al., 1981). Both of the former antibodies were visualized with FITC-conjugated secondary antibodies (green). Rabbit pAb specific for ZO-1 (Zymed) was used at 1:100 and detected with Rhodamine-conjugated secondary antibody (red). Images were captured using a Nikon Eclipse E800 microscope, SPOT camera (Diagnostic Instruments Inc), Image-Pro Plus 3.0.01.00 software (Media Cybernetics), and a Nikon Plan Fluor Ph1 DLL 20× objective (0.50 NA). Bar, 60 μm.

• Supplemental Figure 2 -

  Fig. S2. DG protein levels in DG^+/+ and partial DG^−/− MEC populations generated by adenoviral infection of the MEpL cell line. (A) Western blot of cell extracts (10 μg protein) prepared on different days after infection of immortalized floxed DG mouse MEpL cell line with control or Cre-recombinase-expressing adenovirus to generate DG^+/+ or partial DG^−/− cell populations, respectively. Lane 1 represents uninfected cells at time 0. Antibodies are described in the legend for Fig. 1. Sizes of molecular mass markers are given in kDa. (B) Vertically paired immunofluorescent images of DG^+/+ and partial DG^−/− cell populations that were stained using a C-terminal β-DG antibody followed by a FITC-labeled secondary antibody (upper panel). Nuclei were stained with propidium iodide (bottom panel). Bar, 60 μm.

• Supplemental Figure 3 -

  Fig. S3. Diagram of DG mutants. Shown are the structures of full-length DG (wtDG), deletion mutants (DEL A, B, C, D and E) and the transmembrane fusion mutant (DG-tmf), consisting of extracellular DG sequences fused to the transmembrane domain of TACE. Numbers refer to amino acids in human α-DG and β-DG, with deleted sequences shown by dotted lines. PM, plasma membrane; TM, transmembrane domain of β-DG; tm, transmembrane domain of TACE.