Fig. 3. (A) Juxtacrine assay with transiently transfected MDCK II cells. Lane 1: vector transfected cells; 2: proHB-EGF; 3: 5 aa deletion; 4: 12 aa deletion; 5: soluble HB-EGF administration. Subconfluent A431 cells were overlain with formalin fixed MDCK cells transiently transfected with different HB-EGF constructs. After 10 minutes, the MDCK cells were washed away, membranes were prepared from A431 cells, EGF receptor was immunoprecipitated with an anti-EGFR antibody and an in vitro kinase assay was performed. Soluble HB-EGF-induced EGF receptor tyrosine phosphorylation was used as a positive control. (B) Immunofluorescent staining of the control, MDCK^5aa and MDCK^pro cells with the anti-FLAG (M2) antibody. Cells were plated on transwells and stained with a polyclonal anti-FLAG (M2) antibody followed by an anti-rabbit conjugated with Rhodamine-X. Photographs were taken using a Zeiss-410 confocal microscope. The photographs shown are three-dimensional reconstructions of the Z-stack captured by confocal microscope and are representative of observations from two independent clones for each transfected molecule. Original magnification x400. (C) The YZ orthogonal view of the sectioning for the FLAG-immunofluorescent staining for the MDCK^5aa and MDCK^pro cells through the stacks obtained using confocal microscopy.