Fig. 7. NHE3-EGFP, NHERF2, ezrin and F-actin colocalize in the OK/NHERF2/NHE3-EGFP cell apical membrane. OK cells transiently expressing NHE3-EGFP were plated on a glass-bottom 35 mm plastic culture dish and processed for confocal imaging. Cells were fixed and labeled either with polyclonal anti-NHERF2 (A2 and E2), monoclonal anti-ezrin (B2) or phalloidin (C1-3 and D1). Secondary antibodies were either Alexa-568-conjugated goat anti-rabbit IgG for NHERF2 staining or Alexa-595-conjugated goat anti-mouse IgG for ezrin staining. The green channel shows the distribution of NHE3-EGFP and red channels show NHERF2 (A2, E2), ezrin (B2) and F-actin (C1-3, D1). Overlayed images are shown in the right column. XZ sections show colocalization of NHE3-EGFP with NHERF2 (A3), ezrin (B3) and F-actin (D3) in microvilli. C1-3 shows F-actin staining at the basal stress fibers (C1), the lateral cell surface (C2) and apical membrane microvilli (C3) in polarized OK cells. Images D1-D3 show F-actin (D1) and NHE3585-EGFP (D2) staining at the apical plasma membrane. D3 is a composite image between D1 and D2. F-actin colocalized with NHE3585-EGFP only at the microvilli (D3a) but not in the subapical juxta nuclear region (D3b). XZ section images E1-E3 show NHE3585-EGFP (E1) and NHERF2 (E2). E3 is a composite image between E1 and E2. NHERF2 did not colocalize with NHE3585-EGFP at the microvilli. However, NHERF1 still shows normal microvilli distribution at the microvilli (F2) and colocalized with microvillus NHE3585-EGFP (F3). These OK cells endogenously expressed ezrin and NHERF1, stably expressed NHERF2 and transiently expressed NHE3-EGFP. Images were taken with a 100x objective. Bars, 10 µm.