Figure 9. Expression levels of TJ and AJ proteins during a cal- cium switch experiment. (A) Equivalent protein loadings of whole cell lysates of parental (P) and MDCK/NZO-3 (NZO-3) cells were analyzed by immunoblot at 6, 12, 24, and 48 h after the re-addition of calcium. The monoclonal antibody against ZO-3 was used to detect both the NZO-3 construct and endogenous ZO-3; the proteins were distinguished by their difference in mo- lecular mass. NZO-3 is not detected in parental cells. NZO-3 ex- pression in MDCK/NZO-3 cells increases in response to the cal- cium switch and then over time returns to stable, steady-state levels (S). Endogenous ZO-3, ZO-1, ZO-2, occludin, and E-cad- herin levels are unchanged during the course of the calcium switch experiment in both cell lines, and no significant differ- ences in these protein levels are detected between parental and MDCK/NZO-3 cells at any time points. Occludin shows a shift to a higher molecular weight species in both cell lines over time. (B) MDCK/NZO-3 cells have more soluble b-catenin than parental cells. At indicated time points after a calcium switch, parental (P) and MDCK/NZO-3 (N) cells were extracted with CSK buffer (see Materials and Methods) to separate TX-100–soluble and –insoluble pools of b-catenin. Samples containing equal amounts of total cellular protein were analyzed for b-catenin expression by immunoblot. The amount of soluble b-catenin is greater in MDCK/NZO-3 cells than parental cells at 12 and 24 h, and to a lesser degree at 48 h after the re-addition of calcium. There is no detectable difference in the amount of insoluble b-catenin be- tween parental and MDCK/NZO-3 cells. Figure 10. In vitro binding analyses show that NZO-3 binds F-actin and ZO-1 exclusively; both NZO-3 and CZO-3 bind occludin and cingulin. (A) Coomassie blue–stained gel showing the equivalent amounts of NZO-3 (N) and CZO-3 (C) fusion proteins used in all subsequent binding assays. (B) NZO-3 specifically cosediments with actin filaments; CZO-3 does not. Equivalent amounts of NZO-3 or CZO-3 were centrifuged in the presence (1) or absence (2) of F-actin. Stoichiometrically equivalent aliquots of superna- tants (S) and pellets (P) were analyzed by immunoblot. NZO-3, but not CZO-3, is found in the F-actin pellet. (C) NZO-3 binds ZO-1; CZO-3 does not. 35S-labeled in vitro–transcribed/translated ZO-1 was incubated with affinity resin containing equal amounts of NZO-3 or CZO-3. The 6-histidine tag plus 36 nonspecific amino acids served as negative control (2). As detected by autoradio- gram, ZO-1 is retained by the NZO-3-containing resin and does not bind to CZO-3 or the negative control peptide. (D) Both halves of ZO-3 bind occludin with similar affinity. NZO-3 and CZO-3 were incubated with affinity resin containing immobilized GST-occludin (1), or GST alone (2). Bound protein was eluted with glutathione and analyzed by immunoblot with anti–ZO-3 an- tibodies (first four lanes) or Coomassie blue staining (last two lanes). Both NZO-3 and CZO-3 are retained by GST-occludin and not by GST alone. The Coomassie blue–stained lanes show that approximately equal amounts of NZO-3 and CZO-3 (arrows) are bound. (E) Both halves of ZO-3 bind to the amino-terminal head region of cingulin. NZO-3 or CZO-3 were added to an affinity col- umn containing the amino-terminal head region of cingulin (N- cing) fused to GST (1) or GST alone (2). Bound fractions were immunoblotted with antibodies specifically recognizing NZO-3 or CZO-3 (first four lanes) or stained with Coomassie blue (last two lanes). Both NZO-3 and CZO-3 are retained on the GST–N-cingu- lin column but not on GST alone. Coomassie blue staining of the bound fractions shows approximately equal amounts of NZO-3 or CZO-3 (arrows) are bound. The other protein bands visible in the Coomassie blue–stained samples correspond to GST–N-cingulin. Wittchen et al. Delayed Junctional Complex Assembly 833