Figure 7.

PMA increases in vitro and in vivo binding of A7 to SNAP23. A. Left Panel. Affinity binding of cell A7 to GST-SNAP23 is higher in PMA treated type II cells. Following incubation of cells for 30 min without or with 80 nM PMA, the cell lysates were prepared and equal amounts of cell proteins were analyzed for A7 binding to Sepharose4B-bound GST-SNAP23 by pull-down assay without or with 1 μM Ca²⁺. The beads were washed and analyzed for bound A7 by western blot analysis. Right Panel. Results are shown as mean ± SE (n = 3 separate experiments) of densitometry analysis (arbitrary units, a.u.) of western blots. Statistical analysis (Student’s t-test) was performed to analyze for the effects of PMA-treatment and for the presence of Ca²⁺. * P < 0.05 in comparison to PMA-treated cells with no Ca²⁺ or control cells without or with 1 μM Ca²⁺. B. Specificity of SNAP23 antibodies was demonstrated by western blot analysis of samples of homogenates (30 μg protein) from rat type II cells, lung and brain with 10 μl antiserum against recombinant (r) SNAP23 pretreated for 24h without (−) or with (+) 100 μg purified rSNAP23. The recombinant protein (arrow) migrated slightly faster in comparison to the tissue SNAP23. The western blot was then probed for actin. Pre-treatment with rSNAP23 protein diminished the reactivity of antiserum for SNAP23 in all samples. C. Left Panel. In separate experiments, lysates from control and PMA-treated cells (30 min incubation) or 5 μl rabbit serum (RS) were immuno-precipitated using SNAP23 antibodies and analyzed for A7 binding by western blot analysis. Rabbit IgG (R-IgG, 2 μg) was also included as a control. The blots were then probed with SNAP23 antibodies to demonstrate relative loading of SNAP23. Control-lys and PMA-lys denote cell lysates (40 μg protein) from control and PMA-stimulated cells, respectively. Right Panel. Western blots of immuno-precipitates from three separate experiments were analyzed by densitometry scanning and the ratios of A7 and SNAP23 (mean ± SE, n = 3) were compared between two groups. * P < 0.05 by Student’s t-test. In comparison to controls, the PMA-stimulated cells showed increased binding of A7 to SNAP23.