FIGURE 3.

EM analysis of GFP-tH and GFP-tK nanoclustering. Plasma membrane sheets of BHK cells transiently expressing GFP-tH or GFP-tK, were labeled with anti-GFP-5 nm gold, imaged in an electron microscope and analyzed using spatial statistics. Plasma membrane sheets generated from BHK cells expressing GFP-tH (a) or GFP-tK (b), and left untreated or treated with 100 μm indomethacin, were labeled with anti-GFP antibody conjugated to 5 nm gold. The plasma membrane sheets were imaged in an electron microscope and the spatial distribution of the gold labeling was analyzed using Ripley's K-function (Equations 1 and 2). Maximum L(r) − r values above the 99% confidence interval (C.I.) for complete spatial randomness (CSR) indicate clustering at that value of r. Univarate K-functions are weighted means (n = 18) standardized on the 99% C.I. Significant differences from the control pattern for indomethacin-treated cells were assessed using Bootstrap tests. Treatment with indomethacin significantly altered GFP-tH nanocluster formation (p = 1 × 10⁻³), but not GFP-tK nanocluster formation (p = 1).