Fig. 2. ErbB2 clustering in SKBR3 cells. (A-F) Membrane sheets were prepared from serum-starved cells (A,D,F), from cells pretreated with the combination of 1 µM AG1478 + 20 µM AG879 for 2 hours (B), or from cells that were serum starved, then stimulated with 20 nM EGF for 2 minutes (C,E). Distributions of ErbB2 were determined by immunogold labeling from the inside using RB9040 primary antibodies and 5-nm-gold-conjugated secondary antibodies. In D-F, membranes were also double labeled with 10-nm-gold reagents to detect phosphorylated tyrosine 1248 in the ErbB2 cytoplasmic tail. (G) Hopkins test is positive for ErbB2 clustering (applied to the data from image A). (H) Range of cluster size for resting ErbB2 in five images from the same experiment in 2A. (I) Positive Ripley's test for coincidence of label for pan-specific anti-ErbB2 antibodies vs phospho-specific ErbB2 antibodies. Bars, 0.1 µm.