JCS007658 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. ErbB receptors cluster when expressed in CHO cells. Membrane sheets were prepared from CHO cells transfected with EGFR-GFP (A,B), ErbB3-mCit (D-F) or ErbB2-mYFP (G,H) and labeled with specific primary antibodies, followed by secondary antibodies conjugated to 5 nm colloidal gold. CHO-EGFR-GFP cells in B were stimulated for 2 minutes with 20 nM EGF. CHO-ErbB3-mCit cells in F were stimulated for 5 minutes with 3.2 nM Heregulin. EGFR was labeled from the outside with GR-15 antibodies; ErbB3 was labeled from the inside with sc-285 antibodies; ErbB2 was labeled from the inside with RB9040 antibodies. Bars, 0.1 μm. Data in C show estimated cluster size and distribution for EGFR in resting cells expressing 80,000 receptors on the cell surface, as determined by Markov Random Field computer simulations. Data in I show that only the transfected CHO-EGFR-GFP cells express EGFR; lower panel shows EGFR phosphorylation status before and after addition of 20 nM EGF. Data in J-K report the endogenous levels of ErbB3 (none) or ErbB2 (very low) in parental CHO cells and the levels of chimericreceptors in transfected cells before and after flow sorting; lower panels in each figure report levels of basal phosphorylation in serum-starved cells using phosphotyrosine-specific antibodies. Inserts show Hopkins clustering test results. Shifts to the right of the red line indicate that receptors are significantly clustered.

• Supplemental Figure S2 -

  Fig. S2. Effects of kinase inhibitors on receptor phosphorylation status. Where indicated, cell-permeable inhibitors AG1478, AG879, PD153035 or Iressa were applied to cell cultures for 2 hours prior to lysis. Labels indicate samples that were stimulated for 2 minutes with 20 nM EGF or 3.2 nM heregulin. Aliquots of lysates were subjected to SDS-PAGE, followed by immunoblotting with phospho-specific antibodies.

• Supplemental Figure S3 -

  Fig. S3. (A) Membrane sheet prepared from SKBR3 cells after 5 minutes treatment with heregulin, then double-labeled for ErbB3 (large gold) and EGFR (small gold). Arrows point to isolated clusters of EGFR distributed across the image. A very large cluster of ErbB3 is in the center. (B) Digital representation of the same image, where ErbB3 are represented by green dots and EGFR by red dots. (C) Ripley's analysis of the same image, where the value for L(t)-t (solid red line) falls within the confidence interval and fails the Ripley's test. This indicates that, that although some EGFR can be found within the larger ErbB3 clusters, the EGFR label is randomly distributed with respect to ErbB3.

• Supplemental Figure S4 -

  Fig. S4. Summary of spatial statistics performed for figures in main text.