The amount of PECAM protected in the LBRC at 4°C is not affected by IQGAP1 knockdown. (A) HUVECs were chilled to 4°C and incubated with the monoclonal anti-PECAM antibody hec7 for the indicated time before being washed extensively and lysed. Lysates were incubated with recombinant Protein-G agarose beads to recover the surface PECAM that had been exposed and was bound to antibody (PECAM IP). PECAM that was not bound at 4°C (which had either not had time to bind PECAM or was protected in the LBRC) was recovered in the flow through (FT). The amount of PECAM in the various fractions was analyzed using Western blotting for PECAM (PECAM relative mobility, 135 kD; all blots in this figure are for PECAM). Equivalent amounts of each sample were loaded to allow for direct comparison of signal intensity for each time point (i.e., the amount of PECAM recovered in the immunoprecipitate (IP) corresponds directly to the amount reduced in the FT). The time course shows that the accessible surface pool of PECAM is quickly bound and saturated at 4°C. MW, molecular weight. (B) Control and IQGAP1 knockdown HUVECs were incubated with the anti-PECAM antibody hec7 at 4°C for 2 h as in A. After extensive washing and lysis, surface PECAM that was able to bind hec7 was recovered using Protein-G beads. To make sure the hec7-bound PECAM was fully recovered, the supernatant from the first IP was transferred to tubes with fresh Protein-G beads. This was repeated three total times (additional empty beads 1–3) before recovering the remaining LBRC-protected PECAM with additional Protein-G beads and hec7 (last lane, IP − beads + fresh antibody [aB]). The remaining FT was checked to make sure that all of the PECAM in the sample was recovered in the IPs (not shown). Equivalent amounts of each sample were loaded to allow for direct comparison of signal intensity. (C) The amount of PECAM recovered in each IP was quantified using densitometry. The amount that was protected from antibody at 4°C was calculated by dividing the amount recovered in the last IP by the total amount recovered in all IPs. Knockdown of IQGAP1 had no effect on the amount of PECAM that was protected at 4°C. The result was the same when calculated using the lysate (first lane) for the total amount of PECAM. Data shown are representative of three independent experiments, and error bars denote the standard deviation. (D) Because recently internalized (e.g., in endosomes) and intracellular (e.g., newly synthesized, but not yet trafficked to the surface) PECAM would be indistinguishable from LBRC-protected PECAM in the assays detailed in A–C, the amount of PECAM on the surface and the LBRC was examined for control and IQGAP1 knockdown HUVECs. Confluent monolayers were incubated for 15 min at 37°C with hec7 to label total plasma membrane PECAM (surface and LBRC). Cells were washed extensively to remove unbound antibody, lysed, and incubated with Protein-G beads to recover antibody-bound PECAM. The relatively small amount of PECAM left in the FT suggests that ablation of IQGAP1 does not alter the expression or intracellular distribution of PECAM.