Figure 2

Transfected SK-N-AS cells express ICAM-2 transcripts and proteins. A) RNA from control human dermal microvascular endothelial cells (HDMVEC) generated RT-PCR products of the predicted 631 base pairs. All ICAM-2 transfectants contained readily detectable ICAM-2 RNA. B) Bar graph depicting quantitation of the RT-PCR products shown in “A”. RNA expression levels were within 20% among the transfectants. This level of variability was not statistically significant. C) Immunoblots of whole cell lysates (40 μg protein/lane) demonstrated that control HBMEC-28 cells and WT transfectants expressed immunoreactive protein having an apparent molecular mass of 55-60 kDa. ICAM-2 variants expressed proteins of appropriately lesser apparent masses. Transfectants expressed equivalent levels of actin and α-actinin. D) Deglycosylation of proteins in whole cell lysates and subsequent immunoblots for ICAM-2 confirmed that all variants displayed the expected apparent molecular mass of ~30kDa. The “nonspecific” band that appears at ~36kDa in all lanes marked + PNGase F is the PNGase F protein itself. E) Quantitation of ICAM-2 variants (lanes indicated as + PNGase F) was done to compare relative amounts of ICAM-2 protein expressed by transfectants. Results were normalized to the level of actin expression for each cell line. F) ICAM-2 WT and variants localized to cell membranes. Experimental details are included in Methods. G) Membrane localization of ICAM-2 WT and gsv4,5 was confirmed by fluorescence activated cell sorting (FACS) of intact cells, incubated with an antibody recognizing the extracellular domain (ED) of ICAM-2 (CBR-IC2/2) and a PE-conjugated secondary antibody. Non-intact cells were gated out using light scatter parameters and propidium iodide uptake. FACS profiles shown are for PE-positive cells generated using negative control IgG (blue line) or anti-ICAM-2 (red line). H) Biotinylation experiments also demonstrated that ICAM-2 WT and variants localized to cell membranes. Experimental details are included in Methods.