Figure 5.

Enrichment of cDNA library by MEC. A. Fraction of HEK293 cells that immunolabel with the BMSPA as defined by falling into the fluorescence sorting gate (illustrated in panel B). Values are normalized to that of the positive control, Lutheran cDNA-transfected cells (~25% of the total number of Lutheran-transfected cells fall into the sorting gate). Open bars indicate pools sorted without any plasmid dilution prior to transfection, whereas the striped bars indicate those that were sorted with a 100-fold dilution of cDNA pool prior to transfection. Error bars denote SEM for 3 measurements each; * = p<0.05 compared to previous MEC round as determined by a two-tailed Student's t-test. B. Flow cytometry dot plots indicating the fluorescent labeling of transfected HEK293 cells for some representative samples from Figure 5A. Black events (dots) indicate the non-transfected (or not recognized by BMSPA) cell population whereas the gray events (dots) indicate cells that fall within the cell sorting gate (outlined region). A conservative sorting gate was used to recover even those membrane protein-encoding cDNA that yield low fluorescence signals. Hence, some negative events (cDNA that do not encode membrane proteins, e.g. PCDNA1.1) also fall within the sort gate and are recovered in a given MEC round, but are subsequently lost due to the multiple round nature of the MEC process. Values in parentheses are consistent with Figure 5A, and indicate the fraction of transfected cells that are fall within the sort gate (gray dots), along with SEM. In all cases, the only cell populations evaluated and shown in the flow cytometric dot plots were those that exclude propidium iodide nuclear stain.