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. 2010 Oct;177(4):1710–1724. doi: 10.2353/ajpath.2010.090903

Figure 2.

Figure 2

In vitro, NTD (1-35)-EGFP and mod NTD (1-35)-EGFP are functional substitutes for the synthetic peptides. A: EGFP fusion proteins were detected in the conditioned media of MEFs cells transfected with NTD (1-35)-EGFP or mod NTD (1-35)-EGFP. EGFP-containing proteins were isolated from conditioned media of transfected [EGFP, NTD (1-35)-EGFP or mod NTD (1-35)-EGFP] or control cells (not transfected) 72 hours after transfection by nucleoporation. EGFP containing proteins were isolated by pull-down using mouse anti-GFP coupled beads. Bound proteins were eluted in Laemmli buffer, separated by SDS-PAGE, transferred to nitrocellulose, and then immunoblotted for GFP. B–D: The presence of focal adhesions was assessed by interference reflection microscopy. At least 300 cells were counted per coverslip. Cells with more than five focal adhesions per cell were scored as positive. B: BAECs responded to NTD (1-35)-EGFP in a dose-dependent manner. Conditioned medium from NTD (1-35)-EGFP–transfected cells was diluted 1:10 with conditioned medium from mock-transfected cells. NTD (1-35)-EGFP–conditioned medium (squares), pEGFP-N1-transfected cell conditioned media (circles), and mock-transfected medium (triangles). hep I peptide (10 nmol/L) was used as a positive control for focal adhesion disassembly (dash-dot line). C: Wild-type and CRT (−/−) MEFs were transfected with NTD (1-35)-EGFP constructs. Twenty-four hours after plating, cells were analyzed by interference reflection microscopy for the presence of focal adhesions. Results are expressed as the mean number of focal adhesion positive cells ± SD; n = 3 replicates with P values calculated by two-parameter unpaired Student's t-test. D: NTD (1-35)-EGFP-secreted protein was active in the presence of serum. MEFs were transfected and grown for 24 hours in medium containing 10% FBS. Transfection efficiencies were measured and then transfected cell populations were mixed with mock-transfected cells to normalize the percentage of construct-expressing cells to 10% of each coculture population. Results are the mean percentage of focal adhesion positive cells ± SD; n for each group is indicated on figure. *P < 0.05.