WASp^I294T 300.19 cells demonstrate increased levels of F-actin but express normal levels of surface L-selectin. (A) Flow cytometry histograms showing EGFP expression in murine 300.19 pre-B cells before (UnT) and after transfection with lentivirus encoding EGFP alone (SEW), WASp^WT, or WASp^I294T. WASp^WT- and WASp^I294T-transfected lines expressing EGFP had average MFI values of 61.8 and 106.7, respectively. Average MFI for EGFP-expressing cells (SEW) was 298.2. (B) Western blot of transfected and untransfected cells showing (i) EGFP-WASp fusion proteins and (ii) endogenous WASp. Lanes 1, 3, and 5 represent cell lines sorted for low EGFP MFI. Lanes 2, 4, and 6 represent cell lines sorted for high EGFP expression. GAPDH levels were monitored for equivalent loading (bottom panel). Relative expression levels were measured using densitometry readings and correction for loading variation. Normalized densitometry readings for EGFP-WASp^WT and EGFP-WASp^I294T were 147486 and 549950 (lanes 1 and 2) and 101303 and 177848 (lanes 5 and 6), respectively. Immunoblots are representative of 3 independent experiments. (C) F-actin levels were compared between EGFP⁺ transduced and EGFP⁺ untransduced cells in the same tube by flow cytometry. WASp^I294T cells demonstrate a significant increase in F-actin content compared with cells expressing WASp^WT (P = .001; 2-tailed t test). There was a small but significant increase in F-actin in WASp^WT cells compared with SEW cells (P = .004; 2-tailed t test). Error bars represent SD. ***P = .001. (D) Untransduced and transduced cells were labeled for L-selectin and analyzed by flow cytometry. Comparable levels of surface L-selectin expression were seen for all cell lines; average MFIs for 3 experiments are shown. All experiments in panels A, C, and D were performed in triplicate on 3 independent occasions.