WASp^EMM is degraded more rapidly and binds less WIP than WASp^WT, but after forced expression with lentiviral transduction, can stably express at similar protein levels to endogenous WASp. (A-B) U937 cells were transduced with EGFP-WASp by lentiviral infection at an multiplicity of infection of 5 to generate stable cell lines. Cells were labeled with ³⁵S cysteine/methionine (pulse), followed by 24 hours incubation in full RPMI with additional 2mM methionine and 2mM cysteine (chase). Samples were harvested before and after the chase period by lysis and anti-EGFP immunoprecipitation. Immunoprecipitates were resolved by SDS-PAGE and radio emission of dried gels was detected by phosphor screens and imaged using a Molecular Imager FX (Bio-Rad). Radio emission signals were quantified and the percent change in signal between 0 and 24 hours of chase was calculated. Differences between constructs in percent degradation from 3 experiments were analyzed by ANOVA. Nonadjacent lanes on Western blots are separated by solid gray lines. All lanes within each figure were on the same gel. (C) U937 cell lines stably transduced with EGFP-WASp constructs were sorted for similar EGFP expression. After 5 passages, cells were harvested and resolved lysates were immunoblotted for WASp and GAPDH (loading control). UT indicates untransduced. (D) Endogenous WASp bands were quantified by densitometry and differences between constructs from 4 experiments were analyzed by ANOVA. Absolute values were expressed as sympercentage differences from endogenous WASp levels in untransduced cells. Error bars represent SEM and dotted lines represent SEM of endogenous WASp concentration in untransduced cells. (E) U937 cells with standardized EGFP-WASp expression were harvested and lysates underwent anti-EGFP immunoprecipitation. Pre- and postimmunoprecipitation lysates were resolved by SDS-PAGE and immunoblotted for WIP. Immunoprecipitants were resolved and immunoblotted for EGFP and WIP. (F) EGFP and WIP bands from 6 experiments were quantified by densitometry. Differences between constructs were analyzed by ANOVA. ***P < .005, **P < .01, *P < .05, and ns indicates P > .05.