Overexpressed IL4R-Ibα becomes trapped in the ER and results in the generation of large platelets in vivo. (A) Platelets from 2 different transgenic lines, Tg33 and Tg51, have previously been analyzed for cell surface expression of the IL4R-GPIbα chimeric protein and platelet size,²⁴  and the results are summarized here. Note that surface expression of IL4R-Ibα in Tg51 mouse is approximately 1.6 times higher than that of Tg33 mouse, yet the resulting platelets are 30% larger. (B) Immunoblot analysis of the IL4R-Ibα chimeric protein from IL4R-Ibα Tg33 and Tg51 mouse platelet lysates. The samples were subjected to SDS-PAGE under nonreducing conditions and probed using an anti-hIL4R mAb. The IL4R-Ibα chimeric protein from these platelets exists in 3 forms; as a disulfide-linked dimer with GPIbβ, as a mature uncomplexed protein, and as an immature incompletely glycosylated protein present in the ER of the cell. Note that there is more of the ER-trapped form in the IL4R-Ibα Tg51 mouse platelet lysate. (C) Immunoblot analysis of filamin A and B content in platelets from wild-type, Tg33, and GPIb knockout mice, showing similar expression in all 3 lines. HSC70 was used as a loading control. (D) Analysis of the filamin ubiquitination status in platelets from wild-type, Tg33, and GPIb knockout mice. Filamins A was immunoprecipitated as described in “Methods” and their ubiquitination status probed using mAb P4D1. Overall ubiquitination profiles of platelet lysates among these platelets were similar, although ubiquitination of FlnA was barely detectable.