Fig. 2.

Generation of Arh^−/−;Dab2^−/−;hLDLR^+/+ mouse embryonic fibroblasts and stable cell lines. (A) Isolated embryonic fibroblasts from Arh^−/−;Dab2^flox/flox;hLDLR^+/+ mice were infected with adenoviruses encoding the cre-recombinase and clonally selected for cells that lack Dab2 expression. Cells lacking Dab2 were infected with retroviruses encoding no ARH (Vector), WT ARH (WT), ARH-C199A (C199A), ARH-C286A (C286A), or ARH-C199A/C286A (CC>AA). All introduced ARH cDNAs used the human ARH sequence. The mRNA produced by the viral vector is bicistronic and encodes both ARH and GFP with an internal ribosome entry site separating the two. Thus, ARH-expressing cells also express GFP, allowing transgenic cells to be FACS sorted by GFP fluorescence. (B) Immunoblots showing Dab2, ARH, LDLR, and α-tubulin expression by the parental Arh^−/−;Dab2^flox/flox;hLDLR^+/+ fibroblasts (Dab2) and by the Vector, WT, C199A, C286A, and CC>AA transgenic cells.