Expression of Myr Akt-ER and non-Myr Akt-ER constructs in B lymphocytes.

(A) Schematic depiction of the Myr Akt-ER and non-Myr Akt-ER constructs. Myristoylation sequence from Src, or a G-→ A mutation within this sequence (in the non-Myr Akt-ER version) was added to the N-terminus of PH-domain deleted (region between aa 4-129) Akt. This Akt, followed by a hemagglutinin (HA) tag, was fused to the estrogen receptor hormone-binding domain (ER). (B) A20 cells were transfected with Akt-ER constructs and the expression in different clones of Myr Akt-ER (clones 8, 24, 51, 58) or non-Myr Akt-ER (clones 1M, 13M) was analyzed by anti-HA immunoprecipitation, followed by anti-HA immunoblotting. (C) Lysates from some of the clones were immunoprecipitated with anti-ER antibodies and immunoblotted with anti-HA. Molecular mass standards (in kilodaltons) are indicated to the left. (D) Comparable expression of BCR and FcγRIIB1 in the clones. Parental A20 cells and those transfected with the Myr Akt-ER or the non-Myr Akt-ER were incubated with antibodies specific for BCR (thin trace) or FcγRIIB1 (dotted line trace) and their expression was analyzed by flow cytometry. The background staining on the cells is shown as a solid histogram (Ø).