Activation of endogenous Akt and MAPK and intracellular calcium flux occur normally in Myr Akt-ER–expressing cells.

(A) Parental A20 cells (lanes 1-3), Myr Akt-ER expressing cells (lanes 4-6) or the non-Myr Akt-ER cells (lanes 7-9) were left unstimulated (lanes 1, 4, 7), stimulated by cross-linking the BCR alone (lanes 2, 5, 8), or co–cross-linking the BCR with FcγRIIB1 (lanes 3, 6, 9). Cell lysates were analyzed for Akt phosphorylation by immunoblotting with phosphospecific anti-Akt antibody (Thr³⁰⁸). The same gel was stripped and reprobed with anti-Akt antibody to demonstrate the protein level in all lanes. (B) Myr Akt-ER or non-Myr Akt-ER expressing cells were stimulated with BCR alone or BCR+FcγRIIB1 co–cross-linking for 5 or 20 minutes in the presence or absence of 4-HT. The lysates were run by SDS-PAGE and probed with anti-phospho MAPK antibody. The addition of 4-HT did not alter the MAPK phosphorylation in response to BCR stimulation or the inhibition of phosphorylation by BCR+FcγRIIB1 cross-linking. (C) Myr Akt-ER expressing cells were loaded with Indo-1 and the 398/480-fluorescence ratio of Indo-1 as a measure of rise in intracellular calcium, was analyzed after cross-linking of BCR or BCR+FcγRIIB1. There was no detectable difference in the calcium flux profiles because of tamoxifen addition.