Analysis of autonomous BCR signaling capacity. (A-C,E) Flow cytometry analysis of Ca2+ flux after activation of the ERT2-BLNK fusion protein by 4-OHT in TKO cells expressing representative leukemic BCRs encoded by transgenic or endogenous HCs. Addition of 4-OHT is marked by an arrow. (D) Analysis of BTK phosphorylation in TKO cells expressing an autonomously active anti-PtC BCR. TKO cells expressing the anti-HEL BCR were used as a negative control. (F) Analysis of pBTK, pBLNK, and phospho-ERK (pERK) levels in primary Eμ-TCL1 leukemias expressing a transgenic anti-PtC BCR, a transgenic anti-Sm BCR, or a nontransgenic BCR. Western blot analysis was performed on cellular extracts obtained from untreated cells (unt.) or cells that were treated for 2 hours with 1 μM SYK inhibitor R406.
