Blk is constitutively active in malignant CTCL cell lines. (A) Malignant CTCL cells (MF2000) and Ramos B cells were incubated without (−) or with LckI (10 μM) for 30 minutes. Then, 20 μg/mL goat anti–human IgM F(ab′)2 fragments (α-IgM) were added as indicated, and the cells were incubated for 5 minutes further. Subsequently, the cells were lysed, and Blk was immunoprecipitated (IP) from the total cell lysates. Finally, the levels of pY-Blk and Blk were determined by WB using phosphotyrosine and Blk-specific antibodies, respectively. (B) Blk was immunoprecipitated from total cell lysates of malignant (CTCL) and nonmalignant (NM) cell lines as well as from a psoriatic T-cell line (PS). Both total cell lysates (Lysate) and immunoprecipitated Blk (IP: Blk) were analyzed by WB using antibodies against phosphotyrosine (pY-Blk), Blk, and Erk. (C) Malignant CTCL cell lines were incubated for 30 minutes with LckI (10 μM) or vehicle (−). Then immunoprecipitated Blk and respective total cell lysates were analyzed by WB. (D) MF2000 cells were incubated with LckI (10 μM) for 30 minutes and lysed, and Blk was immunoprecipitated from the total cell lysates. The precipitated Blk was incubated for different time intervals (minutes) in kinase buffer without or with LckI (2 μM) and constitutive active Blk (1 ng/μL) as indicated. The levels of pY-Blk and Blk were analyzed by WB. For pY-Blk, films exposed for 2 or 10 minutes are shown. (E) WB analysis of Csk and Erk expression in the indicated cell lines.
