Xid mutation affects the stability of Btk protein. (A) Expression levels of Btk protein in Xid splenic B cells. The whole-cell lysates of splenic resting B lymphocytes obtained from control CBA/J (CBA) or Xid (Xid) mice were subjected to immunoblot analysis with an anti-Btk mAb (43-3B). The membrane was reblotted with anti-ERK2. Data are representative of 3 independent experiments with similar results. (B) The expression vector (0.5 μg and 1.0 μg per reaction, respectively) for either human wild-type Btk (WT) or XLA-derived mutants (R28P and R28C) was in vitro transcribed and translated in the presence of Transcend tRNA (Promega). The amounts of biotinylated Btk proteins were evaluated with streptavidin-HRP. (C) Jurkat T lymphocytes were transiently transfected with the expression vector for either human wild-type Btk (WT) or XLA-derived mutants (R28P and R28C), which contains EGFP downstream of an IRES. Whole-cell lysates were subjected to immunoblot with an anti-Btk antibody (top panel), followed by reblot with an anti-GFP antibody (bottom panel). (D) In vivo activation of Btk protein with Xid mutation. Splenic B cells obtained from 2 control CBA or 20 Xid mice were stimulated with or without 20 μg/mL anti-IgM F(ab′)2 (anti-μ) at 37°C for 3 minutes. Btk was then immunoprecipitated with an anti-Btk antibody, followed by immunoblot analysis with 4G10 (pY-Btk). The membrane was reblotted with 43-3B (Btk). Data in panels B through D are representative of 2 independent experiments with similar results.
