Figure 2
Knockdown Syk expression confers a competitive disadvantage through the induction of apoptosis. (A) Semiquantitative Western blot analysis of Syk knockdown in murine tumor B cells (DBL-114) with anti-Syk (top panel) and anti–β-actin (bottom panel) of the stripped blot. Cells were infected with empty vector (pLL3.7), control shRNA (shLuc), or Syk-specific shRNA (shmSyk). Percent knockdown determined from 2 separate blots. (B) Plot of in vitro competition assay in TBL-1 or DBL-114 cells infected with either control shLuc or shmSyk. Competition between infected (GFP+) and uninfected cells (GFP−) cells is shown. All samples were normalized to percent of GFP+ cells on day 2 after infection and shown relative to empty vector pLL3.7 thereafter (n = 4). (C) Plot of 7-AAD–positive cells normalized to levels of 7-AAD expression in cells expressing shLuc relative to 7-AAD expression in shmSyk (n = 3). (D) PI staining of DBL-114 tumor B cells expressing shLuc (gray filled, top number) or shmSyk (black trace, bottom number) 2 days after sorting for greater than 95% GFP-expressing cells.

Knockdown Syk expression confers a competitive disadvantage through the induction of apoptosis. (A) Semiquantitative Western blot analysis of Syk knockdown in murine tumor B cells (DBL-114) with anti-Syk (top panel) and anti–β-actin (bottom panel) of the stripped blot. Cells were infected with empty vector (pLL3.7), control shRNA (shLuc), or Syk-specific shRNA (shmSyk). Percent knockdown determined from 2 separate blots. (B) Plot of in vitro competition assay in TBL-1 or DBL-114 cells infected with either control shLuc or shmSyk. Competition between infected (GFP+) and uninfected cells (GFP) cells is shown. All samples were normalized to percent of GFP+ cells on day 2 after infection and shown relative to empty vector pLL3.7 thereafter (n = 4). (C) Plot of 7-AAD–positive cells normalized to levels of 7-AAD expression in cells expressing shLuc relative to 7-AAD expression in shmSyk (n = 3). (D) PI staining of DBL-114 tumor B cells expressing shLuc (gray filled, top number) or shmSyk (black trace, bottom number) 2 days after sorting for greater than 95% GFP-expressing cells.

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