Figure 2. CD5 is required for CK2-induced phosphorylation of STAT3 in CLL cells.

(A) CD5 is constitutively phosphorylated in CLL cells. Western immunoblotting detected CD5 and tyrosine pCD5 in CLL cells from the PB of eight randomly selected CLL patients. C, control (Jurkat cells). (B) CD5, CK2, and serine pSTAT3 co-immunoprecipitate. CLL cell lysates from three patients were immunoprecipitated with anti-CD5 antibodies. The immune complex was separated using SDS-PAGE, and serine pSTAT3, CK2, and CD5 were detected in the immunoprecipitate by Western immunoblotting. HeLa and Jurkat cells were used as controls. I.P., immunoprecipitate; B, beads. (C) CD5-neutralizing antibodies reduce serine pSTAT3 levels in CLL cells. CLL cells were incubated with CD5-neutralizing antibodies, isotype antibodies, or culture media. After 2 hours, the cells were harvested and subjected to Western immunoblotting. As shown, compared with untreated cells, the levels of CD5 and serine pSTAT3 were lower in cells that were incubated with CD5-neutralizing antibodies, but not in cells incubated with the isotype control antibodies, whereas the levels of STAT3 were unchanged by any treatment. Densitometry analysis was used to quantify protein levels. Abs.: antibodies. (D) CD5-siRNA reduces the phosphoserine STAT3 levels in CLL cells. CLL PB cells from two patients were transfected with CD5-siRNA using electroporation. After 48 hours, the cells were harvested and processed. Transfection efficiency was 35%, as assessed by flow cytometry detecting cells with intracellular GFP-conjugated siRNA. Left panel: CD5-siRNA significantly reduced CD5 mRNA levels. qRT-PCR was used to detect CD5 transcripts. The δ-δ cycle threshold method was used to determine the relative fold change in CD5 transcripts after transfection with CD5-siRNA. Right panel: Western immunoblotting of CLL cells from two patients transfected with CD5-siRNA or GAPDH were analyzed using Western immunoblotting. As shown, CD5-siRNA, but not GAPDH, significantly reduced the protein levels of CD5 and serine pSTAT3 compared with levels in untreated CLL cells. K-562, HeLa, and Jurkat cells were used as positive controls.