Figure 5.

An increased level of CD154 stimulation can yield higher tyrosine phosphorylation. CD19⁺ B lymphocytes, containing 42% CD27⁺ cells, were stimulated as described in Fig. 3 with 1000, 5000 and 25 000 CD154 molecules per B lymphocyte. After 3 and 72 hr, B lymphocytes were selected using CD19-positive selection with magnetic beads, protein extracts were prepared, and tyrosine phosphorylation patterns were determined using 4G10 antiphosphotyrosine monoclonal antibodies. A peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG) was used as the secondary reagent, as described in Materials and methods. The blot was stripped and re-probed with an anti-actin antibody. These results are representative of two independent experiments. STD, molecular weight protein standard.