Figure 7. Peritoneal B-1 cells express high pLyn levels.

(A) Peritoneal B-1 and splenic B-2 cells from C57BL/6 mice were resuspended in serum free IF-12, rested for 30 min at 37°C and immunoprecipitated with anti-Lyn antibody and probed for pSFK and Lyn.

(B) Peritoneal B-1 and splenic B-2 cells from wild type C57BL/6 mice were stimulated with biotinylated anti-IgM and/or anti-CD19 + avidin and treated as in panel A above to obtain whole cell lysates and immunoblotted for detection SHP-1 (~60 kDa), membrane was stripped and re-probed for actin as gel loading control.

(C) Whole cell lysates obtained from similar treatment as in panel A were immuno-precipitated with anti-Lyn antibody. The blot was probed for SHP-1, stripped and reprobed for Lyn to correct for gel loading.

(D and E) CD5-SHP-1 association is important for the negative regulatory role of CD5 in B-1 B cells: Total splenocytes from 3–4 week old viable motheaten (me/me^v) and negative littermate control mice were treated similar to the splenocytes from wild type C57BL/6 mice in Fig. 2B. Duplicate set of cell suspensions stained independently for B-1 (B220⁺ CD43⁺) and B-2 (B220⁺ CD43⁻) B cells were stimulated with avidin after 30 second relative calcium concentration baseline determination. This is one of three independent experiments. The responses from motheaten B-1 cells are shown in panel D and those from B-2 in motheaten compared to littermate control mice in panel E.