Calponin-3 is phosphorylated upon stimulation of B cell progenitors.

<p>A. Schematic illustration of the conducted screen designed to identify signaling components downstream of the pre-B cell receptor. B. Coomassie Blue staining of an SDS-PAGE showing constitutive (control) and pervanadate-induced tyrosine-phosphorylation of proteins in B cell progenitors. The position of the band corresponding to calponin-3 is marked by an arrow. C. Western blot indicating Syk-dependent phosphorylation of calponin-3 upon pervanadate stimulation in B cell progenitors. Pre-B cells transduced with an empty control vector or a with a vector encoding an HA-tagged calponin-3 were stimulated with pervanadate in the presence or absence of a Syk inhibitor for 3 min. Untreated cells served as a control. Cellular lysates either directly subjected to SDS-PAGE and western blotting or immunoprecipitated with an anti-HA antibody. Actin was used as a loading control. D. Western blot analysis for tyrosine phosphorylation of calponin-3 in the S2 Schneider cell system. S2 cells were transfected with expression constructs encoding calponin-3 and Syk, Lyn or Btk, respectively. Cellular lysates were subjected to SDS-PAGE and western blotting. E. Confocal image of pre-B cells expressing GFP or a calponin-3-GFP fusion protein, respectively. F. Western blot for analysis of calponin 2 and 3 expression in bone marrow cells cultured with IL-7 for 5d, in sorted CD19- and CD19+ splenic B cells, in total thymocytes and in the brain. Western blotting against actin was used as a loading control.