ail was dispensable 5 / 16 Calponin-3 in B Lymphocyte Development . Lastly, western blot analysis revealed strong expression of calponin-3 in primary B cell precursors as well as in mature B cells, albeit to slightly lower levels compared to the brain. In contrast, calponin-3 was undetectable in non-B cells of the spleen, whereas thymic cells seemed to express low amounts. Family member calponin-2 was abundant in the thymus and in splenic B cells, but only weakly expressed in B cell precursors, whereas calponin-1 was not detectable in any of the analyzed cell types. Taken together, this indicates that calponin-3 is specifically expressed in early B lymphocytes, localizes to the plasma membrane and becomes tyrosine phosphorylated in a Syk-dependent manner upon stimulation of B cell precursors. Targeting of the Cnn3 locus Based on our initial screen and the in vitro analyses in pre-B cells, we were wondering whether calponin-3 plays a role in early B cell development. To investigate this in vivo, a targeting vector Fig 1. Calponin-3 is phosphorylated upon stimulation of B cell progenitors. 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 and pervanadateinduced 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 MedChemExpress 518303-20-3 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. doi:10.1371/journal.pone.0128385.g001 6 / 16 Calponin-3 in B Lymphocyte Development Fig 2. Targeting of ES cells to generate PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19696528 a floxed calponin-3-GFP knock-in. A. Schematic illustration of the Cnn3 locus, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19697401 the targeting construct and the Cnn3 locus after targeting. The targeting strategy aimed at replacing exons 2 to 5 with a floxed mini gene corresponding to exons 2 to 7 fused to a GFPcDNA. Exons and the mini gene are represented by grey boxes, the neomycin-resistance gene is illustrated by a white box. LoxP-sites are depicted as black triangles, FRT-sites as white triangles. Restriction sites for the enzymes used for southern blot analysis are indicated. Please note that the illustration is not in scale. B. Southern blot analysis of the targeted clone used for blastocyst injection. Genomic DNA was digested by NcoI, HindIII, BamHI or KpnI, respectively, separated by agarose gel electrophores
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