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 (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.
Fig 2.
Targeting of ES cells to generate a floxed calponin-3-GFP knock-in.
A. Schematic illustration of the Cnn3 locus, 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 GFP-cDNA. 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 electrophoresis and blotted. The blot was hybridized with the internal 5’- and the external 3’-probe as indicated in A. Arrows indicate the positions of fragments corresponding to the non-targeted as well as the targeted allele.
Fig 3.
Calponin-3-GFP is expressed from the endogenous Cnn3 locus.
A. RT-PCR analysis of the Cnn3 mRNA splicing in targeted cells. Total RNA from the pre-B cell line Oct as well as from pre-B cells derived from a Cnn3 ki f/f mouse was transcribed into cDNA and analyzed by PCR. Primer pairs for PCRs 1 and 2 are depicted in the schematic illustration of the targeted allele. A sample lacking cDNA served as a control. B. Western blot analysis for expression of the calponin-3-GFP fusion protein from the targeted Cnn3 locus. Pre-B cells derived from a Cnn3 ki f/f mouse or a non-targeted littermate were lysed and subjected to SDS-PAGE and blotting. Oct pre-B cells expressing GFP and calponin-3-GFP were used as a control. Equal loading was demonstrated by anti-eIF4α. C. Flow cytometric analysis of cells from a Cnn3 ki f/f mouse or a non-targeted +/+ littermate. Cells were isolated from the bone marrow of the respective mice, stained with an anti-IgM antibody and analyzed for expression of IgM and GFP by flow cytometry. Numbers indicate the percentage of cells in the respective gate.
Fig 4.
High expression of calponin-3 throughout B cell development.
A. Staining pattern and gating strategies for cells isolated from the bone marrow, the spleen, lymph nodes and the peritoneal cavity. Numbers indicate the respective populations as analyzed in B. Expression of calponin-3-GFP in different B cell subsets derived from a Cnn3 ki f/f mouse or a +/+ littermate. Cells were isolated, stained and analyzed as indicated in A. Bar graphs indicate the ratio of the GFP MFI of ki f/f cells versus that of +/+ cells. Data represent 4 independent experiments. For statistical analysis, normalized GFP MFI values of control and ki f/f littermates were compared by a paired t-test (p≤0.05 = *, p≤0.01 = **, p≤0.001 = ***). LN, lymph node; PC, peritoneal cavity.
Fig 5.
Deletion of calponin-3 results in exencephaly.
A. Targeted Cnn3 locus before and after Cre-mediated deletion, illustrated as in Fig 2A. B. Reflected light images of Cnn3 ko d/d embryos their +/+ littermates as a control. C. Southern blot analysis of a control mouse (+/+), a heterozygous Cnn3 (+/d) and a homozygous Cnn3 knockout mouse (d/d). BamHI-digested genomic DNA from the respective mice was separated by gel electrophoresis, blotted and hybridized with the 3’ external probe. Arrows indicate the positions of fragments corresponding to the wild-type (+) as well as the deleted (d) allele. D. Fetal brain tissue of a homozygous Cnn3 knockout and a control littermate was lysed, subjected to SDS-PAGE and western blotting and probed for calponin-3 and actin expression as a control.
Fig 6.
Conditional deletion of calponin-3 does not affect early B cell development and function.
A. Percentages of different developmental stages and cell types derived from the bone marrow of control and B cell-specific Cnn3 knockout mice. Staining and gating of cells were performed according to Fig 4A. Control littermates (+/+ or +/f, positive for mb1-Cre) are depicted as black dots, knockout animals (f/d or f/f, positive for mb1-Cre) as white squares. Black bars mark the averaged percentage of cells for each subgroup. Percentages of cells in control and knockout animals were compared in an unpaired t-test (p>0.05 = not significant, n.s.). B. Western blot indicating induced signaling of control and calponin-3-deficient cells upon pre-BCR crosslinking. BM-derived B cells were starved for 30 min and then stimulated with an anti-μ antibody for 3 min. Cellular lysates were subjected to SDS-PAGE and western blotting. Anti-actin was used as a loading control, anti-calponin-3 confirmed the genotype of the used cultures. The band corresponding to calponin-3 is marked by an arrow, whereas non-specific signals from the polyclonal anti-calponin-3 antibody are labeled with asterisks. C. Induced calcium flux in B cell precursors derived from the bone marrow of control and knockout mice. Bone marrow cells cultured in the presence of IL-7 for 5 d were loaded with Indo-1, stimulated with pervanadate (marked by arrow) and analyzed by flow cytometry.