Frequency of spliced exons in MelJuSo cells.

*<p>A total number of 100 clones was analyzed.</p

Subcellular localization of endogenous BAT3 in % of cells.

<p>Subcellular localization of endogenous BAT3 in % of cells.</p

Identification of BAT3 splice variants.

<p><b>A.</b> Schematic presentation of BAT3 variants. BAT3 full-length containing exons 2 to 25 is shown on top (translation starts with exon 2). Identified BAT3 variants with deleted sequences are shown below. Deleted exons are indicated by triangles. Designation of the variants is shown on the right and the calculated number of amino acids (AA) of the BAT3 proteins is indicated on the left. UBL: ubiquitin-like domain, NLS: nuclear localization signal, BAG: Bcl-2 associated athanogene-domain, <b>B.</b> Expression of BAT3 isoforms. COS-7 cells were transfected with V5-tagged BAT3 cDNAs, lysed, separated by SDS-PAGE and immunoblotted for BAT3 with anti V5 antibody. BAT3 variants are indicated on the top and a molecular weight marker is shown on the left. Compared to the full length BAT3 with a molecular weight of about 130 kDa, the calculated sizes of the variants are the following: BAT3 Δ5 (128 kDa), BAT3 Δ11b (127 kDa), BAT3 Δ24 (125 kDa), BAT3 Δ5, 11b (125 kDa) and BAT3 Δ11b, 24 (122 kDa).</p

Tissue specific expression of <i>BAT3</i> splice variants.

<p>Tissue specific expression of <i>BAT3</i> splice variants.</p

Subcellular localization of BAT3 variants in transfected HeLa cells.

<p>Cells were transfected with BAT3 splice variants and stained after 24 hours with a monoclonal V5 antibody for evaluation by immunofluorescence microscopy. Left panel displays DAPI staining, second panel BAT3 staining, third panel merging of images and right panel shows corresponding phase contrast images. The displayed transfected cells show examples for nuclear (BAT3 full-length, upper panel), for nuclear and cytosolic (BAT3 Δ11B, middle panel) and for enhanced cytosolic staining (BAT3 Δ11B, 24, lower panel). Scale bars = 10 µm.</p

Detection of exon sequences in BAT3 cDNA clones by PCR.

<p><b>A.</b> Exon-intron-structure of the <i>BAT3</i> gene. Exon sequences are indicated as arrows. The start codon for translation in exon 2 is indicated. Potentially spliced exons are numbered. Exon 1 and 7 exist in different fragment lengths, 247 bp or 271 bp for exon 1 (NCBI) and 236, 254 or 278 bp for exon 7 (NCBI, ENSEMBL). Exons highlighted in grey could be deleted without changing the reading frame. The novel exon 11B with a length of 108 bp is labeled by arrow. Exon 11B also can be deleted without a change of the reading frame of the adjacent exon sequences. <b>B.</b> Schematic presentation of BAT3 cDNA. Exon boundaries are indicated and protein-encoding exons are numbered from 2 to 25. Potentially spliced exons are highlighted in grey. The position of complementary primer pairs (a to d) for forward (f) and reverse (r) PCR are indicated by arrows. <b>C.</b> Primers indicated in B were used to characterize BAT3 cDNA clones. Digestion of two BAT3 cDNAs is shown for example. Lane M contains size standards with bp indicated on the left. Lanes a: PCR products of clones 1 and 2 exhibit a band of 280 bp (no exon 11B) or of 380 bp (with exon 11B). Lanes b: Both clones generate a PCR product of 660 bp, indicating the presence of exon 9. Lanes c: The sizes of PCR fragments from clone 1 and clone 2 are 800 bp or 950 bp, respectively. This size is consistent with the presence or absence of exon 24. Lanes d: The presence of exon 5 is verified in clones 1 and 2 by a PCR product of 400 bp.</p

Subcellular localization of the BAT3 variants in % of transfected HeLa cells.

<p>Subcellular localization of the BAT3 variants in % of transfected HeLa cells.</p

Impact of leptomycin B treatment on the subcellular localization of transfected BAT3 splice variants and of endogenous BAT3.

<p><b>A.</b> HeLa cells transfected with BAT3 Δ11B,24 were cultured on coverslips in the presence (lower panel) or absence (upper panel) of leptomycin B (LMB) for 2 h. Cells were subsequently stained with V5 mAb and inspected with standard immunofluorescence microscopy (second panel). Left panel shows DAPI stained nuclei, third panel merging of images and right panel displays phase contrast images. Scale bars = 10 µm. <b>B.</b> Raji cells were cultured for 2 h in the presence (lower panel) or absence (upper panel) of LMB and then plated on coverslips. Cells were subsequently stained with the polyclonal anti-BAT3 serum and with ISCR3 mAb (HLA-DR) for evaluation by immunofluorescence microscopy. Left panel shows DAPI staining, second panel staining for BAT3, third panel staining for HLA-DR and images were merged in the right panel. Scale bars = 5 µm.</p

Subcellular localization of endogenous BAT3 in four cell types.

<p>HeLa cells (Adenocarcinoma), the human melanoma cell line MelJuSo, the B lymphoma Raji and monocyte-derived dendritic cells (moDCs) were plated on coverslips and stained for BAT3 using a polyclonal serum against a C-terminal peptide (middle lane). Cell nuclei (left lane) were visualized with DAPI (A) or 7AAD (B). Merged images are shown in the right lane. <b>A.</b> Immunofluorescence staining was evaluated with a standard fluorescence microscope and <b>B.</b> by confocal microscopy. Scale bars = 10 µm. <b>C.</b> Nuclear and cytosolic staining of endogenous BAT3 in Raji cells was evaluated in 10 single cells using ImageJ. MFI, mean fluorescence intensity per region of interest <b>D.</b> Western blot analysis of subcellular fractions from Raji and HeLa cells. Nuclei (N) and cytoplasm (C) were separated by SDS-PAGE and immunoblotted for BAT3, GADPH (cytosolic marker) and histone H3 (nuclear marker).</p

Characterization of Glucocerebrosides and the Active Metabolite 4,8-Sphingadienine from <i>Arisaema amurense</i> and <i>Pinellia ternata</i> by NMR and CD Spectroscopy and ESI-MS/CID-MS

Sphingolipid metabolites regulate cellular processes such as cell proliferation, differentiation, and apoptosis. In this study, glucocerebrosides (GluCer) from rhizomes of <i>Arisaema amurense</i> and <i>Pinellia ternata</i> were fully characterized using 1- and 2-dimensional nuclear magnetic spin resonance (NMR) and circular dichroism (CD) spectroscopy and tandem collision-induced dissociation mass spectrometry (ESI-MS/CID-MS). Three new acylated and seven known GluCer were elucidated with 4,8-sphingadienine (4,8-SD, d18:2) as backbone. 4,8-SD is a metabolite after enzymatical hydrolysis of GluCer in the gut lumen. In this study, 4,8-SD was hydrolyzed from GluCer and chromatographically purified on silica gel. In contrast to the GluCer, 4,8-SD showed cytotoxic effects in the WST-1 assay. GluCer with 4,8-SD as sphingoid backbone are present in plants consumed as food, such as spinach, soy, and eggplant