Additional Information about TRANSPATH Materials

Materials A1 to A5

In Category A materials, the whole organism or its parts are used. The organism can be wild type, a naturally occurring mutant, or genetically manipulated.

A1 Wild Type

In Material A1 (wild type), signaling is comparable to that of material B3 (primary cells) that have been put into tissue culture media with artificial additives and incubated for at least some hours. A1 materials are mainly used for isolation or purification of large amounts of proteins from whole organism or organs by either immunoprecipitation (M4) or biochemical approaches like gel filtration or anion exchange chromatography (M8, M23), or microscopical analysis (M3) in situ.

Example: TRANSPATH database: XN000033572 amphiphysin2(r) + FAK1(r) <=> amphiphysin2(r):FAK1(r) rat brain: (A1, M4)

Example: PubMed ID: 11826105
"...Immunoprecipitation from rat brain. S-SCAM was immunoprecipitated from the Triton X-100 extract of rat crude synaptosomes as described previously (Hirao et al., 2000). The immunoprecipitates were immunoblotted with either the anti-S-SCAM or the anti-beta-catenin antibody..."

A2 Natural Chromosomal Mutants Leading to a New Phenotype

A3 Knock-out Organisms or Their Organs, Tissues

Example: PubMed ID: 12379224
"...Generation of the EphB6 knockout mice. J1 and RW4 embryonic stem (ES) cells were electroporated with the linearized targeting vector and selected with geneticin on embryonal fibroblast feeder cells generated from the embryos of the cholecystokinin-B receptor knockout mice [24]. Fetal thymus organ culture. Fetal thymus organ cultures (FTOC) were done as described previously [27]. Briefly, the thymic lobes of anembryonic day 15.5 (E15.5) were organ cultured on the surface of Nucleopore filters (0.8 lm pore size, Costar, MA) supported on collagen hemostatic sponges (Integra Life Sciences, NJ) in a 24-well plate containing 0.5 ml RPMI1640 medium supplemented with 10% fetal calf serum, 50 lM 2-mercaptoethanol, 100 U/ml penicillin, 100 lg/ml streptomycin, and 1_ non-essential amino acids (Invitrogen, Netherlands) for 5 days at 37 _C under 5% CO2 in the air..."

A4 Transgenic Animals

Example: PubMed ID: 10973986
"...For nuclear matrix preparations from brain, nuclei were isolated from DRPLA transgenic mice as described elsewhere (Schilling et al. 1999 ), and the nuclear matrix was prepared from the isolated nuclei following the same protocol as for transfected cells, except that the final wash with 2 M NaCl was omitted. The subnuclear fractions were immunoblotted for atrophin-1 and ETO/MTG8 with antibodies AP142 (Wood et al. 1998 ) and 2174 (Sacchi et al. 1998 )..."

Example: PubMed ID: 11442350
"...Six HD transgenic mice at 5 months of age and age-matched nontransgenics (or six DRPLA mice at 12 months and age-matched nontransgenics), were tested.... Animals were killed after decapitation, their brains were immediately removed and bisected sagittally. In the analysis of 4-week-old animals, we homogenized entire hemibrains. We also analyzed the forebrains of older, severely affected, HD (at 6 months), and DRPLA (at 12 months) mice. Tissue was frozen rapidly on dry ice, and stored at 270°C until assayed. Frozen tissues were homogenized in 10-20 vol of chilled 50 mM sodium phosphate buffer, pH 7.0. An aliquot was then adjusted to 0.1 M sodium acetate, cooled, and centrifuged to sediment particulate matter prior to HPLC analysis. Monoamines were measured by HPLC with electrochemical detection, with sensors set at 150, 250, 350, and 500 mV (ESA Inc., Chelmsford, MA). Exactly 10 ml of tissue extract was injected onto a C18 reverse phase MD-150 column (ESA Inc.) and eluted at a flow of 0.6 ml/min for 20 min. The mobile phase consisted of 1.7 mM 1-tetraethylammonium and 8% acetonitrile in 75 mM sodium phosphate buffer, pH 2.9..."

A5 Induced Mutations (Undefined)

Materials B1 to B4

B1 Primary Cultures of Embryonic Cells

Example: PubMed ID: 11826105
"...Hippocampal neuron culture and hippocampal slice culture. Hippocampal neuron cultures were performed from embryonic day 18 embryos as described previously (Takeuchi et al., 1997; Goslin et al., 1998). Hippocampal slices were obtained from postnatal day 6 (P6) or P8 rats and cultured on Millicell CM culture plate inserts (Millipore, Bedford, MA) in Eagle's MEM containing 25% (v/v) HBSS, 6.5 gm/ l glucose, 100 U/ml penicillin, 100 _g/ml streptomycin, and 25% (v/v) horse serum at 32°C under 5% CO2..."

B2 Primary Cultures of Pluripotent Stem Cells

Not found in our annotated references so far, possibly obsolete.

B3 Primary Cultures (Other than Embryonic Cells or Pluripotent Stem Cells)

Example: PubMed ID: 12097473
"...Neuronal staining. Cultured hippocampal neurons were fixed with 4% paraformaldehyde, permeabilized by methanol, and incubated with primary antibodies [KIF1B_ (1189; 3 _g/ml), PSD-95 (SM55; 3 _g/ml), SAP97 (B9591; 3 _g/ml), S-SCAM (3 _g/ml), and MAP2 (1:200)], followed by Cy3- or FITC-conjugated secondary antibodies. Neuronal images were captured by confocal laser scanning microscopy (LSM510; Zeiss, Oberkochen, Germany)..."

B4 Transfected Primary Cultures (Recombinant Cells, Tissues, Organisms)

Example PubMed ID: 14764452
"...Pooled human umbilical venous endothelial cells (HUVECs) were purchased from CellSystems and cultured in endothelial basal medium (EBM; CellSystems) supplemented with hydrocortisone (1_g/mL), bovine brain extract (3 _g/mL), gentamicin (50 _g/mL), amphotericin B (50 _g/mL), epidermal growth factor (10 _g/mL), and 10% fetal calf serum (FCS) until the third passage. After detachment with trypsin, cells (4_105 cells) were grown in 6-cm cell culture dishes for at least 18 hours. Figure 3. Homeobox A9 regulates EphB4 expression. A and B, HUVECs were transfected with HoxA9 antisense oligonucleotides or with HoxA9 siRNA for 24 hours, lysed, and EphB4 expression was detected by Western blot analysis HUVECs were transfected with a myc-tagged HoxA9 wild-type construct. Cells were lysed 20 or 40 hours after transfection and expression of EphB4 was detected by Western blot analysis..."

Example: PubMed ID: 11114890
" ...Figure 4. ...(B) ...The natural CYP3A23 promoter DR3 (WT) or its mutant variants (M1 and IR6) were transfected into primary rat hepatocytes in the presence of expression vectors for CAR or SXR. Cells were subsequently mock treated or treated with indicated compounds. Results are shown as fold induction over solvent, and represent the averages and standard error from triplicate assays. The concentrations of compound were the same as in A..."

Materials C1 and D1

Materials C1 and D1 are permanent, highly manipulated cells such as permanent cell lines. Permanent cell lines are immortalized and therefore transformed (many cancer cell lines) and differ in their regulation/signaling in comparison to primary cells of the same healthy tissue.

C1 Permanent Tissue Culture Cells

Example: PubMed ID: 9214622
"..For morphological analysis, HT1080 and Swiss 3T3 cells were seeded onto glass coverslips at a density of 13105 cells per 35 mm dish, cultured overnight and fixed. sMDCK2 cells were seeded onto glass coverslips at a density of 13104 cells per 35 mm culture dish DMEM containing 10% FCS, and incubated for 16 h. The medium was then changed to DMEM without FCS, and the cells were incubated another 24 h. Serum-starved cells were stimulated with or without 10-7 M TPA (Sigma) for 15 min at 37°C, and fixed. For indirect immunofluorescence, cells were fixed in phosphatebuffered saline (PBS) containing 3.7% paraformaldehyde for 20 min at room temperature, and then permeabilized with 0.2% Triton X-100 in agarose gel containing 2.1% formaldehyde, and transferred to a Biodyne A filter (Pall BioSuport, NY). The filter was then hybridized with the PBS for 10 min..."

D1 Permanent Transfected/Infected Mammalian Tissue Culture Cells

Example: PubMed ID: 11826105
"...Pull-down assay. COS cells were transfected with various constructs of beta-catenin, and the extracts were prepared as described above. For each extract, 160 _l was incubated with either 200 pmol of GST-S-SCAM-10 containing PDZ4 and PDZ5 or 200 pmol of GST fixed on 14.5 _l of glutathione beads. After the beads were washed, the proteins on the beads were immunoblotted with the anti-Myc antibody..."

Materials D2, E1, and F1

Materials D2, E1, and F1 include various non-mammalian cell types that have been transfected, transformed, or infected to produce recombinant proteins.

D2 Recombinant Proteins Produced in Higher Eukaryotic Cells

D2 Materials produce recombinant proteins from cDNAs incorporated in plasmids (Baculovirus vectors, for example) and are non-mammalian high eukaryotic cells such as insect cells or transfected/microinjected Xenopus oocytes. 

Example PubMed ID: 10748061
" ...Baculoviruses allowing the expression of single subunits of the TFIIH core (XPB, XPD, p62, p52, p44, and p34) and of CAK (cdk7, cyclin H, and MAT1) were as described (17). Baculovirus encoding for hRARgDAB as an His-tagged fusion protein was constructed in the pVL 1392 vector (Pharmingen). Baculovirus expressing hRARa1 as a flag fusion protein was constructed in the pSK278 vector (28)..."

E1 Recombinant Proteins Produced in Yeast

Example: PubMed ID: 15265006
" ...The oligonucleotides that encoded the last 7 amino acid residues and a stop codon of rat HCN1-4 were cloned into a bait plasmid, pAS2-1 (BD Biosciences Clontech). The pACT2 prey plasmid containing the full-length rat tamalin was co-transfected with the above bait plasmids into yeast Y190 cells (BD Biosciences Clontech) and ß-galactosidase reporter gene assays were performed as previously described (Kitano et al. 2002)..."

F1 Recombinant Proteins Produced in Bacteria

Example: PubMed ID: 15265006
"...GST and MAL fusion proteins were expressed in E. coli and purified by glutathione Sepharose 4B beads (Amersham Biosciences) and amylose resin (New England Biolabs), respectively. The purified proteins were dialysed against PBS at 4 °C. GST and MAL fusion proteins were immobilized on glutathione-Sepharose 4B beads and amylose resin, respectively, and incubated with supernatants..."

Material F2

F2 Materials are produced in vitro, either by in vitro translation of proteins or production of synthetic peptides. 

Example: PubMed ID: 12464607
" vitro Translation Reaction-Atrophin-1 constructs were transcribed using T7 polymerase and then translated using the TNT system (Promega) in the presence of [35S]methionine. Translations were incubated with 10-50 ng of each of the caspases for 2 h in the following buffer: 20 mM PIPES, 100 mM NaCl, 1% CHAPS, 10% sucrose, 10 mM dithiothreitol, and 0.1 mM EDTA, pH 7.2, at 37 °C..." 

Example: PubMed ID: 10973986
"...Antibody APG840 was raised against synthetic peptide DRPLA-425 corresponding to residues 425-439 of human atrophin-1 at Cocalico Biologicals Inc., Reamstown, PA. This peptide is 100% conserved between human and mouse and was previously used to produce antibody AP142 in rabbit (Wood et al., 1998)..."

Material X1 Recombinant Proteins Produced in Unspecified Cells

Material X1 describes recombinant proteins produced in unspecified cells. This Material term is used when the expressing cell system has not been specified in the reference.

Material X2 Unspecified Material

Material X2 is used in cases where no information about the material could be obtained from the reference. This term is used as last choice, when all other efforts have failed.

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