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
"...in 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.