Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Translin coactivates steroidogenic factor-1-stimulated transcription.

doi: 10.1210/me.2005-0355

Figure Lengend Snippet: Fig. 2. Antibody Supershift of StF-IT-2;DNA Complexes with a Translin Antibody Extracts from MA-10 cells (left panel, lanes 1–5; right panel, lanes 9–12) or fraction 28 (middle panel, lanes 6–8) from the StF-IT-2 purification (Fig. 1) were incubated with the 447/ 419 oligonucleotide (lanes “Probe”). Two protein:DNA com- plexes, SF-1:DNA and StF-IT-2:DNA, are formed with MA-10 cell extracts (left panel, lane 2), indicated by arrows. A single complex corresponding to StF-IT-2:DNA formed with fraction 28 (middle panel, lane 6). The SF-1:DNA and StF-IT-2:DNA complexes are competed by 100-fold excess 447/419 oli- gonucleotide (lane 3, cold). Addition of an antibody against translin supershifts the StF-IT-2:DNA complex, and forms an- other complex, StF-IT-2 (lanes 4 and 7). Addition of preimmune serum (lanes 5 and 8) or addition of antibodies against 14-3-3 or 14-3-3 (right panel, lanes 11 and 12) has no effect.

Article Snippet: Antisera were affinity-purified polyclonal rabbit antibodies directed against recombinant mouse TB-RBP (75) or were polyclonal rabbit antibodies directed against the C-terminal amino acids of translin or TRAX (118, 119), or commercially available antibodies against 14-3-3 proteins (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Purification, Incubation

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Translin coactivates steroidogenic factor-1-stimulated transcription.

doi: 10.1210/me.2005-0355

Figure Lengend Snippet: Fig. 5. DNA Binding and Transcriptional Activation by Mu- tant Translin A, Gel shift assay. Recombinant mouse translin (wild type) or mutant translin (Mut) were incubated with a double- stranded, kinased 32P-labeled 447/419 oligonucleotide probe (DS), or with a single-stranded sense (SS-S) or anti- sense (SS-AS) oligonucleotide probe. Wild-type translin, but not mutant translin, forms a protein:DNA complex with these probes. B, Luciferase activity in HeLa cells after transfection with luciferase reporter constructs (1 g) 447/419 p36Luc, and expression vectors containing wild-type trans- lin, mutant translin, SF-1, or a combination of translin (wild type or mutant) plus SF-1. Transfected cells were assayed 48 h after transfection. Transfection efficiencies were con- trolled using the dual Renilla/firefly luciferase assay system. Results are means SE from triplicate wells from three ex- periments.

Article Snippet: Antisera were affinity-purified polyclonal rabbit antibodies directed against recombinant mouse TB-RBP (75) or were polyclonal rabbit antibodies directed against the C-terminal amino acids of translin or TRAX (118, 119), or commercially available antibodies against 14-3-3 proteins (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Binding Assay, Activation Assay, Gel Shift, Recombinant, Mutagenesis, Incubation, Labeling, Luciferase, Activity Assay, Transfection, Construct, Expressing

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Translin coactivates steroidogenic factor-1-stimulated transcription.

doi: 10.1210/me.2005-0355

Figure Lengend Snippet: Fig. 7. GST Pull-Down Assays A, SF-1 binds to translin. [35S]SF-1 was incubated with GST (lane GST) or GST-translin (lane GST-translin) bound to the glutathione- Sepharose matrix, and specifically bound proteins separated on 12% SDS polyacrylamide gels. The lane [35S]SF-1 contains 10% of the [35S]SF-1 applied to the beads. Similarly, [35S]translin was incubated with GST (lane GST) or with GST-translin (lane GST-translin) bound to the glutathione-Sepharose matrix, and specifically bound proteins were separated on 12% SDS polyacrylamide gels. The lane [35S]translin contains 10% of the [35S]translin applied to the matrix. Arrows indicate the 35S-labeled SF-1 and the 35S-labeled translin. B, Regions of SF-1 that bind to translin. Various regions of SF-1 used in the pull-down assays were cloned as indicated by the amino acid numbers below the cartoon; the regions of SF-1 included in these constructs are indicated by horizontal lines below the SF-1 line cartoon, on the left. Each of these constructs was labeled with [35S]methionine in vitro, and incubated with GST-translin bound to the glutathione-Sepharose matrix. The lanes input contain 10% of the 35S-labeled SF-1 fragments applied to the beads. DBD, DNA binding domain (amino acids 1–79); LBD, ligand binding domain (amino acids 225–451); AF2, activating function 2 domain (amino acids 451–462). C, Translin activation of SF-1 hinge mutants. Three deletional hinge mutants of SF-1 were cloned into p36Luc and transfected with the 447/419 luciferase reporter into HeLa cells in the presence of nothing or translin expression vector. SF-1 mutants had deletions in amino acids 101–169 (SF-1 101–169), amino acids 101–225 (SF-1 101–225), or amino acids 170–225 (SF-1 170–225). D, Translin activation of SF-1 serine 203 mutant. A serine 203 to alanine mutant of SF-1 (Ser 203 Mut) was transfected with the 447/419 luciferase reporter into HeLa cells in the presence of nothing or translin expression vector. Transfected cells were assayed 48 h after transfection. Transfection efficiencies were controlled using the dual Renilla/firefly luciferase assay system. Results are means SE from triplicate wells from three experiments.

Article Snippet: Antisera were affinity-purified polyclonal rabbit antibodies directed against recombinant mouse TB-RBP (75) or were polyclonal rabbit antibodies directed against the C-terminal amino acids of translin or TRAX (118, 119), or commercially available antibodies against 14-3-3 proteins (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Incubation, Labeling, Clone Assay, Construct, In Vitro, Binding Assay, Ligand Binding Assay, Activation Assay, Transfection, Luciferase, Expressing, Plasmid Preparation, Mutagenesis

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Translin coactivates steroidogenic factor-1-stimulated transcription.

doi: 10.1210/me.2005-0355

Figure Lengend Snippet: Fig. 9. TRAX Is Part of the StF-IT-2:DNA Complex A, Gel shift assay using MA-10 cell extracts, incubated with a 32P-labeled 447/419 oligonucleotide. Addition of an anti- body against TRAX (66) supershifts the StF-IT-2 complex, and forms another complex, StF-IT-2. Addition of preimmune se- rum has no effect. B, Gel shift assay using recombinant TRAX and translin prepared in bacteria. Recombinant TRAX or translin were incubated with a 32P-labeled 447/419 rP450c17 dou- ble-stranded (DS) or single-stranded (SS) oligonucleotide probe; TRAX does not bind to DNA because there is no protein: DNA complex, whereas translin prepared in a similar fashion binds to the 447/419 oligonucleotide. C, Luciferase activity in HeLa cells. HeLa cells were transfected with the 447/419 p36Luc reporter construct together with expression vectors for translin, TRAX, SF-1, or a combination of the three vectors. Transfections with translin or TRAX alone contained 0.15 g of plasmid; transfections with both translin and TRAX contained either 0.075 g of each plasmid [SF-1/(Trans/Trax)/2], or 0.15 g of each plasmid (SF-1/Trans/Trax). Transfected cells were assayed 48 h after transfection. Results are means SE from triplicate wells from three experiments.

Article Snippet: Antisera were affinity-purified polyclonal rabbit antibodies directed against recombinant mouse TB-RBP (75) or were polyclonal rabbit antibodies directed against the C-terminal amino acids of translin or TRAX (118, 119), or commercially available antibodies against 14-3-3 proteins (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Gel Shift, Incubation, Labeling, Recombinant, Bacteria, Luciferase, Activity Assay, Transfection, Construct, Expressing, Plasmid Preparation

Journal: Cell and Tissue Research

Article Title: Expression of choline and acetylcholine transporters in synovial tissue and cartilage of patients with rheumatoid arthritis and osteoarthritis

doi: 10.1007/s00441-014-2036-0

Figure Lengend Snippet: Human primer pairs used for qualitative an real-time RT-PCR ( CHT high-affinity choline transporter, CTL choline transporter-like, OCTN organic cation transporters novel, OCT organic cation transporter, VAChT vesicular acetylcholine transporter, βMG β2-microglobulin)

Article Snippet: Rabbit anti-human OCT1 (1:150); Aviva Systems Biology (#ARP41516_T100) , OCT1 blocking peptide (200 μg/ml); Aviva Systems Biology (#AAP41516).

Techniques: Quantitative RT-PCR, Sequencing

Journal: Cell and Tissue Research

Article Title: Expression of choline and acetylcholine transporters in synovial tissue and cartilage of patients with rheumatoid arthritis and osteoarthritis

doi: 10.1007/s00441-014-2036-0

Figure Lengend Snippet: Antibodies and blocking peptides used for single-stain immunohistochemistry ( CHT high-affinity choline transporter, CTL choline transporter-like, OCT organic cation transporter)

Article Snippet: Rabbit anti-human OCT1 (1:150); Aviva Systems Biology (#ARP41516_T100) , OCT1 blocking peptide (200 μg/ml); Aviva Systems Biology (#AAP41516).

Techniques: Blocking Assay, Immunohistochemistry, Recombinant

Journal: Cell and Tissue Research

Article Title: Expression of choline and acetylcholine transporters in synovial tissue and cartilage of patients with rheumatoid arthritis and osteoarthritis

doi: 10.1007/s00441-014-2036-0

Figure Lengend Snippet: Expression of organic cation transporter mRNA in the human joint. a , b RT-PCR for organic cation transporter 1-3 ( OCT1-3 ) and organic cation transporter novel ( OCTN1 ) mRNA in synovia ( a ) and cartilage ( b ) from the hip of OA and RA patients ( lanes M 100 bp ladder, lanes + RT+, lanes − RT-). CaCo-2 cells ( CC ) were used as a positive control for OCT2 mRNA expression and water ( H 2 O ) was employed instead of cDNA template as a negative control

Article Snippet: Rabbit anti-human OCT1 (1:150); Aviva Systems Biology (#ARP41516_T100) , OCT1 blocking peptide (200 μg/ml); Aviva Systems Biology (#AAP41516).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control

Journal: Cell and Tissue Research

Article Title: Expression of choline and acetylcholine transporters in synovial tissue and cartilage of patients with rheumatoid arthritis and osteoarthritis

doi: 10.1007/s00441-014-2036-0

Figure Lengend Snippet: CTL1- and CTL2-positive cells express the ACh-synthesizing enzyme, carnitin acetyltransferase ( CarAT ). a Single-staining for CTL1 in synovial tissue. b , c Double-staining for CarAT ( blue ) and CTL1 ( brown ) in synovial tissue. The boxed area in b indicates the region shown at higher magnification in c . d Single-staining for CTL2 in synovial tissue. e , f Double-staining for CarAT ( blue ) and CTL2 ( brown ). The boxed area in e indicates the region shown at higher magnification in f . g-i Single-staining for CHT1 ( g ), OCT1 ( h ) and OCT3 ( i ) on synovial tissue

Article Snippet: Rabbit anti-human OCT1 (1:150); Aviva Systems Biology (#ARP41516_T100) , OCT1 blocking peptide (200 μg/ml); Aviva Systems Biology (#AAP41516).

Techniques: Staining, Double Staining

Journal: Cell and Tissue Research

Article Title: Expression of choline and acetylcholine transporters in synovial tissue and cartilage of patients with rheumatoid arthritis and osteoarthritis

doi: 10.1007/s00441-014-2036-0

Figure Lengend Snippet: Representation of the expression of choline and acetylcholine transporters in the human joint. The transporters CTL1-5, OCT1, OCT3, and OCTN1 are expressed in the synovia and the cartilage of the human joint. The classic neuronal choline transporter CHT1 is only expressed in synovial tissue, and not in the cartilage of the joint

Article Snippet: Rabbit anti-human OCT1 (1:150); Aviva Systems Biology (#ARP41516_T100) , OCT1 blocking peptide (200 μg/ml); Aviva Systems Biology (#AAP41516).

Techniques: Expressing