Journal: Urology

Article Title: Preliminary immunohistochemical characterization of a monoclonal antibody (pro:4-216) prepared from human prostate cancer nuclear matrix proteins

doi: 10.1016/s0090-4295(97)00337-3

Figure Lengend Snippet: FIGURE 2. NMP composition of normal human prostate (A) and human prostate cancer (C) taken from a radical prostatectomy specimen from a patient with clinical/y localized prostate cancer. High resolution two-dimensional gel electrophoresis of NMP preparations were isolated and analyzed as described in Methods. Panels B and D represent Western immunoblots using PRO:4-2 16 monoclonal antibody; immunoblotting technique as described in Methods. Arrowhead represents the localization of NMP(s) with immunoreactivity to the PRO:4-2 16 mouse monoclonal anti- body generated against human prostate cancer nuclear matrix preparations. kD = molecular weight in thousands; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis; pl = isoelectric point.

Article Snippet: The mouse monoclonal antibodies were UROLOGY 50 (51, 1997 801 then detected with a CY3 conjugated goat anti-mouse antibody (Jackson ImmunoResearch, West Grove, Pa) and DNA was visualized with DAPI (4,6-diamidino-2-phenylindole) at 2 /.

Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Isolation, Western Blot, Generated, Molecular Weight, SDS Page, Polyacrylamide Gel Electrophoresis

Journal: Urology

Article Title: Preliminary immunohistochemical characterization of a monoclonal antibody (pro:4-216) prepared from human prostate cancer nuclear matrix proteins

doi: 10.1016/s0090-4295(97)00337-3

Figure Lengend Snippet: FIGURE 3. lmmunohistochemical staining of human prostate tissue with the mouse monoclonal antibody PRO:4- 2 16. Anti-mouse IgM secondary antibody (Vector’s ABC Elite Kit). 3-3’-Diaminobenzidine tetrahydrochloride (DAB) was used as a chromogen following ethyl green counterstain. (A) Histologically normal prostate tissue (nuclei dem- onstrate an IHC intensity score of 0). (B) Histologically normal prostate tissue with the emphasis on stromal nuclei (weakly positively staining stromal nuclei represent an IHC intensity score of 1). (Cl Adenocarcinoma of the prostate from a tumor with Gleason grade 3 + 3 = 6 (IHC intensity score of 3). The cancerous glands surround a noncancerous gland. (0) A prostatic gland demonstrating PIN (positively staining nuclei represent an IHC intensity score of 1 to 2). The arrow in panel B identifies an area of prostatic stroma. The small arrows in panel C identify prostatic ade- nocarcinoma glands and the large arrow in panel C identifies a histologically normal gland found among cancerous glands. The arrow in panel D represents histologic PIN.

Article Snippet: The mouse monoclonal antibodies were UROLOGY 50 (51, 1997 801 then detected with a CY3 conjugated goat anti-mouse antibody (Jackson ImmunoResearch, West Grove, Pa) and DNA was visualized with DAPI (4,6-diamidino-2-phenylindole) at 2 /.

Techniques: Staining

Journal:

Article Title: A Comprehensive Analysis of Allelic Methylation Status of CpG Islands on Human Chromosome 21q

doi: 10.1101/gr.1351604

Figure Lengend Snippet: Methylation and Other Features of CpG Islands on Human Chromosome 21q

Article Snippet: For expression analysis of PPP1R2P2, HSF2BP, H2B-like , and DKFZp434A171-like , total RNA (2.5 μg) from various human tissues (Clontech) was reverse-transcribed and used as templates for PCR using the following thermal cycling parameters: 3 min at 95°C + (30 sec at 95°C + 30 sec at 65°C + 30 sec at 72°C) times 30 cycles.

Techniques: Methylation, Variant Assay, Binding Assay

Journal:

Article Title: A Comprehensive Analysis of Allelic Methylation Status of CpG Islands on Human Chromosome 21q

doi: 10.1101/gr.1351604

Figure Lengend Snippet: Expression of genes methylated in their promoters or 5′-UTRs. RT-PCR was performed using total RNA from various human tissues (1, bone marrow; 2, adrenal gland; 3, thymus; 4, prostate; 5, trachea; 6, thyroid; 7, spleen; 8, small intestine; 9, colon; 10, uterus; 11, placenta; 12, testis; 13, heart; 14, skeletal muscle; 15, liver; 16, lung; 17, kidney; 18, whole brain; 19, salivary gland; 20, peripheral blood cells). The products were resolved by polyacrylamide gel electrophoresis, stained with SYBR Green, and visualized by UV illumination.

Article Snippet: For expression analysis of PPP1R2P2, HSF2BP, H2B-like , and DKFZp434A171-like , total RNA (2.5 μg) from various human tissues (Clontech) was reverse-transcribed and used as templates for PCR using the following thermal cycling parameters: 3 min at 95°C + (30 sec at 95°C + 30 sec at 65°C + 30 sec at 72°C) times 30 cycles.

Techniques: Expressing, Methylation, Reverse Transcription Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Staining, SYBR Green Assay

Journal:

Article Title: Distribution and Kinetics of Lipoprotein-Bound Endotoxin

doi: 10.1128/IAI.69.5.2821-2828.2001

Figure Lengend Snippet: (A) Chromatographic profile of the major plasma lipoprotein classes separated by HPGC with online cholesterol detection. The average molecular masses calculated from retention characteristics were 5 MDa for VLDL, 1 MDa for LDL, and 300 kDa for HDL. Horizontal bars indicate the lipoprotein fractions pooled for further analysis. (B) SDS-PAGE analysis of these lipoprotein fractions with subsequent silver staining, showing the protein composition of the lipoproteins. M, molecular weight in thousands; lane A, VLDL; lane B, LDL; lane C, HDL. Each well was loaded with 20 μl containing a total of approximately 2 μg of protein.

Article Snippet: Protein profiles of the individual lipoprotein classes isolated by HPGC were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) on precast linear, 4 to 15% (wt/vol) acrylamide gradient gels containing a 4% (wt/vol) acrylamide stacking layer (Bio-Rad, Hercules, Calif.) ( 17 ).

Techniques: SDS Page, Silver Staining, Molecular Weight

Journal:

Article Title: Analysis of Receptor Usage by Ecotropic Murine Retroviruses, Using Green Fluorescent Protein-Tagged Cationic Amino Acid Transporters

doi:

Figure Lengend Snippet: (A) Expression of mCAT1-GFP. Protein samples (100 μg) prepared from untransfected human 293 cells (lane 1) and from cells transfected with pEGFP-N1 (lane 2) or pmCAT1-GFP (lane 3) were immunoprecipitated with a rabbit anti-GFP polyclonal antibody (Clontech), fractionated by SDS-PAGE on a 4 to 20% gradient SDS-polyacrylamide gel, transferred to a PVDF membrane, and reacted with anti-GFP monoclonal antibody. Binding of the anti-GFP monoclonal antibody was detected by the chemiluminescence method (Roche Diagnostics). Positions of molecular size markers are shown on the left. (B) N-linked glycosylation of mCAT1-GFP. Protein samples (100 μg) prepared from untransfected 293 cells (control [Ctrl.]; lanes 1 and 2) and from cells transfected with pmCAT1-GFP (lane 3 and 4) were immunoprecipitated with a rabbit anti-GFP polyclonal antibody (Clontech), incubated without (−) or with (+) N-glycosidase F (Roche Diagnostics), and analyzed by immunoblot detection as described above. Larger and smaller arrowheads on the right indicate monomeric and dimeric mCAT1-GFP; solid and shaded arrowheads indicate signals for glycosylated and unglycosylated forms, respectively. (C) Susceptibility of mCAT1-GFP-expressing 293 cells to M-MuLV-pseudotyped BAG vector. Untransfected 293 cells (panel 1) and cells transfected with pmCAT1-GFP (panel 2) or pEGFP-N1 (panel 3) were inoculated with 1,000-fold-diluted supernatant of M-MuLV-based CRE cells producing BAG vector (34). Four days after inoculation, the cells were fixed and stained with X-Gal. Absence of lacZ staining in untransfected 293 cells (panel 1), and cells expressing only GFP (panel 3), and a representative lacZ+ colony of the mCAT1-GFP-expressing cells (panel 2) is shown. (D) The lacZ+ colony-forming titer of BAG vector produced by CRE cells on NIH 3T3, untransfected 293 cells, GFP-expressing 293 cells, and mCAT1-GFP-expressing 293 cells. Data indicate the average titer calculated from the number of the lacZ+ colonies in duplicate experiments.

Article Snippet: Immunoprecipitates were rinsed three times with the lysis buffer, suspended in the Laemmli sample buffer (Bio-Rad), and boiled at 100°C for 5 min. Boiled samples were fractionated by 4 to 20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Novex, San Diego, Calif.), transferred to a polyvinylidene difluoride (PVDF) membrane, and reacted with a mouse monoclonal antibody against GFP (Clontech) or an anti-mCAT1 rabbit serum ( 21 ) given by James Cunningham.

Techniques: Expressing, Transfection, Immunoprecipitation, SDS Page, Binding Assay, Incubation, Western Blot, Plasmid Preparation, Staining, Produced

Journal:

Article Title: Analysis of Receptor Usage by Ecotropic Murine Retroviruses, Using Green Fluorescent Protein-Tagged Cationic Amino Acid Transporters

doi:

Figure Lengend Snippet: Ecotropic receptor function of various CAT proteins. (A) Schematic diagrams of various CAT proteins tagged with GFP. In the diagram of the mCAT1-TED2 chimera, solid and open boxes represent segments derived from mCAT1 and mCAT2A, respectively. (B) Comparison of amino acid sequences of the TED of CAT family proteins. The region of mCAT1 from amino acids 210 to 241 (2) and the corresponding regions of mCAT2 (6) and rCAT3 (15) are aligned. Potential N-linked glycosylation sites are underlined. Dots indicate that corresponding amino acids are missing. (C) Immunoblot detection of various GFP-tagged CAT molecules. Protein samples (100 μg) prepared from untransfected 293 cells (lane 1) or the cells transfected with the plasmid encoding mCAT1-GFP (lane 2), mCAT1-TED2-GFP (lane 3), mCAT2A-GFP (lane 4), or rCAT3-GFP (lane 5) were immunoprecipitated with an anti-GFP polyclonal antibody, fractionated by SDS-PAGE, transferred to a PVDF membrane, and reacted with an anti-GFP monoclonal antibody. Positions of molecular size markers are shown on the left. (D) Susceptibility of the cells expressing various CAT-GFP fusion proteins to F-MuLV and PVC-211 MuLV. Control NIH 3T3 cells and human 293 cells expressing GFP alone or GFP-tagged CAT molecules were inoculated with serially diluted BAG vector pseudotyped with PVC-211 MuLV (solid bars) or F-MuLV (open bars). Four days after inoculation, the cells were fixed and stained with X-Gal. The data indicate the average lacZ+ colony-forming titer determined from the number of the stained cell colonies obtained from duplicate experiments. m1, wild-type mCAT1-GFP; TED2, mCAT1-TED2-GFP; m2A, mCAT2A-GFP; r3, rCAT3-GFP.

Article Snippet: Immunoprecipitates were rinsed three times with the lysis buffer, suspended in the Laemmli sample buffer (Bio-Rad), and boiled at 100°C for 5 min. Boiled samples were fractionated by 4 to 20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Novex, San Diego, Calif.), transferred to a polyvinylidene difluoride (PVDF) membrane, and reacted with a mouse monoclonal antibody against GFP (Clontech) or an anti-mCAT1 rabbit serum ( 21 ) given by James Cunningham.

Techniques: Derivative Assay, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, SDS Page, Expressing, Staining

Journal:

Article Title: Analysis of Receptor Usage by Ecotropic Murine Retroviruses, Using Green Fluorescent Protein-Tagged Cationic Amino Acid Transporters

doi:

Figure Lengend Snippet: Dimerization of CAT protein. Protein samples (100 μg) prepared from 293 cells transfected with plasmid constructs (5 μg) encoding mCAT2A-GFP (lanes 1 and 5), mCAT2A-HA (lanes 2 and 6), mCAT1-GFP and mCAT2A-HA (lanes 3 and 7), or mCAT2A-GFP and mCAT2A-HA (lanes 4 and 8) were immunoprecipitated (I.P.) with an anti-HA monoclonal antibody (12CA5), fractionated by SDS-PAGE, transferred to a PVDF membrane, and reacted with an anti-HA or anti-GFP monoclonal antibody. m1 and m2 represent mCAT1 and mCAT2, respectively. Positions of molecular size markers are shown on the left in kilodaltons; arrowheads on the right indicate the signals for HA- and GFP-tagged mCAT2A.

Article Snippet: Immunoprecipitates were rinsed three times with the lysis buffer, suspended in the Laemmli sample buffer (Bio-Rad), and boiled at 100°C for 5 min. Boiled samples were fractionated by 4 to 20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Novex, San Diego, Calif.), transferred to a polyvinylidene difluoride (PVDF) membrane, and reacted with a mouse monoclonal antibody against GFP (Clontech) or an anti-mCAT1 rabbit serum ( 21 ) given by James Cunningham.

Techniques: Transfection, Plasmid Preparation, Construct, Immunoprecipitation, SDS Page