Antibodies. The list of DS Know-How to be disclosed by DS to Zymeworks as of the Effective Date is specified in the Exhibit 1.23.
Antibodies. Describe the antibodies used and how they were validated for use in the system under study (i.e. assay and species). Well documented HT29 and SW480 colon cancer cell lines have been bought from Sigma. Authentication by Sigma. Before use cell lines were tested in house for mycoplasma and were both negative. HT29 and SW480 colon cancer cell lines have not been listed by ICLAC as contaminated cell lines.
Antibodies. 2.1.4.1.1 Primary antibodies Primary antibodies used are listed in Table 6 below. Table 6 Primary antibodies Antibody Supplier Monoclonal/ Polyclonal Species Working dilution Actin (AC-40) Sigma Monoclonal Mouse 1:10000 (IB) BiP/GRP78 Cell Signaling Technology Polyclonal Rabbit 1:1000 (IB) BiP/GRP78 Abcam Polyclonal Rabbit 1:300 (IF) HA Sigma Polyclonal Rabbit 1:10000 (IB) 1:5000 (IF) Myc 9B11 Cell Signaling Technology Monoclonal Mouse 1:10000 (IB) 1:5000 (IF) TDP-43 Proteintech Polyclonal Rabbit 1:5000 (IB) 1:2000 (IF) TDP-43 phosphorylated on S91/92 X. Xxxxxx /custom made by Proteintech Polyclonal Rabbit 1:1000 (IB) VAPA (VAP33) BD Biosciences Monoclonal Mouse 1:2500 (IB) VAPB (#3504) (Xx Xxx et al., 2012) Polyclonal Rabbit 1:5000 (IB) VAPB (Sk83) (Xx Xxx et al., 2012) Polyclonal Rat 1:250 (IF) PTPIP51 (FAM82A2) Atlas Antibodies Polyclonal Rabbit 1:5000 (IB) Abbreviations: IB= immunoblot; IF= immunofluorescence
Antibodies. To localize mGluR7, Protein A chromatography purified rabbit polyclonal antibody raised against peptide (C-NSPAAKKKYVSYNN) corresponding to amino acids 899-912 of human mGluR7 was used at concentration 1:500 (Upstate/Millipore, Catalog # 07-239). Immunoblotting studies by the manufacturer on rat brain microsomal preparation probed with anti-mGluR7 (0.5 micrograms/ml) showed a band at 97kD. The rabbit polyclonal anti-mGluR4a antibody (Zymed/Invitrogen, 1:200, Catalog #513100, Lot #60103066A2) was raised against a synthetic peptide derived from the C-terminal 200 amino acids of the rat mGluR4 protein specific for the mGluR4a splice variant. Studies by the manufacturer confirmed the reactivity on Western blots (Mr=93,000- 110,000) using rat brain cell lysate. Tissue from mGluR4 knock out mice is completely devoid of immunostaining when probed with this antibody (Xxxxx, Xxxx et al., unpublished data). Two antibodies, rabbit anti-VGluT1 and guinea pig anti-VGluT2, were used as specific markers of terminals from either the VGluT1-positive corticostriatal or VGluT2- positive thalamostriatal glutamatergic projections. To localize VGlut1, we used commercially available Rabbit anti-VGluT1 (MaB Xxxxxxxxxxxx, Xxx # XXX0-0, Xxx # XX000X, 1:5000). To generate this VGluT1 antibody a peptide from the COOH terminus of the rat vesicular glutamate transporter 1 (rvGluT1), corresponding to amino acids 543– 560 (cATHSTVQPPRPPPPVRDY) was synthesized. A cysteine was added to the peptide to aid its conjugation to the protein carrier keyhole limpet hemocyanin (KLH; Xxxxxx, Rockford, IL). Antisera were obtained from rabbits (Covance) immunized with the conjugated peptide, and the IgG fraction was recovered (Raju, xx.xx. 2006). Previous studies from our laboratory and others using immunoblotting of brain lysate revealed single bands at ~60 kDa. Preadsorbtion of primary antibody with synthetic peptide (0.2–
Antibodies. To localize mGluR8 we used a commercially available affinity-purified polyclonal antibody raised in rabbit against the C-terminal domain of human mGluR8 conjugated to KLH (1:750, LifeSpan BioSciences/MBL International Corporporation, Catalog #LS-A925, Lot #5715/16 AP10-1). A simple dot blot examination confirmed that the antibody was in fact specific to the N-terminal peptide against which it was created (data not shown). Additional studies from our laboratory have used striatal tissue from mGluR8a,b knockout mice (Duvoisin et al., 2005; generously given by Xx Xxxxxx
Antibodies. Table 2 Antibodies for Identification of Nicotinic Subunits and Afferent Classes Primary Vendor Dilution Secondary Vendor Dilution alpha 3 Novus Biologicals 1:500 Cy3 anti- rabbit Xxxxxxx Immunoresearch 1:250 alpha 6 Novus Biologicals 1:500 Cy3 anti- rabbit Xxxxxxx Immunoresearch 1:250 alpha 7 abcam 1:1000 Cy3 anti- rabbit Xxxxxxx Immunoresearch 1:250 alpha 9 Santa Xxxx Biotechnology 1:500 Biotin anti- Goat Xxxxxxx Immunoresearch 1:250 CGRP Serotec 1:200 Cy5 anti-Goat Xxxxxxx 1:100 Immunoresearch CGRP Pennsylvania Labs 1:200 Cy5 anti- Guinea pig Xxxxxxx Immunoresearch 1:100 parvalbumin Sigma 1:1000 Cy5 anti- mouse Xxxxxxx Immunoresearch 1:100 somatostatin Xxxxxxx Immunoresearch 1:100 Dylight 488 Xxxxxxx Immunoresearch 1:100 CGRP Serotec 1:200 Dylight 488anti guinea pig Xxxxxxx Immunoresearch 1:100 CGRP Serotec 1:200 Dylight 488anti-goat Xxxxxxx Immunoresearch 1:100 α9 required further amplification with a tertiary label, extravidin conjugated to Cy3 at a dilution of 1:1000.
Antibodies. The term "Antibodies" shall mean monoclonal antibodies and the cell lines from which the Antibodies are produced, either characterized or uncharacterized, including but not limited to those cell lines and Antibodies listed in Transfer Agreement No. M960924 between The Regents and an Affiliate of M-Tech, dated the 11th of June, 1997 and the License Agreement No. R981110 between The Regents and an Affiliate of M-Tech, dated the 17th of January, 1999, that have been developed by Dr. Irie, at UCLA or JWCI, HMI, M-Tech and their respective Affiliates prior to the Effective Date, as evidenced by documentation in Dr. Irie's possession as of the Effective Date, including:
Antibodies. INTRODUCTION Autoimmune thyroid disease is characterized by the production of antibodies to various thyroid proteins. Thyroglobulin (Tg) is one such protein and the detection of circulating antibodies to Tg is indicative of autoimmunity.
Antibodies. Mouse monoclonal antibodies recognizing RT-1A MHC class I molecules (OX18) and rat transferrin receptor (OX26) were purchased from Serotec (UK) 44. Monoclonal antibody RCMV 35, directed against a 29-kDa RCMV protein, has been described previously 45. For flow cytometry experiments, either FITC-conjugated rabbit anti-mouse IgG (Dako A/S, Denmark) or PE-conjugated goat anti-mouse IgG (Xxxxxxx ImmunoResearch Laboratories, West Grove, PA) were used as secondary antibody. Infection The cells were infected at 80-90% confluency, washed with PBS, placed on EMEM containing 2% FCS for 30 min, and washed again with PBS. The virus was diluted in EMEM with 2 % NCS. For flow cytometry experiments, cells were infected with an m.o.i. of 1; for biochemical experiments, cells were infected with an m.o.i. of 3. To increase the efficiency of infection, cells were centrifuged at 700 g at 20 °C for 45 min and placed at 37 °C for another 10-15 min 46. The infection medium was replaced by culture medium containing 2% NCS. For biochemical experiments, RPMI was used for cell culture and infections. Flow cytometry Cell surface expression of MHC class I molecules, transferrin receptor and EGFP were analyzed by flow cytometry using FACSort equipment and Cell Quest software (Becton Xxxxxxxxx, USA). Cells were stained in PBS containing 1% BSA and 10mM NaN3 47. Metabolic labeling, immunoprecipitations and SDS-PAGE For pulse-chase experiments, cells were starved in medium lacking methionine and cysteine at 37 °C for 1 hr. The cells were labeled with 35S Promix (Amersham), and chased in medium with excess of L-cystine and L-methionine for the times indicated in the figures 47. Where indicated, media were supplemented with the proteasome inhibitor carboxybenzyl-leucyl- leucylleucinal (ZL3H). Cells were lysed in Nonidet-P40 lysis buffer containing leupeptin, AEBSF and ZL3H at 4 °C for 30 min. To remove cell debris, lysates were centrifugated at 10,000g for 10 min. Prior to immunoprecipitation, lysates were precleared twice using normal rabbit and normal mouse sera precoupled to mixed (1:1) Protein A- Sepharose and Protein G-Sepharose beads. Immunoprecipitations were performed on precleared lysates at 4 °C for 2-4 h using specific antisera precoupled to Protein A/G Sepharose beads. Subsequently, the beads were washed with NET buffer [50 mM Tris/HCl, pH 7.4/150 mM NaCl/5 mM EDTA/0.5% (v/v) NP-40] supplemented with 0.1% SDS. The immunoprecipitates were boiled in sample buffer [40 mM Tris/HCl, p...
Antibodies. Ep-CAM was detected with mAb 323/A3 (Centocor Europe BV, Leiden, NL), E- cadherin with mAb HECD-1 (Thamer Diagnostica BV, Uithoorn, NL), β-catenin with clone 14 (Transduction Lab., Lexington, KY), involucrin with clone SY5 (Sigma-Aldrich Chemie Gmbh, Steinheim, Germany), and ck13 with clone 1C7 (Thamer Diagnostica BV). For cytoskeletal stainings, TRITC-labeled phalloidin was used (Sigma-Aldrich).
