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. If [***] during the Research Term either Party has a reasonable belief that an Antibody that is being researched or Developed by or on behalf of Prothena (or its Affiliates) Targets a given Collaboration Target, and wishes to confirm whether or not such Antibody Targets such Collaboration Target, then either Party may issue a notice to the other Party with respect thereto (a “Confirmation Notice”). The Parties shall discuss in good faith for a period of [***] ([***]) days whether or not such Antibody Targets the applicable Collaboration Target. In the event that the Parties do not agree in writing within such [***] ([***]) day period whether or not such Antibody Targets the applicable Collaboration Target, either Party may issue a written notice to the other Party requesting the independent evaluation described in Section 2.4.3. [***] [***] Certain information in this document has been omitted and filed separately with the Securities and Exchange Commission. Confidential treatment has been requested with respect to the omitted portions.
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. 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. ChIP was performed using 3 μl per sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), H3K4me3 (Ab858, lot GR240214-4), JARID2 (Novus Biologicals NB100-2214, Lot E2). Western blots were stained with EZH2 (Cell Signaling 52465, lot 7, 1:2,000), Myc (Santa Xxxx sc-789, lot D1715, 1:1,000) or MTF2 (Protein Tech, 16208-1-AP, lot 00-000-0000, 1:2,000) primary antibodies, which were detected using Dako secondary antibodies (P0161, lot 20033538, P0217 20040441). Bioinformatic analysis. Illumina 75-bp sequencing files were mapped using bwa (version 0.7.10-r789) and normalized for sequencing depth before loading in the UCSC Genome Browser track hub (see “Data availability”). Peaks were called with MACS2-2.748 using the –no-model option and manual shift provided with the –extsize parameter. The extent of shifting was calculated with spp R library (see URLs). A q value threshold of 0.001 was applied in all cases and either the –call- summits (MTF2 and EZH2) or the –broad (H3K27me3 and H3K4me3) parameter was used. High-confidence conserved peaks were identified with MAnorm49 allowing a maximum of 1.5-fold change between replicates. Peak summits were defined as the non-overlapping 100-bp region around the summits called by MACS in the high-confidence peaks. Heat maps of ChIP-seq signal were generated using fluff v2.1.050 and clustered for dynamics using the “–g” option. The same analysis pipeline was applied for analysis of published data. Motif search and k-mer analysis was performed with GimmeMotifs v0.8.651 and k-mer-SVM v1.027. k-mers for enrichment analysis were selected among those ending with a GCG trinucleotide to maximize the number of base pairs (and therefore the information content) on the 5′ end of the sequence. The resulting pool of k-mers was filtered by SVM weight, and k-mers scoring higher than 1.5 or lower than –1.5 were used for enrichment analyses. DNA shape predictions were performed on sequences from MTF2 peak summits containing the k-mers (in natural sequence context) using the sliding pentamer model and R package described in refs 52,53. Random forest classification of DNA shape features was performed using the scikit-learn Python package54. Control regions were generated shuffling the sequence of each entry of the positive set while preserving the number of pre-existing GCGs and their position in the sequence. Shape-qualifying ...
Antibodies. Describe the antibodies used and how they were validated for use in the system under study (i.e. assay and species). E14 ref 28, Mtf2GT/GT ref15, Mtf2Δ/Δ ref15, Jarid2-/- ref35 and Eed-/- ref36 Mtf2GT/GT were genotyped by PCR as in ref 15, Mtf2Δ/Δ , Jarid2-/- and Eed-/- by WB All lines are routinely tested for mycoplasma contamination no commonly misidentified cell lines were used
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. The following antibodies were used in this study: RaAHNAK (KIS 1;5,000), MaVSV (P5D4), MaE4/5 (MaAHNAK, Abnova), MaMYC (9E10, Roche), RaMYC (Cell Signalling 1;5,000), MaHistone2, MaSC35, GaSC35 (Santa Xxxx), 3F5 (VHH against PABPN1), 3A (VHH against β-Amyloid), MahnRNP-Q, RahnRNP-A1, RaMyoD, MaM13 and MaM13hrp (both 1;5,000 Novagen) GaMousealexa488 (Molecular Probes), GaRabbitalexa594 (Molecular Probes) at 1;1,000 and 1;2,000, respectively. GaRabbitIRDye800 and GaMouseIRDye680 (Xxxxxxxx) were used at 1;5,000 for western blotting. RNA extraction and cDNA synthesis RNA was extracted from homogenized tissue or cultured cells with a kit (Machery- Xxxxx) according to the manufacturer’s protocol. 1µg RNA was used as input for a cDNA synthesis reaction (Fermentas) according to the manufacturer’s protocol. Genomic DNA was digested on the column and cDNA was purified with a gel- extraction kit (Machery-Xxxxx). Polymerase Chain Reaction (PCR) All primers were designed with the webtool Primer3 (xxxxx.xx.xxx.xxx/xxxxxx0/), with mispriming against human or rodent databases. End-point PCR reactions were performed with Phusion polymerase in HF buffer according to the manufacturer’s protocol. Quantitative PCR reactions were performed with SYBRgreen, in 15µl reaction volume with 3ng cDNA input. Primer sequences are depicted in Table S1, All primers had comparable efficiencies between 95% and 105%. All measurements were performed in triplo. Relative mRNA expression levels were calculated using GAPDH as reference gene, with the method of Pfaffl [200]. Briefly, expression differences were determined with the following formula: 2^dCt and the squareroot of (sdGAPDH^2+sdGOI^2), where GOI means Gene Of Interest. The standardized expression (relative dCt) was calculated by: 2^dCt*sddCt*LN(2)/N technical replicates. Finally, for relative expression levels, one value was set to 1 (100%). To calculate statistical significance in Figure 6, relative ct values of 4 biologically independent experiments were averaged. The square root of the added individually squared standard deviations was plotted as error bar in Figure 8. Finally, a paired t-test with equal variance was performed.
