Revision 3

#3287Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, W-S, IP, IHC-P, IF-IC, FC-FP

REACTIVITY:

H

SENSITIVITY:

Endogenous

MW (kDa):

258, 110, 50-80

Source/Isotype:

Rabbit IgG

UniProt ID:

#P08922

Entrez-Gene Id:

6098

Product Information

Product Usage Information

Application Dilution
Western Blotting 1:1000
Simple Western™ 1:10 - 1:50
Immunoprecipitation 1:50
Immunohistochemistry (Paraffin) 1:250
Immunofluorescence (Immunocytochemistry) 1:200 - 1:800
Flow Cytometry (Fixed/Permeabilized) 1:200 - 1:800

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

ROS1 (D4D6®) Rabbit mAb recognizes endogenous levels of total ROS1 protein. This antibody does not cross-react with other related proteins when analyzed by western blot. Please note that staining may be observed in ROS1 rearranged lung carcinomas, macrophages/giant cells, reactive type II pneumocyte hyperplasia, and the epithelium in areas of bronchiolar metaplasia. Staining of unknown specificity has been observed in cholangiocarcinoma, hepatocellular carcinoma, and kidney tissues.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a protein corresponding to residues in the carboxy terminal domain of the human ROS1 protein.

Background

ROS1, an orphan receptor tyrosine kinase of the insulin receptor family, was initially identified as a homolog of v-ros from the UR2 sarcoma virus (1). ROS1 consists of a large extracellular domain that is composed of six fibronectin repeats, a transmembrane domain, and a C-terminal kinase domain. Being an orphan receptor, the functions of ROS1 are not well known, though it has been shown to play an important role in differentiation of epididymal epithelium (2). The first oncogenic fusion of ROS1, FIG-ROS1, was initially identified by research studies in glioblastoma (3), and subsequent studies have found this fusion in cholangiocarcinoma (4), ovarian cancer (5), and non-small cell lung cancer (NSCLC) (6). Investigators have found additional oncogenic ROS1 fusion proteins in NSCLC (at a frequency of ~1.6%), where the ROS1 kinase domain is fused to the amino-terminal region of several different proteins, including CD74 and SLC34A2 (6-8). ROS1 fusion proteins activate the SHP-2 phosphatase, PI3K/Akt/mTOR, Erk, and Stat3 pathways (3,4,9). There are two autophosphorylation sites (Tyr2274, Tyr2334) downstream of the kinase domain of ROS1, either of which may serve as biomarkers of ROS1 kinase activity, including that of ROS1 fusion proteins (10).

  1. Matsushime, H. et al. (1986) Mol Cell Biol 6, 3000-4.
  2. Yeung, C.H. et al. (1999) Biol Reprod 61, 1062-9.
  3. Charest, A. et al. (2003) Genes Chromosomes Cancer 37, 58-71.
  4. Gu, T.L. et al. (2011) PLoS One 6, e15640.
  5. Birch, A.H. et al. (2011) PLoS One 6, e28250.
  6. Rimkunas, V.M. et al. (2012) Clin Cancer Res 18, 4449-57.
  7. Rikova, K. et al. (2007) Cell 131, 1190-203.
  8. Stumpfova, M. and Jänne, P.A. (2012) Clin Cancer Res 18, 4222-4.
  9. Jun, H.J. et al. (2012) Cancer Res 72, 3764-74.
  10. Zou, H.Y. et al. (2015) Proc Natl Acad Sci U S A 112, 3493-8.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting W-S: Simple Western™ IP: Immunoprecipitation IHC-P: Immunohistochemistry (Paraffin) IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized)

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

使用に関する制限

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Revision 3
#3287

ROS1 (D4D6®) Rabbit mAb

Western Blotting Image 1: ROS1 (D4D6®) Rabbit mAb Expand Image
Western blot analysis of extracts from HCC78 (SLC34A2-ROS1), U-118 MG (FIG-ROS1), and HeLa (ROS1 negative) cells using ROS1 (D4D6®) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). Note: HCC78 cells express the 85, 70, and 59 kDa forms of the SLC34A2-ROS1 fusion protein (7).
Western Blotting Image 2: ROS1 (D4D6®) Rabbit mAb Expand Image
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing wildtype ROS1 (hROS1; +), using ROS1 (D4D6®) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western Blotting Image 1: ROS1 (D4D6®) Rabbit mAb Expand Image
Simple Western™ analysis of lysates (1.0 mg/mL) from HCC-78 cells using ROS1 (D4D6® ) Rabbit mAb #3287. The virtual lane view (left) shows the target bands (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 66-440 kDa separation module.
Immunoprecipitation Image 1: ROS1 (D4D6®) Rabbit mAb Expand Image
Immunoprecipitation of ROS1 from HCC78 cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or ROS1 (D4D6®) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using ROS1 (D4D6®) Rabbit mAb, followed by Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 and Anti-mouse IgG, HRP-linked Antibody #7076.
Immunohistochemistry Image 1: ROS1 (D4D6®) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human lung carcinomas showing distinct localization using ROS1 (D4D6®) Rabbit mAb.
No image available
Flow Cytometry Image 1: ROS1 (D4D6®) Rabbit mAb Expand Image
Flow cytometric analysis of HeLa cells (blue, negative) and HCC78 cells (green, positive) using ROS1 (D4D6®) Rabbit mAb (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.