Revision 1

#68355Store at -20C

1 Kit

(9 x 20 microliters)

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.
Product Includes Product # Quantity Mol. Wt Isotype/Source
MX1 (D3W7I) Rabbit mAb 37849 20 µl 76 kDa Rabbit IgG
OAS1 (D1W3A) Rabbit mAb 14498 20 µl 40, 44 kDa Rabbit IgG
RNase L (D4B4J) Rabbit mAb 27281 20 µl 80 kDa Rabbit IgG
IFITM3 (D8E8G) XP® Rabbit mAb 59212 20 µl 15 kDa Rabbit IgG
BST2 (D5V5Z) Rabbit mAb 19277 20 µl 28-40 kDa Rabbit IgG
TRIM5α (D6Z8L) Rabbit mAb 14326 20 µl 56 kDa Rabbit IgG
Phospho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb 3398 20 µl 38 kDa Rabbit IgG
Phospho-SAMHD1 (Thr592) (D7O2M) Rabbit mAb 89930 20 µl 72 kDa Rabbit IgG
IFITM1 Antibody 13126 20 µl 14 kDa Rabbit 
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

The Host Cell Viral Restriction Factor Antibody Sampler Kit provides an economical means of detecting the expression of various host cell viral restriction factors using phospho-specific and total protein antibodies. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.

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 antibodies.

Background

Viral restriction factors are proteins produced by host cells that function, in part, to negatively impact various stages of viral life cycles in order to prevent propagation. MX1 (Myxovirus resistance protein 1/MxA) is an interferon-inducible antiviral protein that confers resistance to RNA viruses by blocking transcription of the viral genome (1,2).

2’-5’-oligoadenylate synthetase 1 (OAS1) is an antiviral protein induced by type 1 interferon that plays a key role in the cellular innate immune response (3). The OAS1 enzyme produces a second messenger, 2’-5’-linked oligoadenylate, which binds to RNase L, which then degrades viral and cellular RNA (4). Research studies indicate that the OAS1 system inhibits protein synthesis and induces apoptosis in virally infected cells, which limits viral infection (5).

Interferon-induced transmembrane protein (IFITM) family members, IFITM1 and IFITM3, appear to function as viral restriction factors by preventing fusion of viral and host membranes (6,7).

BST2 (CD317, Tetherin, HM1.24) is a type II transmembrane glycoprotein functioning as a major mediator of the innate immune defense against the dissemination of enveloped viruses by tethering viron on the cell surface, preventing viral release (8).

TRIM5α blocks viral infection by interacting with the incoming viral capsid and promoting its premature disassembly (9).

PKR-induced phosphorylation of the eukaryotic initiation factor 2 (eIF2) α subunit at Ser51 is a well-documented mechanism to downregulate protein synthesis upon viral infection (10).

SAM domain and HD domain-containing protein 1 (SAMHD1) prevents autoimmunity and HIV infection by hydrolyzing intracellular deoxynucleoside triphosphates (dNTPs), thereby limiting inappropriate immune activation by self nucleic acid and inhibiting reverse transcription of the HIV genome (11). Phosphorylation of SAMHD1 at Thr592 by cyclin A2/CDK1 was identified as a regulatory mechanism that controls SAMHD1 activity. SAMHD1 is phosphorylated in proliferating cells, which inhibits its ability to block HIV infection (12).

  1. Staeheli, P. et al. (1986) Cell 44, 147-58.
  2. Kochs, G. and Haller, O. (1999) Proc Natl Acad Sci U S A 96, 2082-6.
  3. Schoggins, J.W. et al. (2011) Nature 472, 481-5.
  4. Dong, B. and Silverman, R.H. (1997) J Biol Chem 272, 22236-42.
  5. Castelli, J.C. et al. (1998) Cell Death Differ 5, 313-20.
  6. Brass, A.L. et al. (2009) Cell 139, 1243-54.
  7. Feeley, E.M. et al. (2011) PLoS Pathog 7, e1002337.
  8. Le Tortorec, A. et al. (2011) Viruses 3, 520-40.
  9. Stremlau, M. et al. (2006) Proc Natl Acad Sci U S A 103, 5514-9.
  10. Zamanian-Daryoush, M. et al. (2000) Mol Cell Biol 20, 1278-90.
  11. Powell, R.D. et al. (2011) J Biol Chem 286, 43596-600.
  12. Cribier, A. et al. (2013) Cell Rep 3, 1036-43.

Background References

    Trademarks and Patents

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

    Limited Uses

    Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

    Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

    Revision 1
    #68355

    Host Cell Viral Restriction Factor Antibody Sampler Kit

    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 1 Expand Image

    Simple Western™ analysis of HT-29 + hIFN⍺ -1 (10ng/mL, O/N) + poly(I:C )(2.5µg/mL, 7hr) + MG-132 (10µM, 7hr) lysates (0.1 mg/mL) using OAS1 (D1W3A) Rabbit mAb #14498. The virtual lane view (left) shows the target band (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 12-230 kDa separation module.

    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 2 Expand Image
    Simple Western™ analysis of lysates (0.1 mg/mL) from HeLa cells using IFITM3 (D8E8G) XP® Rabbit mAb #59212. The virtual lane view (left) shows the target band (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 12-230 kDa separation module.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 3 Expand Image
    Western blot analysis of extracts from K-562 cells, CD4+ T cells, and CD8+ T cells using IFITM1 Antibody.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from various cell lines using TRIM5α (D6Z8L) Rabbit mAb.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from HeLa, NCI-H460, and HT-29 cells, untreated (-) or treated with Human Interferon-α1 (hIFN-α1) #8927 (10 ng/ml, 16 hr; +), using OAS1 (D1W3A) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from various cell lines using BST2 (D5V5Z) Rabbit mAb.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from various cell lines using RNase L (D4B4J) Rabbit mAb.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 8 Expand Image
    Western blot analysis of extracts from C2C12 cells, untreated or thapsigargin-treated, using Phospho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb (upper) or eIF2α Antibody #9722 (lower).
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of extracts from HeLa cells, untreated (-) or treated with Human Interferon-α1 (hIFN-α1) #8927 (10 ng/ml, 16 hr; +), using MX1 (D3W7I) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of extracts from various cell lines using IFITM3 (D8E8G) XP® Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 11 Expand Image
    After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 12 Expand Image
    Western blot analysis of extracts from THP-1 cells, untreated (-) or treated with calf intestinal phosphatase (CIP) plus λ phosphatase (+), using Phospho-SAMHD1 (Thr592) (D7O2M) (upper) or SAMHD1 Antibody #12361 (lower).
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 13 Expand Image
    Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing full-length human IFITM1 (hIFITM1; +), using IFITM1 Antibody.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 14 Expand Image
    Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human TRIM5α (hTRIM5α-Myc/DDK), using TRIM5α (D6Z8L) Rabbit mAb.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 15 Expand Image
    Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing human OAS1 isoform p41 (hOAS1 p41; +), using OAS1 (D1W3A) Rabbit mAb.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 16 Expand Image
    Western blot analysis of extracts from 293T cells, untransfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human RNase L (hRNase L-Myc/DDK; +), using RNase L (D4B4J) Rabbit mAb.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 17 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon carcinoma, untreated (left) or λ phosphatase-treated (right), using Phopsho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 18 Expand Image
    Immunoprecipitation of MX1 from extracts of HeLa cells treated with Human Interferon-α1 #8927 (10 ng/ml, 16 hr). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is MX1 (D3W7I) Rabbit mAb. Western blot analysis was performed using MX1 (D3W7I) Rabbit mAb.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 19 Expand Image
    Western blot analysis of extracts from 293T cells untransfected (-), or transfected with a construct expressing Myc-tagged full-length human IFITM3 (hIFITM3-Myc; +), using IFITM3 (D8E8G) XP® Rabbit mAb.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 20 Expand Image
    Western blot analysis of extracts from Jurkat and THP-1 cells, untreated (-) or treated with TPA (12-O-Tetradecanoylphorbol-13-Acetate) #4174 (80 nM, 16 hr; +), using Phospho-SAMHD1 (Thr592) (D7O2M) Rabbit mAb (upper), SAMHD1 Antibody #12361 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 21 Expand Image
    Western blot analysis of extracts from HeLa cells, untreated (-) or treated with Human Interferon-α1 (hIFN-α1) #8927 (10 ng/ml, 16 hr; +), using IFITM1 Antibody (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 22 Expand Image
    Immunoprecipitation of TRIM5α from RL cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or TRIM5α (D6Z8L) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using TRIM5α (D6Z8L) Rabbit mAb.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 23 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Phospho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 24 Expand Image
    Immunohistochemical analysis of paraffin-embedded human ductal carcinoma of the breast using MX1 (D3W7I) Rabbit mAb performed on the Leica® Bond Rx.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 25 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon carcinoma using IFITM3 (D8E8G) XP® Rabbit mAb.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 26 Expand Image
    Immunoprecipitation of phospho-SAMHD1 (Thr592) from THP-1 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Phospho-SAMDHD1 (Thr592) (D7O2M) Rabbit mAb. Western blot analysis was performed using Phospho-SAMDHD1 (Thr592) (D7O2M) Rabbit mAb.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 27 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lymphoma using Phospho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 28 Expand Image
    Immunohistochemical analysis of paraffin-embedded T84 (left, positive) and HCT116 (right, negative) cell pellets using MX1 (D3W7I) Rabbit mAb.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 29 Expand Image
    Immunohistochemical analysis of paraffin-embedded normal human lymph node using IFITM3 (D8E8G) XP® Rabbit mAb.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 30 Expand Image
    Flow cytometric analysis of THP-1 cells, treated with TPA (80 nM, 16 hr; red) or untreated (green), using Phospho-SAMHD1 (Thr592) (D7O2M) Rabbit mAb (solid lines) or 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.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 31 Expand Image
    Immunohistochemical analysis of paraffin-embedded HeLa cell pellets, untreated (left) or treated with Human Interferon-alpha1 (hIFN-alpha1) #8927 (right) using MX1 (D3W7I) Rabbit mAb.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 32 Expand Image
    Immunohistochemical analysis of paraffin-embedded HeLa (left) and RPMI 8226 (right) cell pellets using IFITM3 (D8E8G) XP® Rabbit mAb.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 33 Expand Image
    Flow cytometric analysis of Jurkat cells (blue) and THP-1 cells (green) using Phospho-SAMHD1 (Thr592) (D7O2M) 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.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 34 Expand Image
    Immunohistochemical analysis of paraffin-embedded human non-small cell lung carcinoma using MX1 (D3W7I) Rabbit mAb.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 35 Expand Image
    Immunohistochemical analysis of paraffin-embedded normal human spleen using IFITM3 (D8E8G) XP® Rabbit mAb.
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 36 Expand Image
    Immunohistochemical analysis of paraffin-embedded human endometrioid adenocarcinoma using MX1 (D3W7I) Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
    Host Cell Viral Restriction Factor Antibody Sampler Kit: Image 37 Expand Image
    Immunohistochemical analysis of paraffin-embedded normal human spleen using MX1 (D3W7I) Rabbit mAb.