Revision 1

#8348

Store at -20C

Rig-I Pathway Antibody Sampler Kit

1 Kit

(9 x 20 microliters)

CST Logo
Orders:

877-616-CELL (2355)

[email protected]

Support:

877-678-TECH (8324)

3 Trask Lane | Danvers | Massachusetts | 01923 | USA

For Research Use Only. Not for Use in Diagnostic Procedures.

Product Includes Product # Quantity Mol. Wt Isotype/Source
MDA-5 (D74E4) Rabbit Monoclonal Antibody532120 µl135 kDaRabbit IgG
Rig-I (D14G6) Rabbit Monoclonal Antibody374320 µl102 kDaRabbit IgG
MAVS Antibody399320 µl75, 52 kDaRabbit
IRF-3 (D6I4C) Rabbit Monoclonal Antibody1190420 µl50-55 kDaRabbit IgG
TBK1/NAK (D1B4) Rabbit Monoclonal Antibody350420 µl84 kDaRabbit IgG
Phospho-TBK1/NAK (Ser172) (D52C2) Rabbit Monoclonal Antibody548320 µl84 kDaRabbit IgG
Phospho-IRF-3 (Ser396) (4D4G) Rabbit Monoclonal Antibody494720 µl45-55 kDaRabbit IgG
Phospho-IKK epsilon (Ser172) (D1B7) Rabbit Monoclonal Antibody876620 µl80 kDaRabbit IgG
IKK epsilon (D20G4) Rabbit Monoclonal Antibody290520 µl80 kDaRabbit IgG
Anti-rabbit IgG, HRP-linked Antibody7074100 µlGoat

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

Description

The Rig-I Pathway Antibody Sampler Kit provides an economical means to evaluate the activation state and total protein levels of multiple members of the Rig-I pathway including Rig-I, MDA-5, MAVS, IRF-3, TBK1/NAK, and IKKε. The kit includes enough primary antibody to perform two western blot experiments per 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 antibody.

Background

Antiviral innate immunity depends on the combination of parallel pathways triggered by virus detecting proteins in the Toll-like receptor (TLR) family and RNA helicases, such as Rig-I (retinoic acid-inducible gene I) and MDA-5 (melanoma differentiation-associated antigen 5), which promote the transcription of type I interferons (IFN) and antiviral enzymes (1-3). TLRs and helicase proteins contain sites that recognize the molecular patterns of different virus types, including DNA, single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), and glycoproteins. These antiviral proteins are found in different cell compartments; TLRs (i.e. TLR3, TLR7, TLR8, and TLR9) are expressed on endosomal membranes and helicases are localized to the cytoplasm. Rig-I expression is induced by retinoic acid, LPS, IFN, and viral infection (4,5). Both Rig-I and MDA-5 share a DExD/H-box helicase domain that detects viral dsRNA and two amino-terminal caspase recruitment domains (CARD) that are required for triggering downstream signaling (4-7). Rig-I binds both dsRNA and viral ssRNA that contains a 5'-triphosphate end not seen in host RNA (8,9). Though structurally related, Rig-I and MDA-5 detect a distinct set of viruses (10,11). The CARD domain of the helicases, which is sufficient to generate signaling and IFN production, is recruited to the CARD domain of the MAVS/VISA/Cardif/IPS-1 mitochondrial protein, which triggers activation of NF-κB, TBK1/IKKε, and IRF-3/IRF-7 (12-15).

Trademarks and Patents

Cell Signaling Technology is a 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.

Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

Cell Signaling Technology Logo
Western blot analysis of extracts from control HeLa cells (lane 1) or IRF-3 knockout HeLa cells (lane 2) using IRF-3 (D6I4C) XP® Rabbit mAb, #11904 (upper) or β-actin (13E5) Rabbit mAb, #4970 (lower). The absence of signal in the IRF-3 knockout HeLa cells confirms specificity of the antibody for IRF-3.
undefined Image 1: Rig-I Pathway Antibody Sampler Kit
Western blot analysis of recombinant GST-IKKε #7553 and lysates from Ramos and RL cells using IKKε (D20G4) Rabbit mAb.
undefined Image 2: Rig-I Pathway Antibody Sampler Kit
Western blot analysis of HCT116 cell extracts, untreated (-) or TBK1/NAK knock-out (+), using TBK/NAK (D1B4) Rabbit mAb Antibody #3504 (upper) or β-actin (D6A8) Rabbit mAb #8457 (lower).
undefined Image 3: Rig-I Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

Cell Signaling Technology Logo
Western blot analysis of differentiated THP-1 and RAW 264.7 cells, untreated or LPS-treated (1 ug/ml, overnight), using Rig-I (D14G6) Rabbit mAb.
undefined Image 4: Rig-I Pathway Antibody Sampler Kit
Western blot analysis of extracts from LNCaP, MCF-7, and HT29 cell lines using MAVS Antibody.
undefined Image 5: Rig-I Pathway Antibody Sampler Kit
Western blot analysis of extracts from HT29 and THP1 cells, control or plpC-transfected (1 hour), using Phospho-IRF-3 (Ser396) (4D4G) Rabbit mAb.
undefined Image 6: Rig-I Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

Cell Signaling Technology Logo
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with human MDA-5 (+), using MDA-5 (D74E4) Rabbit mAb.
undefined Image 7: Rig-I Pathway Antibody Sampler Kit
Western blot analysis of extracts from THP-1 cells differentiated with TPA #4174 (80 nM, overnight) followed by treatment with LPS (1 μg/ml), up to 24h, using Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb (upper), or total TBK1/NAK (D1B4) Rabbit mAb #3504 (lower).
undefined Image 8: Rig-I Pathway Antibody Sampler Kit
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.
undefined Image 9: Rig-I Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

Cell Signaling Technology Logo
Western blot analysis of extracts from THP-1 cells, differentiated with TPA #4174 (80 nM, overnight) followed by treatment with LPS (1 μg/ml, indicated times), using Phospho-IKKε (Ser172) (D1B7) Rabbit mAb (upper) or IKKε (D20G4) Rabbit mAb #2905 (lower).
undefined Image 10: Rig-I Pathway Antibody Sampler Kit
Western blot analysis of extracts from various cell lines using IRF-3 (D6I4C) XP® Rabbit mAb.
undefined Image 11: Rig-I Pathway Antibody Sampler Kit
Western blot analysis of extracts from various cell lines using TBK1/NAK (D1B4) Rabbit mAb.
undefined Image 12: Rig-I Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

Cell Signaling Technology Logo
Western blot analysis of extracts from HeLa cells, mock-transfected or transfected with human MAVS, using MAVS Antibody.
undefined Image 13: Rig-I Pathway Antibody Sampler Kit
Western blot anlaysis of extracts from differentiated THP-1 cells, untreated or treated with LPS (1 μg/ml, indicated times), using MDA-5 (D74E4) Rabbit mAb.
undefined Image 14: Rig-I Pathway Antibody Sampler Kit
Western blot analysis of extracts from THP-1 cells differentiated with TPA #4174 (80 nM, overnight) followed by treatment with LPS (1 μg/ml, 1 hour), with or without phosphatase treatment using Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb (upper), or total TBK1/NAK (D1B4) Rabbit mAb #3504 (lower).
undefined Image 15: Rig-I Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

Cell Signaling Technology Logo
Immunoprecipitation of IRF-3 from THP-1 cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or IRF-3 (D6I4C) XP® Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using IRF-3 (D6I4C) XP® Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 and Anti-mouse IgG, HRP-linked Antibody #7076 were used as secondary antibodies.
undefined Image 16: Rig-I Pathway Antibody Sampler Kit
Western blot analysis of extracts from Raw264.7 and CHO cells using Rig-I (D14G6) Rabbit mAb.
undefined Image 17: Rig-I Pathway Antibody Sampler Kit
Confocal immunofluorescent analysis of MCF-7 cells using MAVS Antibody (green) showing colocalization with mitochondria that have been labeled with MitoTracker® Red CMXRos (red). Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).
undefined Image 18: Rig-I Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

Cell Signaling Technology Logo
Confocal immunofluorescent analysis of THP-1 cells differentiated with TPA #4174 (80nM, overnight) (left), followed by treatment with LPS (1μg/ml, 1 hour) (center) or LPS with λ phosphatase treatment (right) using Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 Phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
undefined Image 19: Rig-I Pathway Antibody Sampler Kit
Confocal immunofluorescent analysis of HT-29 cells, untransfected (left) or transfected with Poly(I:C) (2.5 μg/ml, 6 hr; right), using IRF-3 (D6I4C) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
undefined Image 20: Rig-I Pathway Antibody Sampler Kit
Flow cytometric analysis of THP-1 cells differentiated with TPA (80nM, 4 days) #9905, untreated (blue) or treated with LPS (1 ng/mL, 1 hr; green) #14011 using Phospho-TBK1/NAK (Ser172) (D52C2) XP® 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.
undefined Image 21: Rig-I Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

Cell Signaling Technology Logo
Simple Western™ analysis of lysates (1 mg/mL) from HeLa cells using IRF-3 (D6I4C) XP ® Rabbit mAb #11904. The virtual lane view (left) shows the target band (as indicated) at 1:50 and 1:250 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:50 (blue line) and 1:250 (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.
undefined Image 22: Rig-I Pathway Antibody Sampler Kit
Simple WesternTM analysis of lysates (0.1 mg/mL) from HeLa cells using TBK1/NAK (D1B4) Rabbit mAb #3504.  The virtual lane view (left) shows the target band (as indicated) at 1:50 and 1:250 dilutions of primary antibody.  The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:50 (blue line) and 1:250 (green line) dilutions of primary antibody.  This experiment was performed under reducing conditions on the JessTM Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
undefined Image 23: Rig-I Pathway Antibody Sampler Kit
Simple WesternTM analysis of lysates (0.1 mg/mL) from Raw264.7 cells using Rig-I (D14G6) Rabbit mAb #3743. The virtual lane view (left) shows the target (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 JessTM Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
undefined Image 24: Rig-I Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

Cell Signaling Technology Logo
Western blot analysis of extracts from THP-1 cells, undifferentiated (-) or differentiated with TPA (80 nM, overnight) followed by treatment with LPS (1 µg/ml, 8 hrs; +), using TBK1/NAK (E9H5S) Mouse mAb #51872 (Panel A) and Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb #5483 (Panel B). Anti-mouse IgG (H+L) (DyLight 680 Conjugate) #5470 (red) and Anti-rabbit IgG (H+L) (DyLight 800 4X PEG Conjugate) #5151 (green) were used as secondary antibodies.
undefined Image 25: Rig-I Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.