Western blot analysis of extracts from various cell lines, untreated (-) or treated with Human Interferon-α1 (hIFNα) #8927 (10 ng/ml, overnight; +), using LGP2 (D3I3L) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from THP-1 cells differentiated with TPA #4174 (80 nM, overnight), untreated or LPS-treated (1 μg/ml for indicated times), using LGP2 (D3I3L) Rabbit mAb (upper) or β-Actin (D6A8) #8457 (lower).
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human LGP2 protein (hLGP2-Myc/DDK; +), using LGP2 (D3I3L) Rabbit mAb.
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.
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.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
LGP2 (D3I3L) Rabbit mAb recognizes endogenous levels of total LGP2 protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val478 of human LGP2 protein.
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).
The DExD/H-box family helicase laboratory of genetics and physiology 2 (LGP2, DHX58) is a Rig-I-like receptor (RLR) that lacks the CARD domain and associated signaling ability (6,16). Research studies demonstrate that LGP2 helicase binds dsRNA and inhibits the Rig-I-like receptors Rig-I and MDA-5. Expression of LGP2 is induced by interferon, dsRNA, and viral infection (17). Studies using LGP2-deficient animals demonstrate a complicated interaction between LGP2 and the other RLRs that involves both positive and negative effects on interferon regulation (18-20). In addition, LGP2 may regulate apoptosis, contribute to CD8+ T cell survival, and protect cancer cells from ionizing radiation (21,22).
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