Western blot analysis of extracts from C2C12 cells, untreated (-) or treated with Mouse Tumor Necrosis Factor-α #5178 (mTNF-α; 20 ng/ml, 5 min; +) with or without Lambda Phosphatase (λPPase) and Calf Intestinal Phosphatase (CIP) (+) as indicated, using Phospho-RIP (Ser321) Antibody (upper), total RIP (D94C12) XP® Rabbit mAb #3493 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).Learn more about how we get our images.
Western blot analysis of extracts from MEF or MEF/RIP KO cells, untreated (-) or treated with Mouse Tumor Necrosis Factor-α #5178 (mTNF-α; 20 ng/ml, 5 min; +), using Phospho-RIP (Ser321) Antibody (Mouse Specific) (upper), total RIP (D94C12) XP® Rabbit mAb #3493 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower). MEF/RIP KO cells were kindly provided by Dr. Junying Yuan, Harvard Medical School, Boston, MA.Learn more about how we get our images.
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
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
Phospho-RIP (Ser321) Antibody (Mouse Specific) recognizes endogenous levels of RIP protein only when phosphorylated at Ser321.
Polyclonal antibodies are produced by immunizing animals with a synthetic phospho-peptide corresponding to residues surrounding Ser321 of mouse RIP protein. Antibodies are purified by protein A and peptide affinity chromatography.
The receptor-interacting protein (RIP) family of serine-threonine kinases (RIP, RIP2, RIP3, and RIP4) are important regulators of cellular stress that trigger pro-survival and inflammatory responses through the activation of NF-κB, as well as pro-apoptotic pathways (1). In addition to the kinase domain, RIP contains a death domain responsible for interaction with the death domain receptor Fas and recruitment to TNF-R1 through interaction with TRADD (2,3). RIP-deficient cells show a failure in TNF-mediated NF-κB activation, making the cells more sensitive to apoptosis (4,5). RIP also interacts with TNF-receptor-associated factors (TRAFs) and can recruit IKKs to the TNF-R1 signaling complex via interaction with NEMO, leading to IκB phosphorylation and degradation (6,7). Overexpression of RIP induces both NF-κB activation and apoptosis (2,3). Caspase-8-dependent cleavage of the RIP death domain can trigger the apoptotic activity of RIP (8).
Necroptosis, a regulated pathway for necrotic cell death, is triggered by a number of inflammatory signals, including cytokines in the tumor necrosis factor (TNF) family, pathogen sensors such as toll-like receptors (TLRs), and ischemic injury (9,10). The process is negatively regulated by caspases and is initiated through a complex containing the RIP and RIP3 kinases, typically referred to as the necrosome. Necroptosis is inhibited by a small molecule inhibitor of RIP, necrostatin-1 (Nec-1) (11). Research studies show that necroptosis contributes to a number of pathological conditions, and Nec-1 has been shown to provide neuroprotection in models such as ischemic brain injury (12). RIP is phosphorylated at several sites within the kinase domain that are sensitive to Nec-1, including Ser14, Ser15, Ser161, and Ser166 (13).
RIP is also phosphorylated at Ser321(mouse)/Ser320(human) by MAPKAPK-2 (MK-2) and TAK1 in response to inflammatory signals such as TNF-α and LPS (14-17). Phosporylation at this site suppresses RIP mediated apoptosis by inhibiting its interaction with FADD and caspase-8 (14-17).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc. Tween is a registered trademark of ICI Americas, Inc.
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