Confocal immunofluorescent analysis of L-929 (NCTC clone 929) cells, untreated (left) or pre-treated with Z-VAD (20 μM, 30 min) followed by treatment with SM-164 (100 nM) and Mouse Tumor Necrosis Factor-α (mTNF-α) #5178 (20 ng/ml, 2 hr; right), using Phospho-RIP3 (Thr231/Ser232) (E7S1R) Rabbit mAb (Alexa Fluor® 488 Conjugate) (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 fluorescent dye and tested in-house for direct immunofluorescent analysis in mouse cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated Phospho-RIP3 (Thr231/Ser232) (E7S1R) Rabbit mAb #91702.
Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted November 2006
revised November 2013
Protocol Id: 182
Phospho-RIP3 (Thr231/Ser232) (E7S1R) Rabbit mAb (Alexa Fluor® 488 Conjugate) recognizes endogenous levels of RIP3 protein only when phosphorylated at Thr231/Ser232.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic phospho-peptide corresponding to residues surrounding Thr231/Ser232 of mouse RIP3 protein. This antibody may not recognize RIP3 when only singly phosphorylated at Thr231 or Ser232.
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).
Explore pathways + proteins related to this product.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DyLight is a trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.
ProLong is a registered trademark of Life Technologies Corporation.
This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is conditioned on the buyer using the purchased product solely in research conducted by the buyer, excluding contract research or any fee for service research, and the buyer must not (1) use this product or its components for (a) diagnostic, therapeutic or prophylactic purposes; (b) testing, analysis or screening services, or information in return for compensation on a per-test basis; or (c) manufacturing or quality assurance or quality control, and/or (2) sell or transfer this product or its components for resale, whether or not resold for use in research. For information on purchasing a license to this product for purposes other than as described above, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or firstname.lastname@example.org.