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47928
Mouse Reactive Necroptosis Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Mouse Reactive Necroptosis Antibody Sampler Kit #47928

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other Image 1 - Mouse Reactive Necroptosis Antibody Sampler Kit

Immunoprecipitation of phospho-RIP (Ser166) protein from L-929 cell extracts treated with Z-VAD(OMe)-FMK #60332 (20 μM, 30 min) followed by Mouse Tumor Necrosis Factor-α (mTNF-α) #5178 (20 ng/ml, 2 hr) and SM-164 (100 nM, 2 hr). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 Phospho-RIP (Ser166) (E7G6O) Rabbit mAb. Western blot analysis was performed using Phospho-RIP (Ser166) (E7G6O) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody.

other Image 2 - Mouse Reactive Necroptosis Antibody Sampler Kit

Western blot analysis of extracts from wild-type (WT) MEF or MEF/RIPK1 knockout (KO) cells, untreated (-) or treated with Z-VAD(OMe)-FMK #60332 (20 μM, 30 min) followed by Mouse Tumor Necrosis Factor-α (mTNF-α) #5178 (20 ng/ml, 7 hr) and SM-164 (100 nM, 7 hr), using Phospho-RIP (Ser166) (E7G6O) Rabbit mAb (upper), RIP (D94C12) XP® Rabbit mAb #3493 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). MEF/RIPK1 KO cells were kindly provided by Dr. Junying Yuan, Harvard Medical School, Boston, MA.

other Image 3 - Mouse Reactive Necroptosis Antibody Sampler Kit

Flow cytometric analysis of MCF7 cells using RIP (D94C12) XP® Rabbit mAb (blue) compared to Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red).

other Image 4 - Mouse Reactive Necroptosis Antibody Sampler Kit

Confocal immunofluorescent analysis of L-929 cells, untreated (left), 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, 2.25 hr; center), or pre-treated with Z-VAD followed by treatment with SM-164 and hTNF-α and post-processed with λ-phosphatase (right), using Phospho-RIP3 (Thr231/Ser232) (E7S1R) Rabbit mAb (green). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).

other Image 5 - Mouse Reactive Necroptosis Antibody Sampler Kit

Immunoprecipitation of RIP3 from L-929 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is RIP3 (D8J3L) Rabbit mAb. Western blot analysis was performed using RIP3 (D8J3L) Rabbit mAb. A conformation-specific secondary antibody was used to avoid cross reactivity with IgG.

other Image 6 - Mouse Reactive Necroptosis Antibody Sampler Kit

Confocal immunofluorescent analysis of L-929 cells, untreated (left), 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, 2.5 hr; center) and then post-processed with λ-phosphatase (right), using Phospho-MLKL (Ser345) (D6E3G) Rabbit mAb (green). Red = Propidium Iodide (PI)/RNase Staining Solution #4087 (fluorescent DNA dye).

other Image 7 - Mouse Reactive Necroptosis Antibody Sampler Kit

Immunoprecipitation of MLKL protein from BaF3 cells. Lane 1 is 10% input, lane 2 is immunoprecipitated with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is MLKL (D6W1K) Rabbit mAb (Mouse Specific). Western blot was performed with MLKL (D6W1K) Rabbit mAb (Mouse Specific).

other Image 8 - Mouse Reactive Necroptosis 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.

other Image 9 - Mouse Reactive Necroptosis Antibody Sampler Kit

Western blot analysis of extracts from L-929 cells, untreated (-) or treated with Z-VAD(OMe)-FMK #60332 (20 μM, 30 min) followed by Mouse Tumor Necrosis Factor-α (mTNF-α) #5178 (20 ng/ml, indicated times) and SM-164 (100 nM, indicated times), using Phospho-RIP (Ser166) (E7G6O) Rabbit mAb (upper), RIP (D94C12) XP® Rabbit mAb #3493 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).

other Image 10 - Mouse Reactive Necroptosis Antibody Sampler Kit

Confocal immunofluorescent analysis of OVCAR8 cells using RIP (D94C12) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

other Image 11 - Mouse Reactive Necroptosis Antibody Sampler Kit

Western blot analysis of L-929 cells, untreated (-), or treated with combinations of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), SM-164 (100 nM, 3 hr; +), and mouse TNF-α (20 ng/ml, 3 hr; +), using Phospho-RIP3 (Thr231/Ser232) (E7S1R) Rabbit mAb (upper), RIP3 (D8J3L) Rabbit mAb #15828 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).

other Image 12 - Mouse Reactive Necroptosis Antibody Sampler Kit

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing full-length mouse RIP3 (mRIP3; +), using RIP3 (D8J3L) Rabbit mAb.

other Image 13 - Mouse Reactive Necroptosis Antibody Sampler Kit

Western blot analysis of L-929 cells, untreated (-), or treated with combinations of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), Mouse Tumor Necrosis Factor-α (mTNF-α) #5178 (20 ng/ml, 4 hr; +), SM-164 (100 nM, 4 hr; +), and necrostatin-1 (Nec-1, 50 μM, 4 hr; +), using Phospho-MLKL (Ser345) (D6E3G) Rabbit mAb (upper), total MLKL (D6W1K) Rabbit mAb (Mouse Specific) #37705 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).

other Image 14 - Mouse Reactive Necroptosis Antibody Sampler Kit

Western blot analysis of extracts from various cell lines using MLKL (D6W1K) Rabbit mAb (Mouse Specific).

other Image 15 - Mouse Reactive Necroptosis Antibody Sampler Kit

Western blot analysis of extracts from H9c2(2-1) cells, untreated (-) or treated with Z-VAD(OMe)-FMK #60332 (20 μM, 30 min) followed by Mouse Tumor Necrosis Factor-α (mTNF-α) #5178 (20 ng/ml, 4.5 hr) and SM-164 (100 nM, 4.5 hr), using Phospho-RIP (Ser166) (E7G6O) Rabbit mAb (upper), RIP (D94C12) XP® Rabbit mAb #3493 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).

other Image 16 - Mouse Reactive Necroptosis Antibody Sampler Kit

Western blot analysis of extracts from various cell lines using RIP (D94C12) XP® Rabbit mAb.

other Image 17 - Mouse Reactive Necroptosis Antibody Sampler Kit

Western blot analysis of extracts from various cell lines using RIP3 (D8J3L) Rabbit mAb.

other Image 18 - Mouse Reactive Necroptosis Antibody Sampler Kit

Western blot analysis of extracts from HeLa cells, untransfected or transfected with human RIP construct, using RIP (D94C12) XP® Rabbit mAb.

other Image 19 - Mouse Reactive Necroptosis Antibody Sampler Kit

Western blot analysis of extracts from wild-type (+) or RIP3 knockout (-) mouse spleen using RIP3 (D8J3L) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). Data were kindly provided by Dr. Junying Yuan, Harvard Medical School, Boston MA.

To Purchase # 47928T
製品番号 サイズ 価格 在庫
47928T
1 Kit  (6 x 20 µl)

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-RIP (Ser166) (E7G6O) Rabbit mAb 53286 20 µl
  • WB
  • IP
M R 78 Rabbit IgG
RIP (D94C12) XP® Rabbit mAb 3493 20 µl
  • WB
  • IP
  • IF
  • F
H M R Hm Mk 78 Rabbit IgG
Phospho-RIP3 (Thr231/Ser232) (E7S1R) Rabbit mAb 91702 20 µl
  • WB
  • IF
M 46-62 Rabbit IgG
RIP3 (D8J3L) Rabbit mAb 15828 20 µl
  • WB
  • IP
M R 46-62 Rabbit IgG
Phospho-MLKL (Ser345) (D6E3G) Rabbit mAb 37333 20 µl
  • WB
  • IF
M 54 Rabbit IgG
MLKL (D6W1K) Rabbit mAb (Mouse Specific) 37705 20 µl
  • WB
  • IP
M 54 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product Description

The Mouse Reactive Necroptosis Antibody Sampler Kit provides an economical means of detecting total and phosphorylated proteins associated with necroptosis. The kit includes enough antibodies to perform two western blots with each primary antibody.

Specificity / Sensitivity

Each antibody in the Mouse Reactive Necroptosis Antibody Sampler Kit detects endogenous levels of its target protein. Phospho-RIP3 (Thr231/Ser232) (E7S1R) Rabbit mAb may not recognize RIP3 when only singly phosphorylated at either Thr231 or Ser232.

Source / Purification

Monoclonal antibodies are produced by immunizing rabbits with synthetic peptides corresponding to Leu190 of human RIP, His411 of mouse RIP3, and residues near the carboxyl terminus of mouse MLKL protein. Phospho-specific monoclonal antibodies are produced by immunizing rabbits with synthetic phosphopeptides corresponding to Ser166 of mouse RIP, Thr231/Ser232 of mouse RIP3, and Ser345 of mouse MLKL protein.

Background

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), ischemic injury, and neurodegenerative diseases (1-3). 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) (4). RIP is phosphorylated at several sites within the kinase domain that are sensitive to Nec-1, including Ser14, Ser15, Ser161, and Ser166 (5). During necroptosis, RIP3 is phosphorylated at Ser227, leading to recruitment and phosphorylation of MLKL at Thr357 and Ser358 (6). Phosphorylation of MLKL results in its oligomerization and translocation to the plasma membrane, where it affects membrane integrity (7-10).

In mice, activation of RIP3 is associated with phosphorylation at Thr231 and Ser232 (11), and then MLKL is phosphorylated at Ser345 by RIP3 (12).

  1. Christofferson, D.E. and Yuan, J. (2010) Curr Opin Cell Biol 22, 263-8.
  2. Kaczmarek, A. et al. (2013) Immunity 38, 209-23.
  3. Zhou, W. and Yuan, J. (2014) Semin Cell Dev Biol 35, 14-23.
  4. Degterev, A. et al. (2008) Nat Chem Biol 4, 313-21.
  5. Ofengeim, D. and Yuan, J. (2013) Nat Rev Mol Cell Biol 14, 727-36.
  6. Sun, L. et al. (2012) Cell 148, 213-27.
  7. Cai, Z. et al. (2014) Nat Cell Biol 16, 55-65.
  8. Chen, X. et al. (2014) Cell Res 24, 105-21.
  9. Wang, H. et al. (2014) Mol Cell 54, 133-46.
  10. Dondelinger, Y. et al. (2014) Cell Rep 7, 971-81.
  11. Chen, W. et al. (2013) J Biol Chem 288, 16247-61.
  12. Murphy, J.M. et al. (2013) Immunity 39, 443-53.

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