Revision 1

#2903Store at -20C

1 Kit

(6 x 20 microliters)

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb 4858 20 µl 32 kDa Rabbit IgG
Phospho-S6 Ribosomal Protein (Ser240/244) (D68F8) XP® Rabbit mAb 5364 20 µl 32 kDa Rabbit IgG
Phospho-eIF4B (Ser422) Antibody 3591 20 µl 80 kDa Rabbit 
Phospho-eEF2k (Ser366) Antibody 3691 20 µl 105 kDa Rabbit 
Phospho-p70 S6 Kinase (Thr389) (108D2) Rabbit mAb 9234 20 µl 70, 85 kDa Rabbit IgG
p70 S6 Kinase (49D7) Rabbit mAb 2708 20 µl 70, 85 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

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

Description

The p70 S6 Kinase Substrates Antibody Sampler Kit provides a fast and economical means of evaluating several substrates of p70 S6 Kinase. The kit contains enough primary and secondary antibody to perform two Western blot experiments.

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

p70 S6 kinase is a mitogen activated Ser/Thr protein kinase that is required for cell growth and G1 cell cycle progression (1,2). p70 S6 kinase phosphorylates the S6 protein of the 40S ribosomal subunit and is involved in translational control of 5' oligopyrimidine tract mRNAs (1). Important S6 ribosomal protein phosphorylation sites include several residues (Ser235, Ser236, Ser240, Ser244) located wtihin a small, carboxy-terminal region of the S6 protein (3,4). p70 S6 kinase has been shown to phosphorylate eIF4B at the rapamycin-sensitive site Ser422 in vivo, and a Ser422Ala mutant of eIF4B shows diminished activity in an in vitro translation assay (5). Phosphorylation of eEF2K by p70 S6 kinase and p90RSK leads to inactivation of eEF2K (6), facilitating the dephosphorylation of eEF2 and thus promoting translation.

  1. Pullen, N. and Thomas, G. (1997) FEBS Lett 410, 78-82.
  2. Dufner, A. and Thomas, G. (1999) Exp Cell Res 253, 100-9.
  3. Ferrari, S. et al. (1991) J Biol Chem 266, 22770-5.
  4. Flotow, H. and Thomas, G. (1992) J Biol Chem 267, 3074-8.
  5. Raught, B. et al. (2004) EMBO J 23, 1761-9.
  6. Wang, X. et al. (2001) EMBO J 20, 4370-9.

Background References

    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
    #2903

    p70 S6 Kinase Substrates Antibody Sampler Kit

    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 1 Expand Image
    Confocal immunofluorescent analysis of fixed frozen mouse hippocampus, untreated (left) or post-processed with λ phosphatase (middle), using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb (green) and, untreated (right), using S6 Ribosomal Protein (5G10) Rabbit mAb #2217 (green) and ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 2 Expand Image
    Confocal immunofluorescent analysis of fixed frozen mouse pancreas, untreated (left) or post-processed with λ phosphatase (right), using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb (green), DyLight™ 554 Phalloidin #13054 (red), and ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 3 Expand Image
    Simple Western™ analysis of lysates (1 mg/mL) from serum-starved MCF-7 cells treated with human IGF-1 (100 ng/mL, 10 min) using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb #4858. The virtual lane view (left) shows the target band (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 Jess™ ​​​​​​​ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 4 Expand Image

    Simple Western™ analysis of lysates (1 mg/mL) from serum-starved MCF-7 cells treated with hIGF-1 (100 ng/mL; 10 min) using Phospho-S6 Ribosomal Protein (Ser240/244) (D68F8) XP® Rabbit mAb #5364. The virtual lane view (left) shows the target band (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 Jess™ ​​​​​​​ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.

    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 5 Expand Image
    Simple Western™ analysis of lysates (0.1 mg/mL) from serum-starved MCF-7 cells treated with hIGF-1 (100 ng/mL, 10 min) using Phospho-p70 S6 Kinase (Thr389) (108D2) Rabbit mAb #9234. The virtual lane view (left) shows the target band (as indicated) and a band corresponding to Phospho-p85 S6 Kinase (Thr412) (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 Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from various human cell lines using p70 S6 Kinase (49D7) Rabbit mAb (upper) and GAPDH (D16H11)XP® Rabbit mAb #5174 (lower).
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from HeLa cells using Phospho-eIF4B (Ser422) Antibody (upper) or eIF4B Antibody #3592 (lower). 48 hours following siRNA transfection, cells were treated with Rapamycin (50 nM) and U0126 (10 µM) as indicated.
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 8 Expand Image
    Western blot analysis of extracts from SW-13 cells (starved for 18 hours) treated with calf intestinal alkaline phosphatase (CIP) or 20% fetal bovine serum for 30 minutes, and extracts from HeLa cells (starved for 18 hours) treated with 200 nM TPA for 30 minutes, using Phospho-eEF2k (Ser366) Antibody (upper) or eEF2k Antibody #3692 (lower).
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of extracts from serum-starved MCF7 cells, untreated (-) or treated (+) with combinations of the following treatments as indicated: human IGF-1 (100 ng/mL, 10 min) and λ phosphatase, using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb (upper) or S6 Ribosomal Protein (5G10) Rabbit mAb #2217 (lower).
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of extracts from MCF7 and NIH/3T3 cells, treated with 100 nM insulin (10 min) or 20% FBS (30 min) as indicated, using Phospho-S6 Ribosomal Protein (Ser240/244) (D68F8) XP® Rabbit mAb (upper) or S6 Ribosomal Protein (5G10) Rabbit mAb #2217 (lower).
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 11 Expand Image
    Flow cytometric analysis of Jurkat cells, untreated (green) or treated with LY294002 #9901, wortmannin #9951 and U0126 #9903 (50 μM, 1 μM and 10 μM, 2hr; blue) using Phospho-S6 Ribosomal Protein (Ser240/244) (D68F8) 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.
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 12 Expand Image
    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.
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 13 Expand Image
    Western blot analysis of extracts from serum starved or serum treated (20%) 293, NIH/3T3, and PC12 cells, using Phospho-p70 S6 Kinase (Thr389) (108D2) Rabbit mAb (upper), or p70 S6 Kinase (49D7) rabbit mAb #2708 (lower).
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 14 Expand Image
    Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® p70/85 S6 Kinase siRNA I #6566 (+) or SignalSilence® p70/85 S6 Kinase siRNA II #6572 (+), using p70 S6 Kinase (49D7) Rabbit mAb #2708 and α-Tubulin (11H10) Rabbit mAb #2125. The p70 S6 Kinase (49D7) Rabbit mAb confirms silencing of p70/85 S6 kinase expression, while the α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of p70/85 S6 kinase siRNA.
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 15 Expand Image
    Immunohistochemical analysis of paraffin-embedded LNCaP cells, untreated (left) or LY294002-treated (right), using Phospho-S6 Ribosomal Protein (S235/236) (D57.2.2E) XP® Rabbit mAb on SignalSlide® Phospho-Akt (Ser473) IHC Controls #8101.
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 16 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Phospho-S6 Ribosomal Protein (Ser240/244) (D68F8) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 17 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Phospho-S6 Ribosomal Protein (Ser240/244) (D68F8) XP® Rabbit mAb.
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 18 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb.
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 19 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse colon using Phospho-S6 Ribosomal Protein (Ser240/244) (D68F8) XP® Rabbit mAb.
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 20 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb.
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 21 Expand Image
    Immunohistochemical analysis on SignalSlide® Phospho-Akt (Ser473) IHC Controls #8101 (paraffin-embedded LNCaP cell pellets -/+ LY294002) using Phospho-S6 Ribosomal Protein (Ser240/244) (D68F8) XP® Rabbit mAb.
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 22 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse spleen using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb.
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 23 Expand Image
    Immunohistochemical analysis of paraffin-embedded LNCaP cell pellets, control (left) or rapamycin-treated (right), using Phospho-S6 Ribosomal Protein (Ser240/244) (D68F8) XP® Rabbit mAb.
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 24 Expand Image
    Immunohistochemical analysis of paraffin-embedded A549 xenograft, untreated (left) or λ phosphatase-treated (right), using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb.
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 25 Expand Image
    Immunohistochemical analysis of paraffin-embedded Rh30 xenograft, control (left) or rapamycin-treated (right), using Phospho-S6 Ribosomal Protein (Ser240/244) (D68F8) XP® Rabbit mAb.
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 26 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb in the presence of control peptide (left) or Phospho-S6 Ribosomal Protein (Ser235/236) Blocking Peptide #1220 (right).
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 27 Expand Image
    Confocal immunofluorescent analysis of HeLa cells, insulin-treated (left) and LY294002-treated (#9901, right), using Phospho-S6 Ribosomal Protein (Ser240/244) (D68F8) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 28 Expand Image
    Immunohistochemical analysis of paraffin-embedded LNCaP cells, untreated (left) or rapamycin-treated (right), using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb.
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 29 Expand Image
    Confocal immunofluorescent analysis of HeLa cells, rapamycin-treated (left) or 20% serum-treated (right), using Phospho-S6 Ribosomal protein (Ser235/Ser236) (D57.2.2E) XP® Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    p70 S6 Kinase Substrates Antibody Sampler Kit: Image 30 Expand Image
    Flow cytometric analysis of Jurkat cells, untreated (green) or treated with LY294002 #9901, Wortmannin #9951, and U0126 #9903 (50 μM, 1 μM, and 10 μM, 2 hr; blue) using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) 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.
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    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.