Revision 3
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.
Applications:

WB, IHC-P, IF-IC, FC-FP

REACTIVITY:

H M

SENSITIVITY:

Endogenous

MW (kDa):

62

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q13501

Entrez-Gene Id:

8878

Product Information

Product Usage Information

Application Dilution
Western Blotting 1:1000
Immunohistochemistry (Paraffin) 1:1600 - 1:6400
Immunofluorescence (Immunocytochemistry) 1:200 - 1:800
Flow Cytometry (Fixed/Permeabilized) 1:400 - 1:1600

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.

Specificity / Sensitivity

Phospho-SQSTM1/p62 (Ser349) (E7M1A) Rabbit mAb recognizes endogenous levels of SQSTM1/p62 protein only when phosphorylated at Ser349. Staining of mitotic cells is observed by immunohistochemstry. The specificity of this staining is unknown.

Species Reactivity:

Human, Mouse

Species predicted to react based on 100% sequence homology

Rat

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phospho-peptide corresponding to residues surrounding Ser349 of human SQSTM1/62 protein.

Background

Sequestosome 1 (SQSTM1, p62) is a ubiquitin binding protein involved in cell signaling, oxidative stress, and autophagy (1-4). It was first identified as a protein that binds to the SH2 domain of p56Lck (5) and independently found to interact with PKCζ (6,7). SQSTM1 was subsequently found to interact with ubiquitin, providing a scaffold for several signaling proteins and triggering degradation of proteins through the proteasome or lysosome (8). Interaction between SQSTM1 and TRAF6 leads to the K63-linked polyubiquitination of TRAF6 and subsequent activation of the NF-κB pathway (9). Protein aggregates formed by SQSTM1 can be degraded by the autophagosome (4,10,11). SQSTM1 binds autophagosomal membrane protein LC3/Atg8, bringing SQSTM1-containing protein aggregates to the autophagosome (12). Lysosomal degradation of autophagosomes leads to a decrease in SQSTM1 levels during autophagy; conversely, autophagy inhibitors stabilize SQSTM1 levels. Studies have demonstrated a link between SQSTM1 and oxidative stress. SQSTM1 interacts with KEAP1, which is a cytoplasmic inhibitor of NRF2, a key transcription factor involved in cellular responses to oxidative stress (3). Thus, accumulation of SQSTM1 can lead to an increase in NRF2 activity.
Phosphorylation of SQSTM1 at Ser349 (Ser351 in mouse) during oxidative stress increases its binding to KEAP1, thereby increasing NRF2 activity (13).

  1. Kirkin, V. et al. (2009) Mol Cell 34, 259-69.
  2. Seibenhener, M.L. et al. (2007) FEBS Lett 581, 175-9.
  3. Komatsu, M. et al. (2010) Nat Cell Biol 12, 213-23.
  4. Bjørkøy, G. et al. (2006) Autophagy 2, 138-9.
  5. Joung, I. et al. (1996) Proc Natl Acad Sci USA 93, 5991-5.
  6. Sanchez, P. et al. (1998) Mol Cell Biol 18, 3069-80.
  7. Puls, A. et al. (1997) Proc Natl Acad Sci USA 94, 6191-6.
  8. Vadlamudi, R.K. et al. (1996) J Biol Chem 271, 20235-7.
  9. Wooten, M.W. et al. (2005) J Biol Chem 280, 35625-9.
  10. Bjørkøy, G. et al. (2005) J Cell Biol 171, 603-14.
  11. Komatsu, M. et al. (2007) Cell 131, 1149-63.
  12. Pankiv, S. et al. (2007) J Biol Chem 282, 24131-45.
  13. Ichimura, Y. et al. (2013) Mol Cell 51, 618-31.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IHC-P: Immunohistochemistry (Paraffin) IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized)

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
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.

Revision 3
#16177

Phospho-SQSTM1/p62 (Ser349) (E7M1A) Rabbit mAb

Western Blotting Image 1: Phospho-SQSTM1/p62 (Ser349) (E7M1A) Rabbit mAb Expand Image
Western blot analysis of extracts from wild-type MEF and MEF SQSTM1/p62 KO cell lines, untreated (-) or treated with sodium arsenite (15 μM, 18 hr; +), using Phospho-SQSTM1/p62 (Ser349) (E7M1A) Rabbit mAb (upper), SQSTM1/p62 (D1Q5S) Rabbit mAb #39749 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). MEF SQSTM1/p62 KO cells were kindly provided by Dr. Junying Yuan, Harvard Medical School, Boston MA.
Western Blotting Image 2: Phospho-SQSTM1/p62 (Ser349) (E7M1A) Rabbit mAb Expand Image
Western blot analysis of extracts from PC-3 cells treated with sodium arsenite (15 μM, indicated times) using Phospho-SQSTM1/p62 (Ser349) (E7M1A) Rabbit mAb (upper), SQSTM1/p62 (D5L7G) Mouse mAb #88588 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western Blotting Image 3: Phospho-SQSTM1/p62 (Ser349) (E7M1A) Rabbit mAb Expand Image
Western blot analysis of extracts from PC-3 cells treated with sodium arsenite (15 μM, 18 hr) with lysates that were untreated (-) or treated with lambda-phosphatase and calf intestinal phosphatase (λPPase/CIP; +) using Phospho-SQSTM1/p62 (Ser349) (E7M1A) Rabbit mAb (upper), SQSTM1/p62 (D5L7G) Mouse mAb #88588 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Immunohistochemistry Image 1: Phospho-SQSTM1/p62 (Ser349) (E7M1A) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded 293T cell pellet, untreated (left, negative) or treated with sodium arsenite (right, positive), using Phospho-SQSTM1/p62 (Ser349) (E7M1A) Rabbit mAb.
Immunohistochemistry Image 2: Phospho-SQSTM1/p62 (Ser349) (E7M1A) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma, untreated (left) or lambda phosphatase treated (right), using Phospho-SQSTM1/p62 (Ser349) (E7M1A) Rabbit mAb.
Immunohistochemistry Image 3: Phospho-SQSTM1/p62 (Ser349) (E7M1A) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human lung adenocarcinoma, untreated (left) or lambda phosphatase treated (right), using Phospho-SQSTM1/p62 (Ser349) (E7M1A) Rabbit mAb.
Immunofluorescence Image 1: Phospho-SQSTM1/p62 (Ser349) (E7M1A) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of PC-3 cells, untreated (left), treated with sodium arsenite (15 μM, 18 hr; center), or treated with sodium arsenite and post-processed with λ-phosphatase (right), using Phospho-SQSTM1 (Ser349) (E7M1A) Rabbit mAb (green). Actin filaments were labeled with DyLight 650 Phalloidin #12956 (red). Blue = DAPI #4083 (fluorescent DNA dye).
Flow Cytometry Image 1: Phospho-SQSTM1/p62 (Ser349) (E7M1A) Rabbit mAb Expand Image
Flow cytometric analysis of PC-3 cells, untreated (blue) or treated with sodium arsenite (15 µM, 18 hr; green), using Phospho-SQSTM1 (Ser349) (E7I4Z) 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® 647 Conjugate) #4414 was used as a secondary antibody.