Revision 4

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Store at -20C

p62/KEAP1/NRF2 Pathway Antibody Sampler Kit

1 Kit

(7 x 20 microliters)

CST Logo
Orders:

877-616-CELL (2355)

[email protected]

Support:

877-678-TECH (8324)

3 Trask Lane | Danvers | Massachusetts | 01923 | USA

For Research Use Only. Not for Use in Diagnostic Procedures.

Product Includes Product # Quantity Mol. Wt Isotype/Source
SQSTM1/p62 (D1Q5S) Rabbit Monoclonal Antibody3974920 µl62 kDaRabbit IgG
Phospho-SQSTM1/p62 (Ser349) (E7M1A) Rabbit Monoclonal Antibody1617720 µl62 kDaRabbit IgG
LC3A/B (D3U4C) Rabbit Monoclonal Antibody1274120 µl14, 16 kDaRabbit IgG
KEAP1 (D6B12) Rabbit Monoclonal Antibody804720 µl60-64 kDaRabbit IgG
NRF2 (D1Z9C) Rabbit Monoclonal Antibody1272120 µl97-100 kDaRabbit IgG
HO-1 (E3F4S) Rabbit Monoclonal Antibody4396620 µl28 kDaRabbit IgG
NQO1 (D6H3A) Rabbit Monoclonal Antibody6226220 µl29 kDaRabbit IgG
Anti-rabbit IgG, HRP-linked Antibody7074100 µlGoat

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

Description

The p62/KEAP1/NRF2 Pathway Antibody Sampler Kit provides an economical means of detecting the non-canonical mechanism of NRF2 activation involving autophagy. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.

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 antibodies.

Background

The cap ’n’ collar (CNC), leucine zipper (bZIP) transcription factor NRF2 (also called nuclear factor erythroid 2-related factor 2 (NFE2L2)) is the master regulator of the cellular antioxidant response, regulating the expression of over 200 genes that contain antioxidant response elements (AREs) in their regulatory regions by heterodimerizing with small MAF proteins (1). While NRF2 is expressed in all cell types, its basal protein levels are usually kept low during homeostatic conditions, mainly by KEAP1 (Kelch-like ECH-associated protein 1). Under normal conditions, KEAP1 binds to and targets NRF2 for ubiquitination-dependent proteasomal degradation. Upon oxidative stress, KEAP1 is modified on some sensor cysteines, affecting its conformation and thus interfering its binding to NRF2, allowing newly synthesized NRF2 to accumulate and translocate to the nucleus to activate its target genes, including HO-1 (heme oxygenase 1) and NQO1 (NAD(P)H:quinone oxidoreductase 1) (2,3). Another mode of NRF2 regulation involves the autophagy adapter protein p62 (or sequestosome 1 [SQSTM1]) in a KEAP1-dependent but cysteine-independent manner, the so called non-canonical pathway. Autophagy is a tightly regulated cellular quality control system that removes damaged proteins or organelles. Autophagy can also be activated to degrade macromolecules to provide nutrients under cellular starvation stress. p62, especially upon phosphorylation at Ser349 (Ser351 in mouse p62), can compete with NRF2 for binding KEAP1 and, as a result, p62 sequesters KEAP1 into the autophagosome and prevents KEAP1-mediated NRF2 degradation. This process may also require autophagy protein LC3. In addition, studies also found that KEAP1 is a p62-regulated substrate for autophagy-mediated degradation, indicating that p62 also plays a role in controlling KEAP1 turnover (4,5). Dysregulation of autophagy results in prolonged NRF2 activation and this may contribute to many diseases, including cancer and neurodegenerative diseases (6-9).

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 4

Cell Signaling Technology Logo
Chromatin immunoprecipitations were performed with cross-linked chromatin from MEF cells treated with DEM (50 μM, 3 hr) and NRF2 (D1Z9C) XP® Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figure shows binding across NQO1, a known target gene of NRF2 (see additional figure containing ChIP-qPCR data).
undefined Image 1: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
Western blot analysis of extracts from MEF wt and U-2 OS cells, untreated (-) or treated with MG-132 #2194 (10 μM, 10 hr; +), using NRF2 (D1Z9C) XP® Rabbit mAb.
undefined Image 2: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
Western blot analysis of extracts from RD cells, untreated (-) or Torin 1-treated (250 nM, 4 hr; +), using LC3A/B (D3U4C) XP® Rabbit mAb.
undefined Image 3: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
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 4

Cell Signaling Technology Logo
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.
undefined Image 4: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
Western blot analysis of extracts from various cell lines using SQSTM1/p62 (D1Q5S) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
undefined Image 5: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
Western blot analysis of extracts from cell lines and tissues, untreated (-) or treated with arsenite (50 μM, 8 hr; +), using HO-1 (E3F4S) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower)
undefined Image 6: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
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 4

Cell Signaling Technology Logo
Western blot analysis of extracts from various cell lines using NQO1 (D6H3A) Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
undefined Image 7: p62/KEAP1/NRF2 Pathway 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.
undefined Image 8: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
Western blot analysis of extracts from various cell lines using KEAP1 (D6B12) Rabbit mAb.
undefined Image 9: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
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 4

Cell Signaling Technology Logo
Chromatin immunoprecipitations were performed with cross-linked chromatin from MEF cells treated with DEM (50 μM, 3 hr) and NRF2 (D1Z9C) XP® Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across chromosome 8 (upper), including NQO1 (lower), a known target gene of NRF2 (see additional figure containing ChIP-qPCR data).
undefined Image 10: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
Immunoprecipitation of NRF2 from MEF wt cell extracts treated with MG-132 #2194 (10 μM, 10 hr) using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or NRF2 (D1Z9C) XP® Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using NRF2 (D1Z9C) XP® Rabbit mAb (lane 3).
undefined Image 11: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
Western blot analysis of extracts from HeLa, NIH/3T3, and KNRK cells, untreated (-) or chloroquine-treated (50 μM, overnight; +), using LC3A/B (D3U4C) XP® Rabbit mAb.
undefined Image 12: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
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 4

Cell Signaling Technology Logo
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).
undefined Image 13: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
Western blot analysis of extracts from MEF cells, untreated (-) or treated with Earles Basic Salt Solution (EBSS; 4 hr; +), using SQSTM1/p62 (D1Q5S) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
undefined Image 14: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
Immunoprecipitation of HO-1 from arsenite-treated (50 μM, 8 hr) NIH/3T3 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is HO-1 (E3F4S) Rabbit mAb. Western blot analysis was performed using HO-1 (E3F4S) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used for detection.
undefined Image 15: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
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 4

Cell Signaling Technology Logo
Confocal immunofluorescent analysis of DU 145 (positive, left) or Saos-2 (negative, right) cells using NQO1 (D6H3A) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
undefined Image 16: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
Western blot analysis of extracts from OVCAR8 cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® KEAP1 siRNA I #5285 (+) or SignalSilence® KEAP1 siRNA II (+), using KEAP1 (D6B12) Rabbit mAb (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The KEAP1 (D6B12) Rabbit mAb confirms silencing of KEAP1 expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.
undefined Image 17: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
Chromatin immunoprecipitations were performed with cross-linked chromatin from MEF NRF2 wild-type (left) and NRF2 knock-out (right) cells, both treated with DEM (50 μM, 3 hr), and NRF2 (D1Z9C) XP® Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using mouse MafG intron 1 primers, SimpleChIP® Mouse NQO1 Promoter Primers #12635, and SimpleChIP® Mouse RPL30 Intron 2 Primers #7015. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
undefined Image 18: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
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 4

Cell Signaling Technology Logo
Confocal immunofluorescent analysis of wild-type NRF2 MEF cells, untreated (left) or treated with MG-132 #2194 (10 μM, 8 hr; center), and NRF2 knock-out MEF cells treated with MG-132 #2194 (10 μM, 8 hr; right), using NRF2 (D1Z9C) XP® Rabbit mAb (green pseudocolor). Actin filaments were labeled with Alexa Fluor® 488 Phalloidin #8878 (red pseudocolor).
undefined Image 19: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
Immunohistochemical analysis of paraffin-embedded mouse prostate using LC3A/B (D3U4C) XP® Rabbit mAb.
undefined Image 20: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
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).
undefined Image 21: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
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 4

Cell Signaling Technology Logo
Western blot analysis of extracts from MEF cells from wild-type or SQSTM1/p62 knockout mice using SQSTM1/p62 (D1Q5S) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). MEF/p62 KO cells were kindly provided by Dr. Junying Yuan, Harvard Medical School, Boston MA.
undefined Image 22: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
Confocal immunofluorescent analysis of OVCAR8 cells using KEAP1 (D6B12) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
undefined Image 23: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
Flow cytometric analysis of MEF wt cells, untreated (blue) or treated with MG-132 #2194 (10uM, 4 hrs; green) using NRF2 (D1Z9C) XP® Rabbit mAb (solid line) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed line). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
undefined Image 24: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
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 4

Cell Signaling Technology Logo
Immunohistochemical analysis of paraffin-embedded human squamous cell lung carcinoma using LC3A/B (D3U4C) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
undefined Image 25: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
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.
undefined Image 26: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
Western blot analysis of extracts from HeLa and C2C12 cells, untreated (-) or treated with Chloroquine #14774 (50 μM, overnight; +), using SQSTM1/p62 (D1Q5S) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
undefined Image 27: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
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 4

Cell Signaling Technology Logo
Immunohistochemical analysis of paraffin-embedded NIH/3T3 cell pellets, control (left) or chloroquine-treated (right), using LC3A/B (D3U4C) XP® Rabbit mAb.
undefined Image 28: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
Immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma, untreated (left) or lambda phosphatase treated (right), using Phospho-SQSTM1/p62 (Ser349) (E7M1A) Rabbit mAb.
undefined Image 29: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
Immunoprecipitation of SQSTM1 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 SQSTM1/p62 (D1Q5S) Rabbit mAb. Western blot was performed using SQSTM1/p62 (D1Q5S) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody.
undefined Image 30: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
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 4

Cell Signaling Technology Logo
Confocal immunofluorescent analysis of HeLa (upper) and C2C12 (lower) cells, chloroquine-treated (50 μM, overnight; left), nutrient-starved with EBSS (3 hr, middle) or untreated (right) using LC3A/B (D3U4C) XP® Rabbit mAb (green) and β-Actin (13E5) Rabbit mAb (Alexa Fluor® 555 Conjugate) #8046 (red). Blue pseudocolor= DRAQ5® #4084 (fluorescent DNA dye).
undefined Image 31: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
Immunohistochemical analysis of paraffin-embedded human lung adenocarcinoma, untreated (left) or lambda phosphatase treated (right), using Phospho-SQSTM1/p62 (Ser349) (E7M1A) Rabbit mAb.
undefined Image 32: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
Flow cytometric analysis of HeLa cells, untreated (blue) or treated with chloroquine (50 µM, 16 hr; green) using LC3A/B (D3U4C) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody.
undefined Image 33: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
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 4

Cell Signaling Technology Logo
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).
undefined Image 34: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
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.
undefined Image 35: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
Simple WesternTM analysis of lysates (0.1 mg/mL) from U-2 OS + MG-132 using NRF2 (D1Z9C) XP® Rabbit mAb #12721. 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 JessTM Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
undefined Image 36: p62/KEAP1/NRF2 Pathway Antibody Sampler Kit
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.