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48768
p62/KEAP1/NRF2 Pathway Sampler Kit
Primary Antibodies
Antibody Sampler Kit

p62/KEAP1/NRF2 Pathway Sampler Kit #48768

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p62/KEAP1/NRF2 Pathway Sampler Kit: Image 1

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.

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 2

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.

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 3

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.

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 4

Confocal immunofluorescent analysis of OVCAR8 cells using KEAP1 (D6B12) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 5

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 SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across NQO1, a known target gene of NRF2 (see additional figure containing ChIP-qPCR data).

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 6

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.

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 7

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).

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 8

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.

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 9

Western blot analysis of extracts from various cell lines using SQSTM1/p62 (D1Q5S) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 10

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).

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 11

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).

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 12

Western blot analysis of extracts from various cell lines using KEAP1 (D6B12) Rabbit mAb.

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 13

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 SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #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).

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 14

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)

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 15

Western blot analysis of extracts from various cell lines using NQO1 (D6H3A) Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 16

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).

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 17

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.

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 18

Immunohistochemical analysis of paraffin-embedded mouse prostate using LC3A/B (D3U4C) XP® Rabbit mAb.

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 19

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 #5289 (+), 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.

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 20

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.

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 21

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.

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 22

Immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma, untreated (left) or lambda phosphatase treated (right), using Phospho-SQSTM1/p62 (Ser349) (E7M1A) Rabbit mAb.

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 23

Immunohistochemical analysis of paraffin-embedded NIH/3T3 cell pellets, control (left) or chloroquine-treated (right), using LC3A/B (D3U4C) XP® Rabbit mAb.

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 24

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.

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 25

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).

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 26

Immunohistochemical analysis of paraffin-embedded human lung adenocarcinoma, untreated (left) or lambda phosphatase treated (right), using Phospho-SQSTM1/p62 (Ser349) (E7M1A) Rabbit mAb.

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 27

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).

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 28

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).

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 29

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.

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 30

Western blot analysis of extracts from RD cells, untreated (-) or Torin 1-treated (250 nM, 4 hr; +), using LC3A/B (D3U4C) XP® Rabbit mAb.

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 31

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.

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 32

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).

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 33

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.

p62/KEAP1/NRF2 Pathway Sampler Kit: Image 34

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).

To Purchase # 48768T
Product # Size Price
48768T
1 Kit  (7 x 20 µl) N/A

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
SQSTM1/p62 (D1Q5S) Rabbit mAb 39749 20 µl
  • WB
  • IP
H M R 62 Rabbit IgG
Phospho-SQSTM1/p62 (Ser349) (E7M1A) Rabbit mAb 16177 20 µl
  • WB
  • IHC
  • IF
  • F
H M 62 Rabbit IgG
LC3A/B (D3U4C) XP® Rabbit mAb 12741 20 µl
  • WB
  • IHC
  • IF
  • F
H M R 14, 16 Rabbit IgG
KEAP1 (D6B12) Rabbit mAb 8047 20 µl
  • WB
  • IF
H M R 60-64 Rabbit IgG
NRF2 (D1Z9C) XP® Rabbit mAb 12721 20 µl
  • WB
  • IP
  • F
  • ChIP
H M Mk 97-100 Rabbit IgG
HO-1 (E3F4S) Rabbit mAb 43966 20 µl
  • WB
  • IP
H M R 28 Rabbit IgG
NQO1 (D6H3A) Rabbit mAb 62262 20 µl
  • WB
  • IF
H 29 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product Description

The p62/KEAP1/NRF2 Pathway 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.

Specificity / Sensitivity

Each antibody in the p62/KEAP1/NRF2 Pathway Sampler Kit detects endogenous levels of its target protein. Phospho-SQSTM1/p62 (Ser349) (E7M1A) Rabbit mAb recognizes endogenous levels of SQSTM1/p62 protein only when phosphorylated at Ser349.

Source / Purification

Monoclonal antibody is produced by immunizing animals with synthetic peptides corresponding to residues surrounding Leu44 of human LC3B (conserved in LC3A), Ala275 of human NRF2, Leu228 of human NQO1, Leu118 of mouse HO-1, and near the carboxy termini of human KEAP1 and SQSTM1/p62 proteins. Phosphorylation-specific monoclonal antibodies are produced by immunizing rabbits with synthetic phosphopeptides corresponding to Ser349 of human SQSTM1/p62 protein.

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).

  1. Zhu, M. and Fahl, W.E. (2001) Biochem Biophys Res Commun 289, 212-9.
  2. Bellezza, I. et al. (2018) Biochim Biophys Acta Mol Cell Res 1865, 721-33.
  3. Yamamoto, M. et al. (2018) Physiol Rev 98, 1169-203.
  4. Jiang, T. et al. (2015) Free Radic Biol Med 88, 199-204.
  5. Nezis, I.P. and Stenmark, H. (2012) Antioxid Redox Signal 17, 786-93.
  6. Rojo de la Vega, M. et al. (2018) Cancer Cell 34, 21-43.
  7. Sánchez-Martín, P. et al. (2019) FEBS J 286, 8-23.
  8. Shah, S.Z.A. et al. (2018) Front Mol Neurosci 11, 310.
  9. Ichimura, Y. and Komatsu, M. (2018) Front Oncol 8, 210.

Pathways & Proteins

Explore pathways + proteins related to this product.

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