Revision 1

#56175Store at -20C

1 Kit

(9 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
LRSAM1 (D1O5S) Rabbit mAb 28405 20 µl 84 kDa Rabbit IgG
Galectin-3/LGALS3 (E7B6R) Rabbit mAb 89572 20 µl 28 kDa Rabbit IgG
NDP52 (D1E4A) Rabbit mAb 60732 20 µl 52, 60 kDa Rabbit IgG
TAX1BP1 (D1D5) Rabbit mAb 5105 20 µl 92 kDa Rabbit IgG
Atg16L1 (D6D5) Rabbit mAb 8089 20 µl 66, 68 kDa Rabbit IgG
Phospho-Atg16L1 (Ser278) (E7K6H) Rabbit mAb 45511 20 µl 68 kDa Rabbit IgG
Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb 5483 20 µl 84 kDa Rabbit IgG
TBK1/NAK (E8I3G) Rabbit mAb 38066 20 µl 84 kDa Rabbit IgG
LC3B (E7X4S) XP® Rabbit mAb 43566 20 µl 14, 16 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 Xenophagy Antibody Sampler Kit provides an economical means of detecting selected targets involved in the process of xenophagy. 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

Xenophagy provides an important defense against foreign pathogens, such as bacteria and viruses, by targeting them for degradation through autophagy (1-3). Several well-known pathogens can be targeted for degradation through xenophagy, including Salmonella typhimurium, Streptococcus pyogenes, and Mycobacterium tuberculosis, and loss of core autophagy genes can result in enhanced vulnerability to these infections. Pathogens are generally targeted to LC3B or other Atg8 family members localized on autophagosome membranes. Sequestosome 1/p62-like receptors (SLRs) function as cargo receptors through ubiquitin-mediated interactions along with LIR-domain interactions with Atg8 proteins on the autophagosome. The SLR TAX1BP1 is recruited to ubiquitylated Salmonella and plays a key role in xenophagy (4). Another SLR NDP52 has also been reported to be required for xenophagy of several pathogens (5,6). LRSAM1 is an E3 ubiquitin ligase with a leucine-rich repeat recognized by microbial ligands and recruits NDP52 (7). In addition, galectins are a family of cytosolic lectins that are associated with phagophores and bacteria. Galectin-3 is recruited to bacterial membranes and is important for protection against Mycobacterium tuberculosis (8). TBK1 is a serine/threonine kinase that plays a major role in controlling autophagy by targeting substrates in the SLR family and the maturation of autophagosomes. TBK1 is phosphorylated at Ser172 within its activation loop, which is required for its kinase activity (9). Atg16L1 is a component of the Atg12-Atg5 conjugation system required for autophagosome maturation (10,11). ULK1-mediated phosphorylation of Atg16L1 at Ser278 promotes xenophagy (12).

  1. Reggio, A. et al. (2020) Exp Cell Res 396, 112276.
  2. Levine, B. (2005) Cell 120, 159-62.
  3. Deretic, V. et al. (2013) Nat Rev Immunol 13, 722-37.
  4. Tumbarello, D.A. et al. (2015) PLoS Pathog 11, e1005174.
  5. Verlhac, P. et al. (2015) Cell Host Microbe 17, 515-25.
  6. Fan, S. et al. (2020) Int J Mol Sci 21, 2008. doi: 10.3390/ijms21062008.
  7. Huett, A. et al. (2012) Cell Host Microbe 12, 778-90.
  8. Kumar, S. et al. (2017) Autophagy 13, 1086-1087.
  9. Kishore, N. et al. (2002) J Biol Chem 277, 13840-7.
  10. Fujita, N. et al. (2008) Mol Biol Cell 19, 2092-100.
  11. Mizushima, N. et al. (2003) J Cell Sci 116, 1679-88.
  12. Alsaadi, R.M. et al. (2019) EMBO Rep 20, e46885.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    XP is a registered trademark of Cell Signaling Technology, Inc.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

    使用に関する制限

    法的な権限を与えられたCSTの担当者が署名した書面によって別途明示的に合意された場合を除き、 CST、その関連会社または代理店が提供する製品には以下の条件が適用されます。お客様が定める条件でここに定められた条件に含まれるものを超えるもの、 または、ここに定められた条件と異なるものは、法的な権限を与えられたCSTの担当者が別途書面にて受諾した場合を除き、拒絶され、 いかなる効力も効果も有しません。

    研究専用 (For Research Use Only) またはこれに類似する表示がされた製品は、 いかなる目的についても FDA または外国もしくは国内のその他の規制機関により承認、認可または許可を受けていません。 お客様は製品を診断もしくは治療目的で使用してはならず、また、製品に表示された内容に違反する方法で使用してはなりません。 CST が販売または使用許諾する製品は、エンドユーザーであるお客様に対し、使途を研究および開発のみに限定して提供されるものです。 診断、予防もしくは治療目的で製品を使用することまたは製品を再販売 (単独であるか他の製品等の一部であるかを問いません) もしくはその他の商業的利用の目的で購入することについては、CST から別途許諾を得る必要があります。 お客様は以下の事項を遵守しなければなりません。(a) CST の製品 (単独であるか他の資材と一緒であるかを問いません) を販売、使用許諾、貸与、寄付もしくはその他の態様で第三者に譲渡したり使用させたりしてはなりません。また、商用の製品を製造するために CST の製品を使用してはなりません。(b) 複製、改変、リバースエンジニアリング、逆コンパイル、 分解または他の方法により製品の構造または技術を解明しようとしてはなりません。また、 CST の製品またはサービスと競合する製品またはサービスを開発する目的で CST の製品を使用してはなりません。(c) CST の製品の商標、商号、ロゴ、特許または著作権に関する通知または表示を除去したり改変したりしてはなりません。(d) CST の製品をCST 製品販売条件(CST’s Product Terms of Sale) および該当する書面のみに従って使用しなければなりません。(e) CST の製品に関連してお客様が使用する第三者の製品またはサービスに関する使用許諾条件、 サービス提供条件またはこれに類する合意事項を遵守しなければなりません。

    Revision 1
    #56175

    Xenophagy Antibody Sampler Kit

    Xenophagy Antibody Sampler Kit: Image 1 Expand Image
    Western blot analysis of extracts from C2C12 and H-4-II-E cells, untreated (-) or treated with Torin 1 #14379 (250 nM, 2 hr; +), using Phospho-Atg16L1 (Ser278) (E7K6H) Rabbit mAb (upper), Atg16L1 (D6D5) Rabbit mAb #8089 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
    Xenophagy Antibody Sampler Kit: Image 2 Expand Image
    Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing Myc/DDK-tagged full-length human Atg16L1 protein (hAtg16L1-Myc/DDK; +) and/or mouse ULK1 protein (mULK1; +), using Phospho-Atg16L1 (Ser278) (E7K6H) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
    Xenophagy Antibody Sampler Kit: Image 3 Expand Image
    Immunoprecipitation of Atg16L1 from Jurkat cell extracts. Lane 1 is 10% input, lane 2 is precipitated with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Atg16L1 (D6D5) Rabbit mAb, #8089. Western blot was performed using Atg16L1 (D6D5) Rabbit mAb.
    Xenophagy Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from various cell lines using LRSAM1 (D1O5S) Rabbit mAb.
    Xenophagy Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from HCT 116 cells, either wild-type (+/+) or TBK1/NAK knockout (-/-), using TBK1/NAK (E8I3G) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Xenophagy Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from HCT 116 and HCT 116 LC3B knockout cells, untreated (-) or treated with Chloroquine #14774 (50 μM, 18 hr) using LC3B (E7X4S) XP® Rabbit mAb #43566 (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The absence of signal in the HCT 116 knockout cells confirms the specificity of the antibody for LC3B.
    Xenophagy Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from AsPC-1, Calu-1, and HCT 116 cells, untreated (-) or treated with Torin 1 #14379 (250 nM, 2 hr; +) and/or the ULK1 inhibitor SBI-0206965 #29089 (50 μM, 2 hr; +), using Phospho-Atg16L1 (Ser278) (E7K6H) Rabbit mAb (upper), Atg16L1 (D6D5) Rabbit mAb #8089 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
    Xenophagy Antibody Sampler Kit: Image 8 Expand Image
    Western blot analysis of extracts from various cell lines using TAX1BP1 (D1D5) Rabbit mAb.
    Xenophagy Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of extracts from THP-1 cells differentiated with TPA #4174 (80 nM, overnight) followed by treatment with LPS (1 μg/ml), up to 24h, using Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb (upper), or total TBK1/NAK (D1B4) Rabbit mAb #3504 (lower).
    Xenophagy Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of extracts from various cell lines using NDP52 (D1E4A) Rabbit mAb.
    Xenophagy Antibody Sampler Kit: Image 11 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.
    Xenophagy Antibody Sampler Kit: Image 12 Expand Image
    Western blot analysis of extracts from various cell lines using Atg16L1 (D6D5) Rabbit mAb.
    Xenophagy Antibody Sampler Kit: Image 13 Expand Image
    Western blot analysis of extracts from various cell lines using Galectin-3/LGALS3 (E7B6R) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Xenophagy Antibody Sampler Kit: Image 14 Expand Image
    Confocal immunofluorescent analysis of fixed frozen mouse colon labeled with Galectin-3/LGALS3 (E7B6R) Rabbit mAb (left, red). Free secondary binding sites were then blocked with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 prior to colabeling with COL1A1 (E8F4L) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) #72827 (right, cyan pseudocolor) and DAPI #4083 (right, blue).
    Xenophagy Antibody Sampler Kit: Image 15 Expand Image
    Confocal immunofluorescent analysis of fixed frozen mouse subicular cortex from an amyloid mouse model of Alzheimer's Disease labeled with Galectin-3/LGALS3 (E7B6R) Rabbit mAb (left, red). Free secondary binding sites were then blocked with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 prior to colabeling with Iba1/AIF-1 (E4O4W) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) #20825 (right, green) and DAPI #4083 (right, blue).
    Xenophagy Antibody Sampler Kit: Image 16 Expand Image
    Confocal immunofluorescent analysis of SK-MEL-5 cells (left, positive) and SK-N-MC cells (right, negative) using Galectin-3/LGALS3 (E7B6R) Rabbit mAb (green), DyLight 554 Phalloidin #13054 (red), and DAPI #4083 (blue).
    Xenophagy Antibody Sampler Kit: Image 17 Expand Image
    Immunoprecipitation of Galectin-3/LGALS3 protein from C2C12 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Galectin-3/LGALS3 (E7B6R) Rabbit mAb. Western blot analysis was performed using Galectin-3/LGALS3 (E7B6R) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody.
    Xenophagy Antibody Sampler Kit: Image 18 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse thymus using Galectin-3/LGALS3 (E7B6R) Rabbit mAb.
    Xenophagy Antibody Sampler Kit: Image 19 Expand Image
    Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human LRSAM1 protein (hLRSAM1-Myc/DDK; +), using LRSAM1 (D1O5S) Rabbit mAb.
    Xenophagy Antibody Sampler Kit: Image 20 Expand Image
    Western blot analysis of extracts from various cell lines using TBK1/NAK (E8I3G) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Xenophagy Antibody Sampler Kit: Image 21 Expand Image
    Western blot analysis of extracts from HeLa, C2C12, and KNRK cells, untreated (-) or treated with Chloroquine #14774 (50 μM, 18 hr; +), using LC3B (E7X4S) XP® Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Xenophagy Antibody Sampler Kit: Image 22 Expand Image
    Western blot analysis of extracts from 293T cells, either mock transfected (-) or transfected with a Myc/DDK-tagged cDNA expression construct encoding the full-length isoform 1 of human TAX1BP1 (hTAX1BP1-Myc/DDK, +), using TAX1BP1 (D1D5) Rabbit mAb.
    Xenophagy Antibody Sampler Kit: Image 23 Expand Image
    Western blot analysis of extracts from THP-1 cells differentiated with TPA #4174 (80 nM, overnight) followed by treatment with LPS (1 μg/ml, 1 hour), with or without phosphatase treatment using Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb (upper), or total TBK1/NAK (D1B4) Rabbit mAb #3504 (lower).
    Xenophagy Antibody Sampler Kit: Image 24 Expand Image
    Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with either a myc-tagged human Atg16L1β construct (hAtg16L1β-Myc; +) or a mouse Atg16L1α construct (mAtg16L1α; +), using Atg16L1 (D6D5) Rabbit mAb. The myc-tagged human Atg16L1β construct was kindly provided by Dr. Qing Zhong, University of California Berkeley.
    Xenophagy Antibody Sampler Kit: Image 25 Expand Image
    Immunohistochemical analysis of paraffin-embedded wild-type mouse brain (left) or brain from an amyloid mouse model of Alzheimer's Disease (right) using Galectin-3/LGALS3 (E7B6R) Rabbit mAb.
    Xenophagy Antibody Sampler Kit: Image 26 Expand Image
    Immunoprecipitation of TBK1/NAK from Raji cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is TBK1/NAK (E8I3G) Rabbit mAb. Western blot analysis was performed using TBK1/NAK (E8I3G) Rabbit mAb.
    Xenophagy Antibody Sampler Kit: Image 27 Expand Image
    Western blot analysis of U20S extracts from WT (left) or TBK1 KO (right) using TBK1/NAK (E8I3G) Rabbit mAb. Membranes stained with Ponceau S for total protein normalization (lower). These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibodies, as a companion to validation data generated by CST scientists.
    Xenophagy Antibody Sampler Kit: Image 28 Expand Image
    Western blot analysis of extracts from control HeLa cells (lane 1) or LC3B knockout HeLa cells (lane 2) using LC3B (E7X4S) XP® Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The absence of signal in the LC3B knockout HeLa cells confirms specificity of the antibody for LC3B.
    Xenophagy Antibody Sampler Kit: Image 29 Expand Image
    Confocal immunofluorescent analysis of THP-1 cells differentiated with TPA #4174 (80nM, overnight) (left), followed by treatment with LPS (1μg/ml, 1 hour) (center) or LPS with λ phosphatase treatment (right) using Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 Phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Xenophagy Antibody Sampler Kit: Image 30 Expand Image
    Confocal immunofluorescent analysis of EBSS-starved PANC-1 cells using Atg16L1 (D6D5) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Xenophagy Antibody Sampler Kit: Image 31 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse spleen using Galectin-3/LGALS3 (E7B6R) Rabbit mAb.
    Xenophagy Antibody Sampler Kit: Image 32 Expand Image
    Confocal immunofluorescent analysis of HCT 116 cells, wild-type (left, positive) or TBK1/NAK knockout (right, negative), using TBK1/NAK (E8I3G) Rabbit mAb (green). Actin filaments were labeled with Alexa Fluor® 555 Phalloidin #8953 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
    Xenophagy Antibody Sampler Kit: Image 33 Expand Image
    Western blot analysis from KARPAS 299 and VCap cells using LC3B (E7X4S) XP® Rabbit mAb #43566 (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The low signal of LC3B in VCap cells is predicted from RNAseq data and confirms the specificity of the antibody for LC3B.
    Xenophagy Antibody Sampler Kit: Image 34 Expand Image
    Flow cytometric analysis of THP-1 cells differentiated with TPA (80nM, 4 days) #9905, untreated (blue) or treated with LPS (1 ng/mL, 1 hr; green) #14011 using Phospho-TBK1/NAK (Ser172) (D52C2) 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.
    Xenophagy Antibody Sampler Kit: Image 35 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse testis using Galectin-3/LGALS3 (E7B6R) Rabbit mAb.
    Xenophagy Antibody Sampler Kit: Image 36 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse forestomach using Galectin-3/LGALS3 (E7B6R) Rabbit mAb.
    Xenophagy Antibody Sampler Kit: Image 37 Expand Image
    Flow cytometric analysis of TALL-1 cells (blue, negative) and Raji cells (green, positive) using TBK1/NAK (E8I3G) 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.
    Xenophagy Antibody Sampler Kit: Image 38 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse kidney using Galectin-3/LGALS3 (E7B6R) Rabbit mAb.
    Xenophagy Antibody Sampler Kit: Image 39 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse liver using Galectin-3/LGALS3 (E7B6R) Rabbit mAb.
    Xenophagy Antibody Sampler Kit: Image 40 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse lung using Galectin-3/LGALS3 (E7B6R) Rabbit mAb.
    Xenophagy Antibody Sampler Kit: Image 41 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse ovary using Galectin-3/LGALS3 (E7B6R) Rabbit mAb.
    Xenophagy Antibody Sampler Kit: Image 42 Expand Image
    Immunohistochemical analysis of paraffin-embedded A20 syngeneic tumor using Galectin-3/LGALS3 (E7B6R) Rabbit mAb.
    Xenophagy Antibody Sampler Kit: Image 43 Expand Image
    Immunohistochemical analysis of paraffin-embedded GL261 syngeneic tumor using Galectin-3/LGALS3 (E7B6R) Rabbit mAb.
    Xenophagy Antibody Sampler Kit: Image 44 Expand Image
    Immunohistochemical analysis of paraffin-embedded Renca syngeneic tumor using Galectin-3/LGALS3 (E7B6R) Rabbit mAb.
    Xenophagy Antibody Sampler Kit: Image 45 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse small intestine using Galectin-3/LGALS3 (E7B6R) Rabbit mAb (left) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right).
    Xenophagy Antibody Sampler Kit: Image 46 Expand Image
    Immunohistochemical analysis of paraffin-embedded RAW 264.7 cell pellet (left, positive) or Neuro-2a cell pellet (right, negative) using Galectin-3/LGALS3 (E7B6R) Rabbit mAb.
    Xenophagy Antibody Sampler Kit: Image 47 Expand Image
    Confocal immunofluorescent analysis of HCT 116 cells either untreated (left) or treated with Chloroquine #14774 (50 µM, overnight) (center) or LC3B HCT 116 knockout cells treated with Chloroquine #14774 (50 µM, overnight) (right) using LC3B (E7X4S) XP® Rabbit mAb (green). Actin filaments were labeled with β-Actin (8H10D10) Mouse mAb (red) and nuclei were labeled with DAPI #4083 (blue).
    Xenophagy Antibody Sampler Kit: Image 48 Expand Image
    Confocal immunofluorescent analysis of MIA PaCa-2 cells untreated (left), nutrient-starved with EBSS (2 hr; middle), or treated with Chloroquine #14774 (50 µM, 24 hr; right) using LC3B (E7X4S) XP® Rabbit mAb (green) and β-Actin (8H10D10) Mouse mAb #3700 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
    Xenophagy Antibody Sampler Kit: Image 49 Expand Image
    Confocal immunofluorescent analysis of C2C12 cells untreated (left), nutrient-starved with EBSS (2 hr; middle), or treated with Chloroquine #14774 (50 µM, 24 hr; right) using LC3B (E7X4S) XP® Rabbit mAb (green) and β-Actin (8H10D10) Mouse mAb #3700 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).