Epithelial-Mesenchymal Transition (EMT)

1 Store at -20 C. Epithelial-Mesenchymal Transition (EMT). Antibody Sampler Kit 3 1 Kit n Orders n 877-616-CELL (2355). (9 x 20 l) #9782. Support n 877-678-TECH (8324). Web n rev. 12/04/17. For Research Use Only. Not For Use In Diagnostic Procedures. Storage: Supplied in 10 mM sodium HEPES (pH ), 150. Products Included Product # Quantity Mol. Wt. Isotype mM NaCl, 100 g/ml BSA, 50% glycerol and less than Vimentin (D21H3) XP Rabbit mAb 5741 20 l 57 kDa Rabbit IgG sodium azide. Store at 20 C. Do not aliquot the antibodies. N-Cadherin (D4R1H) XP Rabbit mAb 13116 20 l 140 kDa Rabbit IgG Recommended Antibody Dilutions: Western blotting 1:1000. Claudin-1 (D5H1D) XP Rabbit mAb . 13255 20 l 20 kDa Rabbit IgG. Please visit for validation data -Catenin (D10A8) XP Rabbit mAb 8480 20 l 92 kDa Rabbit IgG and a complete listing of recommended companion ZO-1 (D7D12) Rabbit mAb 8193 20 l 220 kDa Rabbit IgG products. Snail (C15D3) Rabbit mAb 3879 20 l 29 kDa Rabbit IgG. Slug (C19G7) Rabbit mAb 9585 20 l 30 kDa Rabbit IgG.

2 TCF8/ZEB1 (D80D3) Rabbit mAb 3396 20 l 200 kDa Rabbit IgG. E-Cadherin (24E10) Rabbit mAb 3195 20 l 135 kDa Rabbit IgG. Anti-rabbit IgG, HRP-linked Antibody 7074 100 l Goat See for individual component applications, species cross-reactivity, dilutions, and additional application protocols. Description: The Epithelial-Mesenchymal Transition (EMT) eton (7,8). Zona occludens proteins ZO-1, 2, and 3 (also recognizes endogenous levels of total N-cadherin protein. Antibody Sampler Kit provides an economical means known as TJP 1, 2, and 3) are peripheral membrane adaptor Claudin-1 (D5H1D) XP Rabbit mAb recognizes endog- of evaluating EMT. The kit includes enough antibody to proteins that link junctional transmembrane proteins such enous levels of total claudin-1 protein. ZO-1 (D7D12) Rabbit perform two western blot experiments with each per primary as occludin and claudin to the actin cytoskeleton (9). ZO-1 mAb detects endogenous levels of total ZO-1 protein. antibody. and -2 are required for tight junction formation and function Vimentin (D21H3) XP Rabbit mAb detects endogenous (10,11); mutations in ZO-1 and Claudin induce EMT (12).

3 Levels of total vimentin protein. Snail (C15D3) Rabbit mAb Background: Epithelial-Mesenchymal Transition (EMT) is Vimentin is an intermediate filament of mesenchymal origin detects endogenous levels of total Snail protein. Slug an essential process during development whereby epithelial and is present at early developmental stages. Vimentin's (C19G7) Rabbit mAb detects endogenous levels of total cells aquire mesenchymal , fibroblast-like properties and dynamic structural changes and spatial re-organization in Slug protein. TCF8/ZEB1 (D80D3) Rabbit mAb detects display reduced intracellular adhesion and increased motility. response to extracellular stimuli helps to coordinate various endogenous levels of total TCF8/ZEB1 protein. -Catenin This is a critical feature of normal embryonic development, signaling pathways (13). -catenin is a key downstream (D10A8) XP Rabbit mAb detects endogenous levels of which is also utilized by malignant epithelial tumors to XP and Cell Signaling Technology are trademarks of Cell Signaling Technology, Inc.

4 Effector in the Wnt signaling pathway (14). It is implicated in total -catenin protein. spread beyond their origin (1-3). This tightly regulated two major biological processes in vertebrates: early embry- process is associated with a number of cellular and molecular Source/Purification: Monoclonal antibodies are onic development (15) and tumorigenesis (16). -catenin events. EMT depends on a reduction in expression of cell produced by immunizing animals with a synthetic peptide also activates Slug. Slug (SNAI2) is a widely expressed adhesion molecules. corresponding to residues surrounding Arg526 of human transcriptional repressor and member of the Snail family of Cadherins mediate calcium-dependent cell-cell adhesion N-cadherin protein, a synthetic peptide corresponding to zinc finger transcription factors (17). Similar to the related and play critical roles in normal tissue development (4). residues near the carboxy terminus of human claudin-1. Snail protein, Slug binds to the E-cadherin promoter region E-cadherin is considered an active suppressor of invasion protein, a synthetic peptide corresponding to residues near to repress transcription during development (18).

5 The binding and growth of many epithelial cancers (4-6). Recent studies the carboxy terminus of human ZO-1 protein, a recombinant of Slug to integrin promoter sequences represses integrin indicate that cancer cells have up-regulated N-cadherin human Snail protein, a recombinant human Slug protein, expression and results in reduced cell adhesion (19). Down in addition to loss of E-cadherin. This change in cadherin a synthetic peptide corresponding to residues surrounding regulation of E-cadherin expression occurs during the EMT. expression is called the cadherin switch and downregula- Arg45 of human vimentin protein, a synthetic peptide during embryonic development (20). ZEB family proteins are tion of E-cadherin is one of the hallmarks of EMT (1). Tight corresponding to residues surrounding Pro780 of human zinc finger and homeobox domain containing transcription junctions, or zonula occludens, form a continuous barrier to E-cadherin, a synthetic peptide corresponding to residues 2014 Cell Signaling Technology, Inc.

6 Factors. One of the targets suppressed by ZEB proteins is fluids across the epithelium and endothelium. They function surrounding Asp868 of human TCF8/ZEB1 protein, or a E-cadherin (1). in regulation of paracellular permeability and in the mainte- synthetic peptide corresponding to residues surrounding nance of cell polarity, blocking the movement of transmem- Specificity/Sensitivity: E-Cadherin (24E10) Rabbit mAb Pro714 of human -catenin protein. brane proteins between the apical and the basolateral cell detects endogenous levels of total E-cadherin protein. The surfaces. Tight junctions are composed of claudin and antibody does not cross-react with related family members, occludin proteins, which join the junctions to the cytoskel- such as N-cadherin. N-Cadherin (D4R1H) XP Rabbit mAb Patent No. 5,675,063. Tween 20 is a registered trademark of ICI Americas, Inc. Applications Key: W Western IP Immunoprecipitation IHC Immunohistochemistry ChIP Chromatin Immunoprecipitation IF Immunofluorescence F Flow cytometry E-P ELISA-Peptide Species Cross-Reactivity Key: H human M mouse R rat Hm hamster Mk monkey Mi mink C chicken Dm D.

7 Melanogaster X Xenopus Z zebrafish B bovine Dg dog Pg pig Sc S. cerevisiae Ce C. elegans Hr horse All all species expected Species enclosed in parentheses are predicted to react based on 100% homology. #9782. Western Immunoblotting Protocol For western blots, incubate membrane with diluted primary antibody in either 5% w/v BSA or nonfat dry milk, 1X TBS, Tween 20 at 4 C with gentle shaking, overnight. NOTE: Please refer to primary antibody datasheet or product webpage for recommended primary antibody dilution buffer and recommended antibody dilution. A. Solutions and Reagents C. Membrane Blocking and Antibody Incubations NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water. NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized mem- 1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml branes, adjust volumes accordingly. 20X PBS to 950 ml dH2O, mix. I. Membrane Blocking 2. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 1.

8 (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room 900 ml dH2O, mix. temperature. 3. 1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) 2. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature. Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3. Wash three times for 5 min each with 15 ml of TBST. 3X SDS loading buffer. Dilute to 1X with dH2O. 4. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add II. Primary Antibody Incubation 1. Incubate membrane and primary antibody (at the appropriate dilution and diluent as 100 ml 10X running buffer to 900 ml dH2O, mix. recommended in the product datasheet) in 10 ml primary antibody dilution buffer with 5. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X transfer buffer: add 100. gentle agitation overnight at 4 C. ml 10X transfer buffer to 200 ml methanol + 700 ml dH2O, mix.

9 2. Wash three times for 5 min each with 15 ml of TBST. 6. 10X Tris Buffered Saline with Tween 20 (TBST): (#9997) To prepare 1 L 1X. 3. Incubate membrane with the species appropriate HRP-conjugated secondary anti- TBST: add 100 ml 10X TBST to 900 ml dH2O, mix. body (#7074 or #7076 at 1:2000) and anti-biotin, HRP-linked Antibody (#7075 at 7. Nonfat Dry Milk: (#9999). 1:1000 1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with 8. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add g nonfat gentle agitation for 1 hr at room temperature. dry milk to 150 ml 1X TBST and mix well. 4. Wash three times for 5 min each with 15 ml of TBST. 9. Wash Buffer: (#9997) 1X TBST 5. Proceed with detection (Section D). 10. Bovine Serum Albumin (BSA): (#9998). 11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA or 5% nonfat dry milk as D. Detection of Proteins indicated on primary antibody datasheet; for 20 ml, add g BSA or nonfat dry milk to 1.

10 Incubate membrane with 10 ml LumiGLO ( ml 20X LumiGLO #7003, ml 20X. 20 ml 1X TBST and mix well. peroxide, and ml purified water) or 10 ml SignalFire #6883 (5 ml Reagent A, 5 ml 12. Biotinylated Protein Ladder Detection Pack: (#7727). Reagent B) with gentle agitation for 1 min at room temperature. 13. Prestained Protein Marker, Broad Range (Premixed Format): (#7720) 2. Drain membrane of excess developing solution (do not let dry), wrap in plastic wrap and 14. Blotting Membrane and Paper: (#12369) This protocol has been optimized for expose to x-ray film. An initial 10 sec exposure should indicate the proper exposure time. nitrocellulose membranes. Pore size m is generally recommended. NOTE: Due to the kinetics of the detection reaction, signal is most intense immediately 15. Secondary Antibody Conjugated to HRP: anti-rabbit (#7074); anti-mouse (#7076) following incubation and declines over the following 2 hr. 16. Detection Reagent: LumiGLO chemiluminescent reagent and peroxide (#7003) or SignalFire ECL Reagent (#6883).