Cells were fully mixed with radioimmunoprecipitation assay (RIPA) lysis buffer (KeyGEN, Jiangsu, China) to extract proteins. After centrifuging at 12,000 rpm speed, liquid supernatant was collected and then separated by equal amounts of SDS-PAGE, followed by electro-transfer onto polyvinylidene difluoride membranes (Thermo Fisher Scientific, MA, USA). Subsequently, membranes were blocked with 5% nonfat milk for 2 h. Primary antibody was diluted to 1:1,000 and then incubated with membrane at room temperature. Secondary antibody was used to combine primary antibody for 2 h. Finally, membranes were developed using an enhanced chemiluminescence detection system (GE Healthcare, Chicago, IL, USA). Immunoblot analysis used the following primary antibodies purchased from Cell Signaling Technology (Danvers, MA, USA): anti-EphB2 (#83029), N-cadherin (#14215), Vimentin (#5741), MMP2 (#40994), MMP2 (#13667), Snail (#3879), Slug (#9585), Sox2 (#3579), Nestin (#33475), Nanog (#8822), Oct-4 (#2750), and GAPDH (#5174) .
Mmp 2
Manufactured by Cell Signaling Technology
Sourced in United States, China, United Kingdom
MMP-2 is a lab equipment product manufactured by Cell Signaling Technology. It functions as a matrix metalloproteinase enzyme that plays a role in the breakdown of extracellular matrix components.
Automatically generated - may contain errors
Lab products found in correlation
Mmp 9, by Cell Signaling Technology (197 mentions)
β actin, by Cell Signaling Technology (69 mentions)
E cadherin, by Cell Signaling Technology (65 mentions)
Vimentin, by Cell Signaling Technology (62 mentions)
N cadherin, by Cell Signaling Technology (58 mentions)
Gapdh, by Cell Signaling Technology (57 mentions)
Bcl 2, by Cell Signaling Technology (38 mentions)
Snail, by Cell Signaling Technology (34 mentions)
Pvdf membrane, by Merck Group (34 mentions)
P akt, by Cell Signaling Technology (31 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (27 mentions)
Cleaved caspase 3, by Cell Signaling Technology (26 mentions)
Cyclin d1, by Cell Signaling Technology (26 mentions)
β catenin, by Cell Signaling Technology (26 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (21 mentions)
P erk, by Cell Signaling Technology (16 mentions)
Gapdh, by Santa Cruz Biotechnology (15 mentions)
Caspase 3, by Cell Signaling Technology (15 mentions)
Timp 2, by Cell Signaling Technology (14 mentions)
Phospho akt, by Cell Signaling Technology (14 mentions)
325 protocols using mmp 2
1
Protein Extraction and Western Blot Analysis
2
Immunoblotting of Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cultured cells were lysed and the protein extraction was performed as described previously [13 (link)]. Lysates were immunoblotted with antibodies specific for ETS1 (Cell Signaling Technology, Cat# 6258BC, Lot# 1), α-tubulin (Cell Signaling Technology Cat# 2125, RRID:AB_2619646, Lot# 11), β-actin (Cell Signaling Technology Cat# 4967, RRID:AB_330288, Lot# 6), MMP2 (Cell Signaling Technology Cat# 4022, RRID:AB_2266622, Lot# 2) and p-AKT (Cell Signaling Technology Cat# 9271, RRID:AB_329825, Lot# 12). The chemiluminescent bands were detected using horseradish peroxidase-labelled secondary antibodies (1:5000) (Cell Signaling Technology Cat# 7074, RRID: AB_2099233, Lot# 28) and ECL luminescence substrate (Bio-Rad, Cat# 170-5061). The expression of proteins was normalized with α-tubulin or β-actin.
3
Molecular Profiling of Nodal Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RT4 and KMBC2 cells transfected with siNC and siNodal were washed three times with cold PBS and then lysed using RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing phenylmethane sulfonyl fluoride. Proteins (40 µg per sample) were separated on 10% sodium dodecyl sulfate–polyacrylamide gels and then transferred electrophoretically onto a PVDF membrane. The membranes were blocked with 5% bovine serum albumin diluted in TBST and then incubated with appropriate antibodies against Nodal, CDC6, E-cadherin, MMP-2, MMP-9, ALK4, ALK7, Smad2, Smad4, and GAPDH (1:1,000; Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. The membranes were then washed three times with TBST, immediately followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:1,000; Cell Signaling Technology) for 1 hour at room temperature. GAPDH was used as the internal control. Protein bands were detected using an enhanced chemiluminescence kit (ECL kit, New York, NY, USA) and visualized by autoradiography on an X-ray film.
4
Investigating EMT Signaling Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies against E-cadherin and N-cadherin were obtained from BD Bioscience (NJ, USA); ZEB1/TCF8, MMP-2, MMP-9, phospho-Akt (Ser 473), phospho-Akt (Thr 308), Akt, phospho-GSK3β (Ser9), GSK3β, phospho-PI3K, PI3K p85α, β-catenin, β-actin and lamin A/C antibodies were from Cell Signaling Technology (MA, USA); fibronectin and α-smooth muscle actin (SMA) antibodies were from Sigma-Aldrich Biotechnology (LP, USA); vimentin, green fluorescent protein (GFP), Twist, and Slug antibodies were from Santa Cruz Biotechnology (CA, USA); and TCTP antibodies were obtained from LabFrontier (Seoul, Korea). 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), rapamycin, and PP242 were obtained from Sigma-Aldrich Biotechnology (LP, USA).
5
Inhibition of FAK and MMPs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DIM was purchased from Sigma-Aldrich (St Louis, MO) and dissolved in DMSO to make a 100 mmol/L stock solution and stored at −20°C in multiple aliquots. PF-562271(N-methyl-N-{3-[({2-[(2-oxo-2, 3-dihydro-1H-indol-5-yl)amino]-5-(trifluoromethyl)pyrimidin-4-yl}amino) methyl]pyridin-2-yl}methane sulfonamide), was obtained from Selleckchem (Houston, TX) and dissolved in DMSO to make a 10 μmol/L stock solution and stored at −20°C. Vitronectin was obtained from Life Technologies, Inc. (Grand Island, NY) and dissolved in phosphate buffered saline with calcium and magnesium (0.90 mM CaCl2, 0.49 mM MgCl2, 2.67 mM KCl, 1.47 mM KH2PO4, 137.93 mM NaCl, 8.06 mM Na2HPO4) to make a 0.5 ug/ml stock solution. Antibodies against Phospo-FAK (Tyr397), FAK, MMP-2, MMP-9, pTEN, β-actin were purchased from Cell Signaling.
6
Protein Characterization and Wnt Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from the sample, and the protein concentrations were determined using the Bradford Protein Assay (Bio‐Rad, Hercules, CA). A total of 50 μg of protein from each sample was separated by 8%–12% SDS‐PAGE and transferred onto a polyvinylidene fluoride membrane (Bio‐Rad). The proteins were detected by incubation of the membranes with antibodies against CLDN3, CITED4 (Abcam), β‐catenin, MMP‐2 and GAPDH (Cell Signaling). WNT‐ and control‐conditioned medium (Wnt‐CM [ATCC number: CRL‐2647] and L‐CM) were collected according to the directions from ATCC and incubated with the cells for 24 h during the experiments. Wnt signaling activity was then determined by performing various assays, such as Western blotting.
7
Western Blot Analysis of EMT Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Harvested cells were lysed with heat denaturation in RIPA cell lysis buffer. Protein lysates (20 μg) were loaded and separated on SDS-PAGE, and the proteins were transferred to polyvinylidene difluoride (PVDF) membranes. The blots were incubated with primary antibodies against LMO1(ab137599, Abcam),NGFR(55014-1-AP, proteintech),MMP2(#40994,Cell Signaling Technology), NF-κB Pathway Sampler Kit antibodies (#9936, Cell Signaling Technology), Epithelial-Mesenchymal Transition (EMT) Antibody Sampler Kit antibodies (#9782, Cell Signaling Technology),GAPDH(AP0066-200,Bioworld); Specific proteins were visualized with enhanced chemiluminescence (ECL, Millipore, Bredford, USA). The intensity of the protein bands was measured (ImageJ software) and normalized to GAPDH.
8
Western Blot Analysis of Signaling Molecules
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed in PBS and lysed in RIPA buffer (Elpis Biotech, Daejeon, Korea) supplemented with a protease inhibitor cocktail (Sigma-Aldrich). Protein phosphorylation states were preserved through the addition of phosphatase inhibitors (Cocktail II, Sigma-Aldrich) to NP-40 buffer. Protein concentrations were determined using a BCA assay kit (Pierce, Rockford, IL, USA). The proteins (10 μg/sample) were resolved through SDS-PAGE and subsequently transferred to a nitrocellulose membrane (Millipore Corp., Billerica, MA, USA). The membranes were blocked with 5% skim milk prior to Western blot analysis. Chemiluminescence was detected using an ECL kit (Advansta Corp., Menlo Park, CA, USA) and the Amersham Imager 600 (GE Healthcare Life Sciences, Little Chalfont, UK). Primary antibodies against the following proteins were used: phospho-STAT3 (Tyr705), STAT3, β-actin, MMP2, MMP9, PARP, p105/p50, p100/p52, p65, and Rel-B (Cell Signaling Technology, Beverly, MA, USA); phospho-JAK2 (Tyr221) and JAK2 (Bioss, Woburn, MA, USA); CD55 (Biorbyt, Woburn, MA, USA); β-tubulin (BD Biosciences, San Diego, CA, USA); and E-cadherin, N-cadherin, Snail, and CD97 (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
9
Comprehensive Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot reagents were obtained from GE Healthcare (Pittsburgh, PA, USA). Fluorescent probes for caspase activity, caspase inhibitors, and annexin V assay kit were from BD Biosciences (San José, CA, USA). Culture media were from Lonza (Basel, Switzerland). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and p38MAPK inhibitor (SB202190) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Primary monoclonal rabbit antibodies against caspase 8 (dilution, 1:1,000; #4927), cleaved-caspase 9 (dilution, 1:1,000; #7237), MMP-2 (dilution, 1:1,000; #4022), MMP-9 (dilution, 1:1,000; #3852), p-p38 (dilution, 1:1,000; #9211), p38 (dilution, 1:1,000; #9212), and TIMP-1 (dilution, 1:1,000; #8946) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Caspase 3 (dilution, 1:1,000; sc-7148), Bid (dilution, 1:1,000; sc-11423), Bcl-2 (dilution, 1:1,000; sc-783), Bcl-xL (dilution, 1:1,000; sc-634), and Bax (dilution, 1:1,000; sc-526) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), and β-actin (dilution, 1:5,000; #A5441) was obtained from Sigma-Aldrich (St. Louis, MO, USA).
Anti-Ki67 (dilution 1:200) and Click-iT Tunel colorimetric IHC detection kit were from Thermo Fisher (Waltham, MA USA). DAB kit was provided from Vector laboratories (Burlingame, CA, USA).
Anti-Ki67 (dilution 1:200) and Click-iT Tunel colorimetric IHC detection kit were from Thermo Fisher (Waltham, MA USA). DAB kit was provided from Vector laboratories (Burlingame, CA, USA).
10
Protein Isolation and Western Blotting Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein isolation and Western blotting analysis were performed as previously described.37 (link) Primary antibodies for ATF4, eukaryotic translation initiation factor 2A (EIF2A), phosphorylated EIF2A(Ser51) [p-EIF2A(Ser51)], and MMP-2 were from Cell Signaling Technology (New England Biolabs, Hitchin, UK), HSPA5 (alias GRP78) from Transduction Lab (BD Biosciences, Wokingham, UK), and TIMP1 from Abcam. The results are shown as follows: Relative Ratio (%) = (Density of Treatment Group/Density of Control) × 100%.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!