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.
Mmp 9
Manufactured by Cell Signaling Technology
Sourced in United States, China, United Kingdom
MMP-9 (Matrix Metalloproteinase-9) is a lab equipment product offered by Cell Signaling Technology. MMP-9 is an enzyme involved in the degradation of the extracellular matrix. It plays a role in various physiological and pathological processes.
Automatically generated - may contain errors
Lab products found in correlation
Mmp 2, by Cell Signaling Technology (197 mentions)
β actin, by Cell Signaling Technology (97 mentions)
E cadherin, by Cell Signaling Technology (96 mentions)
Vimentin, by Cell Signaling Technology (85 mentions)
Gapdh, by Cell Signaling Technology (78 mentions)
Bcl 2, by Cell Signaling Technology (65 mentions)
N cadherin, by Cell Signaling Technology (64 mentions)
P akt, by Cell Signaling Technology (50 mentions)
Pvdf membrane, by Merck Group (46 mentions)
Cleaved caspase 3, by Cell Signaling Technology (45 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (36 mentions)
Snail, by Cell Signaling Technology (36 mentions)
Cyclin d1, by Cell Signaling Technology (36 mentions)
β catenin, by Cell Signaling Technology (31 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (27 mentions)
P erk, by Cell Signaling Technology (24 mentions)
Erk1 2, by Cell Signaling Technology (24 mentions)
P erk1 2, by Cell Signaling Technology (21 mentions)
P jnk, by Cell Signaling Technology (20 mentions)
Timp 1, by Cell Signaling Technology (18 mentions)
482 protocols using mmp 9
1
Molecular Profiling of Nodal Signaling
2
Immunohistochemical Analysis of Brain Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Resected brain tissues (n = 3 per experiment group) were fixed by immersion in 4% formaldehyde (Beyotime, Zhejiang, China) for 60 min at 37°C before paraffin embedding. These samples were then sectioned at 5 µm thickness via microtome (Model HM310, Microm Inc., USA), dewaxed with xylene, and cleared with a series of changing ethanol concentrations. Blocking was done by incubation in 5% bovine serum albumin (Thermo Fisher Scientific, Carlsbad, CA, USA) and 0.3% Triton- X-100 (Thermo Fisher Scientific) for 1 hour at room temperature (RT). Antibodies used for IHC were glial fibrillary acidic protein (GFAP) (1:100, Abcam, Cambridge, USA), ET-1 (1:50, Abcam), VEGF-A (1:100, Cell Signaling Technology, Cambridge, MA, USA), or MMP-9 (1:100, Cell Signaling Technology). All incubation with primary antibodies were done in 1 BSA overnight at 4°C. Samples were viewed at 100× magnification by light microscopy (BX61, Olympus Optical GmbH, Hamburg, Germany) and imaged (Color View II; Soft Imaging System, Olympus Optical GmbH, Hamburg, Germany).
3
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
hVSMCs and tissues were lysed with radioimmunoprecipitation assay (RIPA) buffer (Beyotime) and centrifuged at 4°C to extract total protein. Protein concentration was detected using a bicinchoninic acid (BCA) kit (Bio-Rad). The protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated with Bcl-2 (cat. no. 4223; 1: 1000; Cell Signaling Technology, Inc.), Bax (cat. no. 5023; 1: 1000; Cell Signaling Technology, Inc.), MMP9 (cat. no. 13,667; 1: 1000; Cell Signaling Technology, Inc.), SIRT1 (cat. no. 2496; 1: 1000; Cell Signaling Technology, Inc.), or GAPDH (cat. no. 5174; 1: 1000; Cell Signaling Technology, Inc.) antibody at 4°C overnight after blocking with 5% nonfat milk phosphate buffer saline (PBS)-0.1%Tween 20 solution for 1 h. The membrane was then incubated with secondary antibodies (cat. no. ab96899; 1:2,000; Abcam, Cambridge, USA) for 1 h at room temperature. The protein bands were visualized using enhanced chemiluminescence luminescent substrate (Amersham ImageQuant800UV; Cytiva, MA, USA) and quantified using ImageJ software.
4
Immunohistochemical Analysis of Vaginal Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 10 sections from each group were incubated with antibodies to Von Willebrand Factor (1:100) (Abcam, Cambridge, MA, USA), VEGF (1:100) (Abcam, Cambridge, MA, USA) and MMP-9 (1:200) (Cell Signaling, Beverly, MA, USA) overnight at 4 degrees Celsius. Slides were rinsed in PBS, followed by treatment using the second antibody using the Max Vision HRP-Polymer anti-Rabbit IHC Kit, (Maxim Co. China). The morphological changes of vaginal tissue were observed under the microscope and photographed (German Leica binocular microscope DM500, × 400). The vaginal tissue in each group is 10, that is, (n = 10). When P < .05, the difference was statistically significant.
5
Protein Expression Analysis of Anterior Vaginal Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anterior vagina tissue protein samples were prepared by homogenizing in RIPA lysis buffer. Equal protein (20 μg/lane) was electrophoresed on 10% SDS-PAGE and then transferred to polyvinylidene fluoride membrane (Millipore Corp, Bedford, MA, USA). Western blot was performed with antibodies against MMP-9 (1:400) (Cell Signaling, Beverly, MA, USA), VEGF (1:800) (Abcam, Cambridge, MA, USA), SMA (1:1000) (Santa Cruz Biotechnology, CA, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1000) (Santa Cruz Biotechnology, CA, USA).The tissue from each animal kept separate, and 1 sample from each treatment group was run on each gel, so 4 lanes were run per gel. More than 3 times were this repeated on different gels.
6
Protein Expression Analysis in UVB-irradiated Skin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein was extracted from the skin tissue of UVB-irradiated hairless mice. An equal concentration of protein samples (20 µg) were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to polyvinylidene fluoride (PVDF) membranes. After transfer, the PVDF membranes were blocked for 1 h at room temperature with a 5% blocking solution (ATTO, Tokyo, Japan). After blocking, they were incubated at 4 °C overnight with indicated primary antibodies (MMP-1 (#54376), MMP-9 (#13667), pERK (#9101), ERK (#9102), pMEK (#9154), MEK (#9126), pp38 (#9215), p38 (#9212), pJNK (#9251), JNK (#9252), and β-actin (#4970) were purchased from Cell signaling, diluted (1:1000), washed three times for 10 min each in Tris-buffered saline (TBS), and then incubated for 2 h at room temperature with the anti-rabbit secondary antibody (#7074, Cell signaling). The proteins were developed by an enhanced chemiluminescence (ECL) solution and detected with a LAS-4000 mini luminescent image analyzer (GE Heathcare, UK).
7
Western Blot Analysis of MMP2 and MMP9
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from cell pellet using
RIPA lysis buffer (Thermo Fisher Scientific, USA)
supplemented with protease inhibitors and phosphatase
inhibitors (Roche) according to the manufacturer’s
protocol. Concentration of total protein was determined
by using BCA™ Protein Assay Kit (Thermo Fisher
Scientific, USA). Then, 40 μg of protein per lane was
separated by 8% sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) and transferred to
polyvinylidene fluoride (PVDF) membranes. 5% skim
milk was used to block PVDF membranes for 1 hour at
room temperature. Next, membranes were incubated
with primary antibodies overnight at 4˚C, followed by
incubation of secondary antibodies for 1 hour at room
temperature. Next, protein bands were visualized using the
enhanced chemiluminescence system (Bio-Rad Clarity
Western ECL, USA). Primary antibodies, including
MMP2 (1:1000), MMP9 (1:1000) and β-actin (1:1000)
and HRP-conjugated secondary antibodies (1:5000) were
obtained from Cell Signaling Technology (CST Inc.,
USA). β-actin was regarded as the internal control.
RIPA lysis buffer (Thermo Fisher Scientific, USA)
supplemented with protease inhibitors and phosphatase
inhibitors (Roche) according to the manufacturer’s
protocol. Concentration of total protein was determined
by using BCA™ Protein Assay Kit (Thermo Fisher
Scientific, USA). Then, 40 μg of protein per lane was
separated by 8% sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) and transferred to
polyvinylidene fluoride (PVDF) membranes. 5% skim
milk was used to block PVDF membranes for 1 hour at
room temperature. Next, membranes were incubated
with primary antibodies overnight at 4˚C, followed by
incubation of secondary antibodies for 1 hour at room
temperature. Next, protein bands were visualized using the
enhanced chemiluminescence system (Bio-Rad Clarity
Western ECL, USA). Primary antibodies, including
MMP2 (1:1000), MMP9 (1:1000) and β-actin (1:1000)
and HRP-conjugated secondary antibodies (1:5000) were
obtained from Cell Signaling Technology (CST Inc.,
USA). β-actin was regarded as the internal control.
8
Acetyshikonin Modulates Inflammatory Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Acetyshikonin (purity >98%) was purchased from Biopurify phytochemicals Ltd (Chengdu, China). CellTiter-Glo® Lumine scent Cell Viability Assay and Proteome Profiler Human Cytokine Array were purchased from Promega Corporation (Madison, WI, USA) and R&D systems (Minneapolis, MN, USA), respectively. DMEM and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA, USA). Phorbol 12-myristate 13-acetate (PMA) and β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Polyclonal antibodies against total NF-κB (p65), phospho-specific p65 (Ser536), total IκBα, phospho-specific IκBα (Ser32), MMP-2 and MMP-9 were obtained from Cell Signaling Technology (Beverly, MA, USA). All other chemicals used in our experiments are molecular biology grade.
9
Synthesis and Evaluation of Small Molecule Inhibitors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ISJ and its intermediate, compound 7 , were synthesized in our laboratory. GF 109203X (GFX) and Go 6976 (GO) were purchased from Tocris Bioscience (Ellisville, MO). 5-Fluorouracil (5-Fu) was obtained from Sigma (St. Louis, Mo). Primary antibodies were purchased from Cell Signaling Technology (Beverly, MA), including those specific for β-actin (#4970), MMP-9 (#2270), E-Cadherin (#3195), FGFR-1 (#9740), PKCε (#2683), c-Raf (#9422), MEK1/2 (#9122), ERK1/2 (#9102), Phospho-c-Raf (#9427), Phospho-MEK1/2 (#9154), and Phospho-ERK1/2 (#4370). The PKC Isoform Antibody Sampler Kit (#9960) and the Apoptosis Antibody Sampler Kit (#9915) were also purchased from Cell Signaling Technology. Antibodies to p53 (AP062) and p21 (AP021) were purchased from Beyotime (Shanghai, China).
10
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).
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!