Leelamine (LEE, Figure 1 A) was purchased from Cayman Chemical (Ann Arbor, MI, USA). LEE stock solution (10 mM) was prepared in EtOH, storage at −20 °C and finally diluted in cell culture medium for use. Anti-CXCR7 and anti-CXCR4 antibodies were purchased from abcam (Cambridge, UK). Anti-MnSOD, anti-fibronectin, anti-vimentin, anti-MMP-9, anti-MMP-2, anti-N-cadherin, anti-E-cadherin, anti-twist, anti-snail, anti-occludin, and anti-b-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-STAT3(Tyr705), anti-STAT3, anti-phospho-JAK1(Tyr1022/1023), anti-JAK1, anti-phospho-JAK2(Tyr1007/1008), and anti-JAK2 were purchased from Cell Signaling Technology (Beverly, MA, USA). Alexa Fluor® 488 donkey anti-rabbit IgG (H+L) antibody and Alexa Fluor® 594 donkey anti-mouse IgG (H+L) antibody was obtained from Life Technologies (Grand Island, NY, USA). The GSH/GSSG-Glo™ Assay kit was purchased from Promega (Madison, WI, USA).
Anti mmp2
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-MMP2 is a laboratory reagent that specifically binds and detects the MMP2 (Matrix Metalloproteinase 2) protein. MMP2 is an enzyme involved in the breakdown of extracellular matrix proteins. This antibody can be used to measure or identify the presence of MMP2 in various experimental and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti mmp 9, by Santa Cruz Biotechnology (34 mentions)
Anti β actin, by Santa Cruz Biotechnology (14 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (11 mentions)
Anti e cadherin, by Santa Cruz Biotechnology (10 mentions)
Anti cyclin d1, by Santa Cruz Biotechnology (9 mentions)
Anti n cadherin, by Santa Cruz Biotechnology (7 mentions)
Anti gapdh, by Santa Cruz Biotechnology (7 mentions)
Anti vimentin, by Santa Cruz Biotechnology (6 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Anti bax, by Santa Cruz Biotechnology (5 mentions)
Anti β catenin, by Santa Cruz Biotechnology (5 mentions)
Anti bcl xl, by Santa Cruz Biotechnology (4 mentions)
Anti c myc, by Santa Cruz Biotechnology (4 mentions)
Anti vegf, by Santa Cruz Biotechnology (4 mentions)
Anti snail, by Santa Cruz Biotechnology (3 mentions)
Anti twist, by Santa Cruz Biotechnology (3 mentions)
Anti occludin, by Santa Cruz Biotechnology (3 mentions)
Anti akt, by Santa Cruz Biotechnology (3 mentions)
Gapdh, by Santa Cruz Biotechnology (3 mentions)
Anti bcl 2, by Abcam (3 mentions)
63 protocols using anti mmp2
1
Leelamine Modulates CXCR7/CXCR4 Signaling
2
Protein Extraction and Western Blot Analysis
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Tissue protein was extracted with a protein extracting reagent (BioTeke Corporation, Beijing, China) according to the manufacturer's directions, while cell protein was extracted through RIPA buffer supplemented with 1% protease inhibitors (Roche, Basel, Switzerland). After centrifugation, the protein content was measured by a BCA Protein Assay Kit (Solarbio, Beijing, China). According to the standard procedures of western blot, total proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA), and then blocked with 5% skim milk in TBST. After being incubated with antibodies, the membranes were visualized with the ECL procedure (Millipore, USA) to get protein bands, which were analyzed by ImageJ software. The primary antibodies included anti-HMGN5, anti-Bcl-2, anti-Bax, anti-Cyclin D1, anti-p21, anti-MMP-2, anti-MMP-9, anti-AKT, anti-p-AKT, anti-ERK1/2, anti-p-ERK1/2 (Santa Cruz Biotechnology, Inc., USA), and anti-β-actin (WanleiBio, Shenyang, China). The secondary antibodies included HRP-conjugated goat anti-rabbit IgG and goat anti-mouse IgG (OriGene Technologies, USA).
3
Protein Expression Analysis in PDAC Cells
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Total protein from human PDAC cells was extracted by protein lysis buffer (Invitrogen) containing 2 μL protease inhibitor, according to the instruction. Protein concentration was determined by a BCA Protein assay kit (Millipore, Darmstadt, Germany). 30 mg of protein in each sample was separated by polyacrylamide gel electrophoresis via 10% separating gel and transferred to 0.22 µm PVDF membranes (Millipore). Then, membranes were blocked with 5% nonfat milk for 2 h at 37°C and followed incubated with anti-MMP-2 (no. sc-13594; 1 : 1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MMP-9 (no. sc-393859; 1 : 750; Santa Cruz Biotechnology), anti-N-cadherin (no. sc-59987; 1 : 1500; Santa Cruz Biotechnology), anti-E-cadherin (no. sc-8426; 1 : 2000; Santa Cruz Biotechnology), anti-Vimentin (no. sc-6260; 1 : 1000; Santa Cruz Biotechnology), anti-Snail (no. sc-271977; 1 : 500; Santa Cruz Biotechnology), and GAPDH (no. sc-47724; 1 : 5000; Santa Cruz Biotechnology) antibodies overnight at 4°C. Goat anti-mouse horseradish peroxidase-conjugated IgG (no. sc-2005; 1 : 2500; Santa Cruz Biotechnology) was used as the secondary antibody and incubated with the membranes for 1 h at 37°C. Finally, protein bands were observed using the enhanced chemiluminescence kit (Millipore) on Chemidoc XRS Gel Imaging System (Bio-Rad, Hercules, CA, USA).
4
Corilagin Attenuates EMT and Wnt Signaling in Cancer
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Corilagin (CLG) was procured from Sigma-Aldrich (St. Louis, MO). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Alexa Fluor® 488 donkey anti-mouse IgG (H+L) antibody and Fluor® 594 donkey anti-rabbit IgG (H+L) antibody was obtained from Life Technologies (Grand Island, NY, USA). Anti-MnSOD(sc-137254), anti-Fibronectin(sc-6952), anti-Vimentin(sc-6260), anti-E-cadherin(sc-8426), anti-N-cadherin(sc-271386), anti-Occludin(sc-5562), anti-Twist(sc-15393), anti-MMP-2(sc-53630), anti-MMP-9(sc-393859), anti-Wnt3a(sc-136163), anti-FZD-1(sc-398082), and anti-β-actin(sc-47778) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Snail(3879S), anti-Axin-1 anti-Azin-1(3323S), anti-β-catenin(9562S), anti-p-GSK3β(9322S), and anti-GSK3β(9315S) antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA).
5
Ginkgolic Acid Inhibits STAT3 Signaling
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Ginkgolic acid C 17:1 (GAC 17:1) (95% purity) and ginkgolic acid C 15:1 (GAC 15:1) (98% purity), anti-STAT3, anti-SHP-1, anti-PTEN, anti-Bcl-2, anti-Bcl-xL, anti-survivin, anti-IAP-1, anti-COX-2, anti-MMP-9, anti-MMP-2, anti-caspase-3, anti-PARP, anti-β-actin, and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Tris base, glycine, NaCl, sodium dodecylsulfate (SDS), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI 1640, MEM, and fetal bovine serum (FBS) were obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Annexin V was obtained from BD Biosciences (Palo Alto, CA, USA). Anti-p-STAT3 (Tyr705), anti-p-JAK1 (Tyr1022/1023), anti-JAK1, anti-p-src (Tyr416), anti-src, anti-cyclin D1, and anti-cleaved caspase-3 were purchased from Cell Signaling Technology (Beverly, MA, USA). pMXs-gw (#18656) and pMXs-STAT3C (#13373) were obtained from Addgene (Cambridge, MA, USA).
6
Immunoblotting of Cell Proteins
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After extraction, proteins were separated via SDS-PAGE and transferred to
polyvinylidene difluoride membranes (MilliporeSigma, Burlington, MA, USA). The
membrane was blocked in 5% BSA and incubated with specific primary antibodies
against CEACAM5 (1:1000, 1:1000, 11077-R327, Sino Biological, Beijing, China
Sino Biological, Beijing, China), anti-Ki67, anti-PCNA (1:1000, Cell Signaling
Technology, Danvers, MA, USA), anti-MMP2 (1:1500, Santa Cruz Biotechnology,
Santa Cruz, CA, USA), anti-MMP9 (1:1500, Santa Cruz Biotechnology), anti-cyclin
D1 (1:1500 Santa Cruz Biotechnology), and β-actin (1:5000, ab8227, Abcam) at 4°C
overnight. After washing, the membrane was incubated with HRP-conjugated
secondary antibodies, and signals were detected using a Novex™ ECL
Chemiluminescent Substrate Reagent kit (Thermo Fisher Scientific). The relative
protein levels were quantified using ImageJ (US National Institutes of Health,
Bethesda, MD, USA).
polyvinylidene difluoride membranes (MilliporeSigma, Burlington, MA, USA). The
membrane was blocked in 5% BSA and incubated with specific primary antibodies
against CEACAM5 (1:1000, 1:1000, 11077-R327, Sino Biological, Beijing, China
Sino Biological, Beijing, China), anti-Ki67, anti-PCNA (1:1000, Cell Signaling
Technology, Danvers, MA, USA), anti-MMP2 (1:1500, Santa Cruz Biotechnology,
Santa Cruz, CA, USA), anti-MMP9 (1:1500, Santa Cruz Biotechnology), anti-cyclin
D1 (1:1500 Santa Cruz Biotechnology), and β-actin (1:5000, ab8227, Abcam) at 4°C
overnight. After washing, the membrane was incubated with HRP-conjugated
secondary antibodies, and signals were detected using a Novex™ ECL
Chemiluminescent Substrate Reagent kit (Thermo Fisher Scientific). The relative
protein levels were quantified using ImageJ (US National Institutes of Health,
Bethesda, MD, USA).
7
Immunohistochemistry Procedure for Tumor Markers
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Immunohistochemistry was done using a single-staining procedure. Anti-WFCD2, anti-Ecadherin,anti-Vimentin monoclonal antibody (Cell Signaling Technology), anti-CD44,anti-MMP2,anti-MMP9,and anti-ICAM-1 rabbit polyclonal antibody (Santa Cruz Biotechnology), were applied to the slides at a dilution of 1:1,00 ~ 1:150 in blocking buffer overnight at 4 °C. The slides were then washed and stained by the avidin-biotin method. The slides were lightly counter stained with hematoxylin. Tumor cells were considered positive for the antigen if there was brown color staining. The intensity was scored as negative (0), weak (1), medium (2), and strong (3),and the proportion of staining was scored as 1 (≤10%), 2 (11–50%), 3 (51–75%), and 4 (>75%). An overall expression score was calculated by multiplying the scores for intensity and proportion, ranging from 0 to 12. For ICAM-1, at least 500 tumor cell for each xenograft sample (n = 5) were randomly selected and counted. The number of positive cell was counted and the positive index was calculated as follows: ICAM-1 index = (number of stained cells/total cell number) × 100%.
8
Comprehensive Protein Expression Analysis
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Western blot was performed similarly to previously described methods [34 (link)], using anti-CtBP2 (1:500), anti-p16INK4A (1:1000), anti-PCNA (1:1000), anti-EGFP (1:1000), anti-CDK2 (1:500), anti-CDK4 (1:500), anti-CyclinD (1:500), anti-CyclinE (1:500), anti-p21 (1:1000), anti-Bax (1:1000) anti-E-cadherin (1:1000), anti-vimentin (1:1000), anti-MMP-2 (1:500) and anti-Glyceraldehyde-3-phosphate dehydro-genase (GAPDH) (1:1000, all the above antibodies from Santa Cruz Biotechnology, America). ImageJ (NIH) was used to compare the density of bands on western blot. Mean densitometry data from independent experiments were normalized by GAPDH.
9
Protein Expression Analysis in Cell Extracts
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Total cell extracts were obtained, and the protein content was quantitatively analyzed using a bicinchoninic acid protein assay and separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The separated proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). Samples were blocked in 5% bovine serum albumin for 2 h at room temperature and incubated with appropriate primary antibodies overnight at 4°C. The primary antibodies were as follows: 1) anti-ARHI (1:1,000; Santa Cruz Biotechnology Inc., Dallas, TX, USA), 2) anti-LC3 (1:1,000; Abcam, Cambridge, UK), 3) anti-GAPDH (1:5,000; Sigma-Aldrich Co., St Louis, MO, USA), 4) anti-bcl-2 (1:1,000; Santa Cruz Biotechnology), 5) anti-Mmp-2 (1:1,000; Santa Cruz Biotechnology), 6) anti-Mmp-9 (1:1,000; Santa Cruz Biotechnology), 7) anti-E-cadherin (1:1,000; Abcam), 8) anti-N-cadherin (1:1,000; Abcam). The membrane was washed in PBST and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG at room temperature for 1 h. The membrane was washed with PBST again, and the ECL kit was used for western blot detection. For whole images, ImageJ was used to estimate the band intensity. To normalize protein loading, monoclonal GAPDH antibody was used.
10
Immunohistochemical Analysis of Tissue Samples
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Tissues were fixed with 4% paraformaldehyde solution for at least 4 h at room temperature, followed by dehydration, dipping in wax, paraffin embedding and cut into sections. Then, these sections were treated with HE staining. For Immunohistochemistry, sections were incubated with serum or BSA for 30 min at room temperature, and then were dipped in diluted primary antibody for 2 h and then incubated with secondary antibody. The antibodies used in this research: primary antibodies: Proteintech (Wuhan, China): anti-SSRP1 (1:200; 15696-1-AP), anti-BCL2 (1:200; 26593-1-AP), anti-BAX (1:200; 50599-2-Ig), anti-MMP2 (1:200; 10373-2-AP), anti-MMP9 (1:200; 10375-2-AP) and PCNA (1:50; SC-56) from Santa Cruz Biotechnology; secondary antibody: goat anti-Rabbit IgG (H+L) (1:200; SA0000I-2) from Proteintech Group Inc. The sample was observed under BX53 fluorescence microscope (Olympus, Japan). Cells stained brown were positive cells.
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