Dimethyl sulfoxide, hydroxylamine·hydrochloride, phenylmethane sulfonylfluoride, thiobarbituric acid, metaphosphoric acid, o-phthaldialdehyde, bovine serum albumin, HEPES, digitonin, 2′,7′-dichlorofluorescein diacetate (DCF-DA), tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbo-cyanine iodide (JC-1), iron (III) chloride hexahydrate, egtazic acid (EGTA), malate, pyruvate, phosphoric acid, metaphosphoric acid, protease inhibitor, polyvinylidene difluoride (PVDF) membrane, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, penicillin, streptomycin, 2′,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and solvents were purchased from Millipore (Billerica, MA, USA). A superoxide dismutase (SOD) determination kit was purchased from Dojindo Molecular Technologies (Kumamoto, Japan). An ATP bioluminescence assay kit was purchased from Promega Corp. (Madison, WI, USA). PM2.5 (mean diameter: 1.06 μm) was purchased from Power Technology INC. (Arizona Test Dust (ATD), Arden Hills, MN, USA). The components of PM2.5 are presented in Table 1 .
Polyvinylidene difluoride
Manufactured by Merck Group
Sourced in United States, Germany, Italy
Polyvinylidene difluoride (PVDF) is a type of synthetic polymer material. It is known for its excellent chemical resistance, thermal stability, and mechanical properties. PVDF is commonly used in the manufacturing of various lab equipment and components, such as membranes, filters, and tubing, due to its unique characteristics.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (9 mentions)
Bca kit, by Thermo Fisher Scientific (9 mentions)
Ripa buffer, by Beyotime (5 mentions)
Peroxidase linked goat anti rabbit igg secondary antibody, by Santa Cruz Biotechnology (4 mentions)
Protease inhibitor, by Merck Group (4 mentions)
Nitrocellulose, by Bio-Rad (4 mentions)
Methanol, by Merck Group (4 mentions)
Potassium hydroxide, by Merck Group (4 mentions)
N methyl 2 pyrrolidone, by Merck Group (4 mentions)
Polyvinyl alcohol (pva), by Merck Group (4 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Horseradish peroxidase conjugated goat anti mouse igg antibody, by Thermo Fisher Scientific (3 mentions)
Enhanced chemiluminescence reaction, by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Digitonin, by Merck Group (2 mentions)
Hepes, by Merck Group (2 mentions)
2 7 dichlorofluorescein diacetate dcf da, by Merck Group (2 mentions)
Superoxide dismutase sod determination kit, by Dojindo Laboratories (2 mentions)
86 protocols using polyvinylidene difluoride
1
Characterization of PM2.5-Induced Oxidative Stress
2
Western Blot Analysis of EMT Markers
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Cells were lysed with lysis buffer radioimmunoprecipitation assay (RIPA) (Beyotime Institute of Biotechnology, China) and a mixture of protease inhibitors phenylmethanesulfonyl fluoride (PMSF) (Beyotime Institute of Biotechnology, China). The protein concentration was estimated by bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology, China). Equal amounts of protein were separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). After being transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA), membranes were blocked with 5 % non-fat milk and then incubated with primary antibodies at 4 °C overnight. Secondary antibodies were incubated for 2 h at room temperature. Protein bands were visualized using the enhanced chemiluminescence detection kit (Thermo scientific, USA). The primary antibodies were: CNTN-1 mAb (1:1000, Abcam, UK), E-cadherin rabbit mAb (1:1000, Cell Signaling Technology, USA), Slug rabbit mAb (1:1000, Cell Signaling Technology, USA), Snail rabbit pAb (1:1000, Abcam, UK), N-cadherin rabbit mAb (1:1000, Cell Signaling Technology, USA) and GAPDH mAb-HRP (1:5000, Bioworld Technology, USA).
3
Western Blot Analysis of Cultured EPCs
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The cultured EPCs were cleaned and hatched in radioimmunoprecipitation assay buffer containing inhibitors of proteases (Roche, China). For protein level determination, we used a bicinchoninic acid (BCA) kit (Thermo Scientific, Waltham, USA). Proteins were electrophoretically separated by 10% sodium dodecyl sulfate-polyacrylamide gel and were transferred to polyvinylidene difluoride (Millipore, Bedford) membranes. The membrane was then blotted with 5% bovine serum albumin and overnight with antibodies against p-p38 total p38, p-Akt, total Akt and β-actin (Cell Signaling Technology) at dilutions indicated by the manufacturer. Anti-rabbit or anti-mouse secondary antibodies and an ECL chemiluminescence detection system (Pierce Biotechnology Inc, Rockford, IL) were applied to scan and semi-quantitatively analyze the proteins according to the manufacturer’s instructions.
4
Western Blot Analysis of HVRP1 in Transfected HEK293A Cells
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Total protein from transfected and non-transfected HEK293A cells was collected with lysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% Triton-X 100, and protease inhibitor cocktail [Sigma]). The cell lysates were centrifuged at 13,000 rpm for 10 minutes at 4°C. Proteins from supernatants were separated by SDS-PAGE (4–20%, Tris-Glycine, Life Technologies). After gel electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were incubated with anti-HVRP1 antibody at a 1∶1000 dilution, or anti-HVRP1 antibody neutralized by antigen peptide (1∶1000 dilution of a 1 µg/µl solution). Mouse anti-rabbit secondary antibody was conjugated with horseradish peroxidase (Chemicon International) and used at a 1∶50,000 dilution. The membranes were stripped by washing with a mild stripping buffer (1.5% glycine, 0.1% SDS, 0.5% Tween-20, pH 2.2) for 2×10 minutes, then PBS 2×10 minutes, and TBST 2×10 minutes. Membranes were then re-probed with anti-β-actin antibody conjugated to HRP (Abcam) at a 1∶5000 dilution. All proteins interacting with primary antibodies were visualized with Super Signal West Pico chemiluminescent substrate (Thermo Fisher).
5
Exosomal Protein Analysis by Western Blot
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Western blotting was performed using the standard SDS-PAGE separation technique as previously reported18 (link),19 . The harvested cells were disrupted with RIPA cleavage buffer (Cell Signaling Technology). Proteins were extracted from exosomes using a Total Exosome Protein Isolation Kit (Invitrogen, Carlsbad, CA, USA) following the instructions supplied. The lysates were collected after centrifugation, and the protein concentration was quantified with BCA kit (Thermo Fisher Scientific). 50 mg of protein was added to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were incubated overnight at 4 °C with antibodies against phosphor-JNK (ab124956), phospho-MAPK (ab195049), phospho-ERK (ab201015), phospho-NF-κB (ab76302), TRAF-6 (ab33915) and GAPDH (ab8245) purchased from Abcam. Subsequently, the membranes were incubated with a secondary antibody for 1.5 h at room temperature. Immunoreactive protein bands were detected using an Odyssey scanning system (USA), quantified by Image J, and normalized to the corresponding amount of total protein. The experiment was repeated in triplicate with 3 replicates.
6
Western Blot Analysis of NMD Pathway Proteins
7
Western Blot Analysis of CBMP-Treated SGC-7901 Cells
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SGC-7901 cells (1.5 × 105 cells/well) in the logarithmic growth phase were cultured in 6-well cell culture plates for 24 h and then treated with different concentrations of CBMP for 24 h. We used RIPA mixed with protease and phosphatase inhibitors to lyse to gain total proteins of SGC-7901 cells. The BCA protein assay kit (Fudebio, Hangzhou, China) was used to measure protein concentration. The extracted protein was packed and stored at −80°C. We used 10% or 12% SDS-PAGE to separate proteins and then transfer them onto polyvinylidene difluoride (PVDF) membranes (Millipore, Atlanta, USA). 5% skim milk was used to block PVDF membranes at room temperature for 1 h, and then, primary antibodies were incubated overnight in a refrigerator at 4°C. After that, the PVDF membranes were incubated with secondary antibodies for 2 h at room temperature. The signals were detected by ImmobilonWestern Chemiluminescent HRP Substrate (Millipore, Atlanta, USA), and the images of the band were evaluated using ImageJ software.
8
Western Blot Analysis of Signaling Proteins
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The 32D cells with different treatments were washed and and the protein sample from the cells were extracted using radio-immunoprecipitation assay buffer containing proteinase inhibitor (Roche, Basel, Switzerland). The protein levels were determined using a BCA kit (Thermo Fisher Scientific). The proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by transferring to polyvinylidene difluoride (Millipore, Bedford) membranes. The membranes were then blocked with 5% bovine serum albumin and incubated overnight with antibodies against TPO (1:1000; #ab180728), p-STAT3 (1:1000; #ab32143), t-STAT3 (1:1500; #ab119352), p-STAT5 (1:800; #ab32364), t-STAT5 (1:1000; #ab32364), Bcl-2 (1:1500; #ab32124), survivin (1:2000; #ab649), Bax (1:2000; #ab32503), c-myc (1:1500; #32072), Bcl-xL (1:1500; #32370), and GAPDH (1:1000; Abcam, Cambridge, UK) at dilutions specified by the manufacturer’s instructions. After incubating with horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h, an ECL chemiluminescence detection system (Pierce Biotechnology Inc, Rockford, IL, USA) were used according to the manufacturer’s instructions to detect and semi-quantitatively analyze the proteins.
9
KRAS Activation Assay Protocol
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Cells were lysed using RIPA buffer (Sigma) containing Protease Inhibitor Cocktail (Sigma) with phosphatase inhibitors (Calbiochem) for separation and blotting on polyvinylidene difluoride (Millipore). The following antibodies were used for immunoblotting: NADK (Santa Cruz; sc-100347); GAPDH (Santa Cruz; sc-25778); Vinculin (Santa Cruz; sc-25336), phospho-ERK1/2 (Cell Signaling; 9101 S) and KRAS (Santa Cruz; sc-30). All antibodies were diluted to 1:1,000 in 1% BSA. Full immunoblot scans are shown in Supplementary Figs 7 and 8 . KRAS activity assay was performed by RAF pulldown using a Ras Activation Assay Kit (EMD-Millipore) by using 650 μg of cell lysate and 10 μg of RAF beads per reaction following the manufacturer's protocol.
10
Western Blot Analysis of HepG2 Cells
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HepG2 cells were lysed in RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China). Cell lysates (20 μg protein/lane) were loaded and separated on gradient polyacrylamide gels and then transferred to polyvinylidene difluoride membranes by electroblotting (Millipore Corp., Boston, MA, USA). Following blocking with 5% non-fat milk containing 0.3% Tween 20 for 1 h, the membranes were incubated overnight with primary antibodies at 4°C, including anti-hSulf-1 (1:250), -stat3 (1:500), -phospho-stat3 (1:500), -phospho-c-met (1:500), -bcl-2 (1:1000) and -cyclin D1 (1:500) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The membranes were washed three times with Tris-buffered saline containing Tween 20 and membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (R&D Systems China Co., Ltd., Shanghai, China) at 4°C for 1 h. Subsequently, membranes were exposed to enhanced chemiluminescent reagents for detection of protein bands. β-actin was used as an internal control.
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