Overnight serum-starved LECs were treated with 100 ng/ml VEGF-C (R&D System) for 20 min at 37 °C, lysed (20 min) in cold buffer (20 mM Tris-HCL, pH 7.5, 150 mM NaCL, 1% Triton X-100, 60 mM octyl β-D-glucopyranoside, 200 μM sodium orthovanadate, 50 mM NaF, 1 mM PMSF, and 1 μg/ml protease inhibitor mix) and the cleared lysates (10,000 × g, 10 min) assayed for protein (Pierce Biotech). Proteins separated on 4–20% gradient gels were transferred to polyvinylidene fluoride membranes (Merk Millipore), blocked (Li-COR Biosciences) 1 h at room temperature before adding primary antibodies (Supplementary Table 1 ) overnight at 4 °C. Infrared dye–labeled secondary antibodies were added for 1 h at room temperature. Protein signals were detected using the Li-Cor Odyssey Infrared (Li-COR Biosciences) and quantified using Image Studio Lite v5.2 Software.
Polyvinylidene fluoride membrane
Manufactured by Merck Group
Sourced in United States, Germany, China, United Kingdom, Morocco, Ireland, Japan, Canada
Polyvinylidene fluoride (PVDF) membranes are a type of lab equipment used for a variety of filtration and separation applications. They are made from a thermoplastic fluoropolymer material and offer high chemical and thermal resistance. PVDF membranes are commonly used in processes such as microfiltration, ultrafiltration, and sample preparation.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (226 mentions)
Ripa lysis buffer, by Beyotime (207 mentions)
Bca protein assay kit, by Beyotime (154 mentions)
Ripa buffer, by Beyotime (120 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (109 mentions)
Protease inhibitor cocktail, by Roche (92 mentions)
Gapdh, by Cell Signaling Technology (86 mentions)
Ripa buffer, by Thermo Fisher Scientific (79 mentions)
Bca kit, by Thermo Fisher Scientific (71 mentions)
Quantity one software, by Bio-Rad (68 mentions)
β actin, by Abcam (68 mentions)
β actin, by Santa Cruz Biotechnology (67 mentions)
β actin, by Cell Signaling Technology (65 mentions)
Bca protein assay, by Thermo Fisher Scientific (64 mentions)
Gapdh, by Abcam (62 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (61 mentions)
Image lab software, by Bio-Rad (61 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (61 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (56 mentions)
Ab205718, by Abcam (51 mentions)
3 925 protocols using polyvinylidene fluoride membrane
1
VEGF-C Signaling Pathway Analysis
2
Quantitative Protein Analysis in Infected Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein isolation was carried out from infected cells by employing the phenylmethanesulfonyl fluoride (Beyotime) mixed with RIPA lysis solution (Beyotime). Protein concentration was measured by the BCA Protein Assay Kit (Beyotime). 25 μg protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (Merk Millipore, Billerica, MA, USA). After blocked with 5% skimmed milk, membranes were incubated with the antibodies against RUNX1 (1:400; Affinity, Changzhou, China) or β-actin (1:1000; Santa Cruz) at 4 °C overnight and then with the secondary antibodies horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000; Beyotime) or goat anti-mouse IgG (1:5000; Beyotime). Protein blot bands were visualized with WD-9413B gel imaging system (Liuyi biotechnology, Beijing, China).
3
Protein Expression Analysis of Anlotinib-Treated SKOV-3 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After treatment with anlotinib at indicated concentrations for 48 h, SKOV-3 cells were washed with cold PBS twice and lysed with radio-immunoprecipitation assay lysis buffer containing protease and phosphatase inhibitor cocktails (MedChemExpress, Monmouth Junction, NJ, USA) on ice for 30 min. To remove insoluble materials, the cell lysate was centrifuged at 13,000× g for 15 min at 4 °C. The total protein concentration of the supernatants was measured by BCA protein assay kit (Thermo Fisher Scientific, Osterode am Harz, Niedersachsen, Germany). Protein was separated by 7.5% or 10% SDS-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (Merk Millipore, Darmstadt, Hessen, Germany). The membranes were reacted with specific primary antibodies overnight at 4 °C with gentle shaking after blocked by 5% nonfat milk for 1 h at room temperature. The immune complexes were reacted with HRP-conjugated secondary antibodies and made visible with chemiluminescence agents (Thermo Fisher Scientific, Osterode am Harz, Niedersachsen, Germany). The immunoblots were detected using the electrochemiluminescence imaging system (Bio-Rad, Berkeley, CA, USA).
4
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For western blot analysis, cells at confluence were washed twice with PBS and harvested by trypsinization. Cells were lysed in RIPA supplemented with cocktail and PMSF (Solarbio), and the protein concentration was determined using a Bradford assay (Bio-Rad Laboratories). Total protein extract (50 µg) of each sample was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroblotted on polyvinylidene fluoride membranes (Merk Millipore). The transferred membranes were probed with specific primary Ab at 1:1000 dilutions: anti-E-cadherin, anti-N-cadherin, anti-vimentin, anti-catenin, anti-claudin. Anti-GAPDH at a dilution of 1:1000 (Cell Signaling Technology) was used as an internal control. After 3 washes, anti-mouse and anti-rabbit horseradish peroxidase-conjugated secondary Ab (1:5000, Cell Signaling Technology) were added to the membranes. Bands were visualized by incubating the membranes with enhanced chemiluminescence reagent (Thermo Fisher Scientific) and exposing the membranes to X-ray film.
5
Macrophage and Chondrocyte Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total proteins from different macrophages and chondrocytes were extracted with the RIPA lysis buffer (MCE, NJ, USA) containing a mixture of phosphatase and protease inhibitors. Then, the proteins were separated with 8–12% SDS-PAGE (Febio science, Hangzhou, China) and transferred to the polyvinylidene fluoride membranes (Merk Millipore, Darmstadt, Germany). The membranes were blocked with 5%BSA and then incubated with the primary antibodies overnight. The membranes were washed next day with PBST and then incubated with the HRP-conjugated secondary antibodies (Beyotime, Shanghai, China) for 90 min. These results on the membranes were captured and documented by Molecular Imager (Bio-Rad, CA, USA).
The primary antibodies used include those against IL-1β (R&D Systems, MN, USA), NLRP3 (Adipogen, CA, USA), ASC (Adipogen), Caspase-1 (Adipogen), P-jak2 (Abcam, MA, USA), P-Stat3 (Abcam), P-bad (Cell Signaling Technology, MA, USA), Cleaved Caspase-3 (Cell Signaling Technology), Jak2 (ABclonal, Wuhan, China), Stat3 (ABclonal), Bad (ABclonal), Clic1 (ABclonal), Clic4 (ABclonal), ADAMTS5 (ABclonal), MMP13 (ABclonal), Bax (HUABIO, Hangzhou, China), Bcl-2 (HUABIO), Clic5 (HUABIO), PIM-1 (HUABIO), Collagen II (ProteinTech, Wuhan, China), Aggrecan (ProteinTech), β-actin (ProteinTech), GADPH (ProteinTech).
The primary antibodies used include those against IL-1β (R&D Systems, MN, USA), NLRP3 (Adipogen, CA, USA), ASC (Adipogen), Caspase-1 (Adipogen), P-jak2 (Abcam, MA, USA), P-Stat3 (Abcam), P-bad (Cell Signaling Technology, MA, USA), Cleaved Caspase-3 (Cell Signaling Technology), Jak2 (ABclonal, Wuhan, China), Stat3 (ABclonal), Bad (ABclonal), Clic1 (ABclonal), Clic4 (ABclonal), ADAMTS5 (ABclonal), MMP13 (ABclonal), Bax (HUABIO, Hangzhou, China), Bcl-2 (HUABIO), Clic5 (HUABIO), PIM-1 (HUABIO), Collagen II (ProteinTech, Wuhan, China), Aggrecan (ProteinTech), β-actin (ProteinTech), GADPH (ProteinTech).
6
Western Blot Analysis of EMT Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins from cultured cells were lysed with SDS lysis buffer. Protein concentration was assayed using a BCA assay kit (Thermo Fisher Scientific, USA). Equal quantities of protein were separated on 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride membranes (Merk, Germany), and then reacted with primary antibodies against ANLN, CDH1, CDH2, Snail, Vimentin, Slug, and β‐Tubulin (Proteintec, China) at 4°C overnight. The membranes were then washed with TBST Buffer and incubated for one hour with Goat anti‐Mouse/Rabbit IgG(H+L)‐HRP (Ray antibody Biotech, Beijing, China). The bands were visualized using electrochemiluminescent luminous fluid (Merk, Germany). Photographs were taken using by a Tanon 6600 Luminescence imaging workstation (Tanon, China).
7
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples (up to 10 mg) were lysed in lysis buffer containing 1% Nonidet P-40 (Calbiochem, Merk, Darmstadt, Germany) and protease and phosphatase inhibitor cocktails (Roche, IL, USA) overnight at 4°C. Total extracts were centrifuged at 12,000 g for 30 min at 4°C, and protein concentration was determined with the BCA method (Pierce Biotechnology, IL, USA). A total of 40 μg of protein per lane was loaded onto 10% Bis-Tris SDS-PAGE gels, separated electrophoretically, and blotted onto polyvinylidene fluoride membranes (Merk). The blots were incubated with antibodies against anti-JAG1 (1:2,000), anti-MMP2 antibody (1:1,500), anti-VIM (Rabbit monoclonal, ab92547, 1:1,000, Abcam), and anti-SNAI1 (Rabbit polyclonal, 1:2,000, ab180714, Abcam) followed by a secondary antibody (1:8,000) tagged with horseradish peroxidase (Santa Cruz Biotechnology). Blots were visualized by enhanced chemiluminescence, and densitometry was performed using a fluorescence image analyzer (Amersham Imager 600, GE, MA, USA). GAPDH was used as a loading control.
8
Western Blot Analysis of Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples (up to 10 mg) were lysed in lysis buffer containing 1% Nonidet P-40 (Calbiochem, Merk) and protease and phosphatase inhibitor cocktails (Roche) overnight at 4°C. Total extracts were centrifuged at 12,000 × g for 30 min at 4°C, and protein concentration was determined with the BCA method (Pierce Biotechnology). A total of 40 µg of protein per lane was loaded onto 10% Bis-Tris SDS-PAGE gels, separated electrophoretically, and blotted onto polyvinylidene fluoride membranes (Merk). The blots were incubated with antibodies against anti-p16INK4A antibody (1:1,000), anti-CDK4 antibody (1:2,000), anti-EGFR antibody (1:1,000) and anti-CDH2 antibody (1:1,000) followed by a secondary antibody (1:10,000) tagged with horseradish peroxidase (Santa Cruz Biotechnology). Blots were visualized by enhanced chemiluminescence, and densitometry was performed using a fluorescence image analyzer (Amersham Imager 600, GE Healthcare). GAPDH was used as a loading control.
9
Western Blot Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples were separated on 8–10% Bis-Tris SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (Merk, Kenilworth, NJ, USA). All primary antibodies (Table S4 ) were diluted in TBST containing 1% bovine serum albumin (BSA) and were incubated with the membranes overnight at 4 °C. Immunoreactive bands were visualized using chemiluminescence.
10
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples underwent separation on 8%–10% Bis‐Tris SDS‐PAGE gels and were subsequently transferred onto polyvinylidene fluoride membranes (Merk). The primary antibodies were diluted in TBST solution supplemented with 1% bovine serum albumin (BSA) and were incubated with the membranes overnight at a temperature of 4°C. The visualization of immunoreactive bands was achieved through the utilization of chemiluminescence. The following antibodies were used in this study: CDK5 (ab40773, 1/200), pPBK‐T9 (ab184953, 1/1000), Snail (ab216347, 1/1000), N‐cadherin (ab18203, 1/1000), PBK (ab236872, 1/1000), E‐cadherin (ab76319, 1/1000) obtained from Abcam. FLAG (YN5598 1/5000), GFP (YM3009 1/5000), and HA (MY3003 1/5000) were obtained from Immunoway. JNK (9252P 1/1000), p‐JNK (4668S 1/1000), p38 (8690P 1/1000), p‐p38 (4511P 1/1000), ERK (4695P 1/1000), p‐ERK (4370P 1/2000) was sourced from Cell Signaling Technology.
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!