Total proteins were extracted by RIPA buffer supplemented with PMSF (Solarbio, Beijing, China) and quantified by a BCA kit. Then, the proteins were separated in SDS-PAGE gels and transferred into PVDF membranes (Millipore, Massachusetts, USA). Membranes were blocked with TBST with 5% skim milk powder and incubated overnight at 4 °C with primary antibodies against YAP1 (1:1000), P-YAP1 (1:1000), VEGFA (1:1000), Hexokinase II (HK2; 1:5000), LDHA (1:2000), HIF1A (1:1000), GAPDH (1:5000), Tublin (1:1000), and β-actin (1:1000) (Proteintech, Wuhan, China). The next day, blots were washed with PBS and then secondary antibodies were incubated with the membrane at room temperature for 1 h. The membrane was visualized using a chemiluminescence kit (Absin, Shanghai, China) and quantified by densitometry analysis using ImageJ software. GAPDH and tubulin were used as loading controls.
Tublin
Manufactured by Proteintech
Sourced in China
Tublin is a laboratory equipment product designed for the isolation and purification of tubulin, a key structural protein found in the cytoskeleton of eukaryotic cells. The core function of Tublin is to facilitate the extraction and separation of tubulin from cell samples, allowing researchers to study its properties and role in cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Proteintech (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
Chemiluminescence kit, by Absin (1 mentions)
β actin, by Proteintech (1 mentions)
P yap1, by Proteintech (1 mentions)
Vegfa, by Proteintech (1 mentions)
Hif1a, by Proteintech (1 mentions)
Nuclear protein extraction kit, by Beyotime (1 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions)
Keap1, by Proteintech (1 mentions)
Lamin b, by Proteintech (1 mentions)
Matrix metalloproteinase 9 mmp 9, by Proteintech (1 mentions)
Oridonin, by Yuanye Bio-Technology (1 mentions)
Acsl4, by Proteintech (1 mentions)
Cl caspase3, by Proteintech (1 mentions)
Slc7a11, by Proteintech (1 mentions)
Ferrostatin 1, by MedChemExpress (1 mentions)
Bcl2 associated x (bax), by Proteintech (1 mentions)
Bcl 2, by Proteintech (1 mentions)
6 protocols using tublin
1
Protein Expression Analysis in Cell Metabolism
2
Chondrocyte Protein Analysis: Evaluating Cellular Responses
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After 24 h of treatment, total proteins of chondrocytes were extracted using radioimmunoprecipitation assay buffer (beyotime) while nuclear proteins were extracted using nuclear protein extraction kit (beyotime). Protein concentrations were measured using a BCA protein assay kit. Total protein (20 µg per sample) was separated by SDS-PAGE on a 12% gel and subsequently transferred to a nitrocellulose membrane (EMD Millipore). The membranes were incubated overnight at 4°C with the following antibodies: Nrf2 (1:1000; Cell Signaling Technology Inc., USA), NQO1 (1:1000; Proteintech, Wuhan, China), Keap1 (1:1000; Proteintech), Matrix metalloproteinase 9 (MMP 9, 1:1000; Proteintech), Matrix metalloproteinase 13 (MMP13, 1:1000; Proteintech), aggrecan (1:1000; CST), collagen II (1:1000; CST), cleaved caspase-3 (1:1000; CST), Bax (1:1000; CST), Bcl-2 (1:1000; CST), GAPDH (1:2000; Proteintech), Tublin (1:2000; Proteintech), LaminB (1:2000; Proteintech). Membranes were washed with TBS-Tween (0.05%) for 30 min, followed by incubation with anti-rabbit secondary antibody (1:5000; CST) for 2h at room temperature. The protein signals were visualized using Enhanced chemiluminescence Plus (Tanon Science and Technology Co., Ltd.).
3
Oridonin and Ferrostatin-1 Protocols
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Oridonin (HPLC ≥98%) was purchased from Shanghai yuanye Bio-Technology Co., Ltd. (#B20310), then the drug was dissolved in DMSO and stored in − 20 °C for usage. Ferrostatin-1 (Fer-1) was purchased from MedChemExpress (#HY-100579, MCE), and stored in − 20 °C for usage. All primary antibody used in the experiments included Actin (1:10000, #60008–1-Ig, Proteintech), Bcl-2 (1:1000, #61193, Proteintech), BAX (1:5000, #50599–2-Ig, Proteintech), cl-caspase3 (1:10000, #19677–1-AP, Proteintech), Tublin (1:1000, #66031–1-Ig, Proteintech), GAPDH (1:2000, #CL594–60004, Proteintech), GPX4 (1:5000, #67763–1-IG, Proteintech), SLC7A11 (1:1000, #26864–1-AP, Proteintech), FTH1 (1:5000, #11682–1-AP, Proteintech), and ACSL4 (1:10000, #81196–1-RR, Proteintech), they were stored in − 20 °C for usage. Ferrostatin-1 (Fer-1) (#HY-100579, MCE), an effective ferroptosis inhibitor [32 (link)], was dissolved in dimethylsulfoxide (DMSO) to create stock solutions and stored at − 20 °C.
4
Insulin Signaling Pathway Analysis
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At the indicated time, C2C12 myotubes were lysed in Radio Immunoprecipitation Assay (RIPA) lysis buffer containing protease and phosphatase inhibitors (Roche, Mannheim, Germany). Protein concentrations were measured with the BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, USA). Proteins were loaded and separated by 8%-10% (v/v) SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membrane and blocked with 5% milk. The membrane was incubated with the determined primary antibodies, respectively overnight at 4 °C as follows: Insulin receptor substrate 1 (Irs-1) (1:1000 in dilution, Cat No: 2382; Cell Signaling Technology, Danvers, MA, USA), Phospho-Irs-1(ser307) (1:1000 in dilution, Cat No: 2381; CST), RAC-alpha serine/threonine-protein kinase (Akt)(1:1000 in dilution, Cat No: 4685; CST), Phospho-Akt (Ser473)(1:1000 in dilution, Cat No: 4060; CST), Pgc1α(1:1000 in dilution, Cat No: ab54481; Abcam, Cambridge, UK), Tublin (1:1000 in dilution, Cat No: 10094-1-AP; Proteintech, Rosemont, USA). Then the membrane was incubated with goat anti-rabbit HRP secondary antibody (1:5000 in dilution, CAT: BL003A; Biosharp, Hefei, China). Proteins bands were visualized using a chemiluminescence kit and analyzed using Image J software.
5
Western Blot Analysis of Mouse Cortical Samples
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Mouse cortical samples were lysed with RIPA containing 1% phenylmethanesulfonyl fluoride (PMSF) (100 mM). The buffered lysis samples were collected into an EP tube and centrifuged at 12,000 rpm at 4 °C for 15 min. The precipitate was discarded and the protein concentration was determined using the BCA Protein Assay Kit (Beijing, China). We performed Western blotting as described previously. In short, proteins were separated and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, MA, USA). The membrane was incubated with primary antibodies overnight at 4 °C. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Proteintech, Wuhan, China) for 1h at room temperature. Finally, the immunoblots were developed by ECL Western blotting detection reagent (Millipore, USA). The primary antibodies against COX2 (dilution 1:1000, Abcam, Cambridge, UK) DP1 (dilution 1:1000, Abcam, USA) and were purchased from Abcam. DP2 (dilution 1:500, Aiffinity, Changzhou, China) was purchased from Aiffinity. Tublin (dilution 1:5000, Proteintech, China) was purchased from Proteintech.
6
Investigating HuR-mediated regulation of lipid metabolism
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Bovine serum albumin (BSA, #B2064), oleic acid (OA, #O1008), palmitic acid (PA, #P0500), MG-132(#M7449), chloroquine (CQ, #C6628), actinomycin D (#SBR00013) and TG assay kit (MAK266) were purchased from Sigma. HuR-ARE interaction inhibitor CMLD-2 was purchased from Millipore (#5.38339.0001). The antibodies used were speci c for HuR (Millipore, #07-468), Akt (Cell Signaling Technology, #4691), phospho-Akt at Ser473 (Cell Signaling Technology, #4060), PTEN (Cell Signaling Technology, #9188), Tublin (Proteintech, #11224-1-AP), and GAPDH (Cell Signaling Technology, cat #2118). Lentivirus encoding PTEN (Lenti-PTEN) or Lenti-LacZ were from Jikai (Shanghai, China).
Adenovirus expressing GFP (AdGFP) and HuR (AdHuR) were from Vigenebio (MD, USA). The insulin ELISA kit was from Mercodia (10-1249-01). The assay kits used to measure serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), nonesteri ed fatty acids (NEFAs) and total cholesterol (TC) were purchased from Jiancheng Bioengineering Institute (Nanjing, China).
Adenovirus expressing GFP (AdGFP) and HuR (AdHuR) were from Vigenebio (MD, USA). The insulin ELISA kit was from Mercodia (10-1249-01). The assay kits used to measure serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), nonesteri ed fatty acids (NEFAs) and total cholesterol (TC) were purchased from Jiancheng Bioengineering Institute (Nanjing, China).
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