Vessels homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer (BOSTER, Wuhan, China) were clarified by centrifugation (14,000r/min, 5min, 4°C). Standard Western blotting protocol was followed. Samples separated by 4% stacking gel and 10% separating gel were transferred onto polyvinylidene fluoride membranes (Millipore, USA). Membranes were blocked for 1h at room temperature or overnight at 4 °C with 5% nonfat milk (Sangon Biotech, Shanghai, China). Then incubated with primary antibody as follows: rabbit anti-rabbit CD147 (1:400, BIOSS, Beijing, China), cavia anti-rabbit IL-6 (1:400, Uscn Life science Inc, Wuhan, China), mouse anti-rabbit IL-10 (1:500, Global Biotech, Shanghai, China), rabbit anti-rabbit TGF-β1 (1:500, Global Biotech), mouse anti-rabbit β-Actin (1:500, BOSTER) for 2h at room temperature or overnight at 4 °C. After three washes, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody as follows: goat anti-rabbit (1:1000, BOSTER), goat anti-guinea pig (1:1000, Global Biotech), goat anti-mouse (1:1000, BOSTER) for 1h at room temperature. Protein bands were visualized by enhanced chemiluminescence with an ECL Plus chemiluminescence detection kit (Sangon Biotech). Optical density of the protein bands was quantified by Gel Analysis V2.02 software (Clinx Science Instruments).
Goat anti mouse
Manufactured by Boster Bio
Sourced in China, United States
Goat anti-mouse is a secondary antibody product designed for use in various immunological techniques. It is produced by immunizing goats with mouse immunoglobulins, resulting in the generation of antibodies that specifically recognize and bind to mouse antibodies. This product is commonly used as a detection reagent in applications such as Western blotting, ELISA, and immunohistochemistry, where it allows for the visualization and quantification of mouse primary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit, by Boster Bio (10 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Ripa lysis buffer, by Solabio (2 mentions)
Protease and phosphatase inhibitor, by Roche (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Ripa lysis buffer, by Boster Bio (1 mentions)
Non fat milk, by Sangon (1 mentions)
Gel analysis v 2, by Clinx (1 mentions)
Chemiluminescence kit, by Merck Group (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Anti gsk3β, by Cell Signaling Technology (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Anti wnt3a, by Cell Signaling Technology (1 mentions)
Anti pten, by Merck Group (1 mentions)
Anti pi3k, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Boster Bio (1 mentions)
Anti p pi3k, by Cell Signaling Technology (1 mentions)
Anti n cadherin, by Abcam (1 mentions)
Anti fibronectin, by Abcam (1 mentions)
13 protocols using goat anti mouse
1
Quantitative Western Blot Analysis of Inflammatory Markers
2
Western Blot Analysis of Protein Expression
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Tissues or cells were treated with lysis buffer containing 1% PMSF. Protein quantitation was determined using the bicinchoninic acid assay. Thirty micrograms of protein was run on 10% SDS‐PAGE gel. Subsequently, the gel‐separated proteins were transferred onto PVDF membranes by wet transfer. The membranes were blocked with 5% non‐fat milk for 2 h at RT and then incubated overnight with the primary antibody at 4°C, followed by incubation with goat anti‐mouse (Boster Biological Technology Co. Ltd, Cat no: BA1075, 1:2000) or goat anti‐rabbit (Boster Biological Technology Co. Ltd, Cat no: BA1054, 1:2000) IgG‐HRP secondary antibody, for 1 h at RT. Immunoblots were visualised using a chemiluminescence kit (Millipore). β‐actin or GAPDH was selected as the internal control for gene expression normalisation based on the molecular weight of protein. The primary antibodies and corresponding secondary antibodies used in present study and their appropriate working dilutions are listed in Table 2 .
3
Immunohistochemical Analysis of Bone Markers
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Immunohistochemistry (IHC) was performed with reference to a previous procedure [27 (link)]. The primary antibodies used were goat anti-rabbit osteocalcin (OCN; 1:400), runt-related transcription factor 2 (RUNX2; 1:200), β-catenin (1:300), and PTH1R (1:300), and anti-mouse matrix metalloproteinase 9 (MMP9; 1:200), vascular endothelial growth factor (VEGF; 1:200), and goat anti-rat BrdU (1:200). All antibodies were purchased from Santa Cruz Biotechnology Corporation (Santa Cruz, CA, USA). The secondary biotinylated goat anti-mouse, rabbit anti-goat, and goat anti-rabbit IgGs were purchased from Boster (Wuhan, China). Positive expression cells were observed the region of interest with an Olympus microscope, and five random regions within the observation range were selected and counted. Results were identified by positive cell values and standard deviations and used for statistical analysis.
4
Protein Expression Analysis in Cells
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Radioimmunoprecipitation assay lysis buffer was used to extract protein from indicated cells. Bradford protein assay kit (Beyotime, Shanghai, People’s Republic of China) was used to measure the protein concentration. Total 60 μg of protein was separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted onto 0.22 μm nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk for 2 hours and incubated with primary antibodies (anti-Sall4, anti-PTEN, anti-phosphorylated PTEN, and anti-Bmi-1 [Sigma-Aldrich]; anti-PI3K, anti-pPI3K, anti-AKT, anti-pAKT, anti-Wnt3a, anti-β-catenin, anti-GSK3β, and anti-GSK3β [Cell Signaling Technology, Danvers, MA, USA]; anti-E-cadherin, anti-N-cadherin, anti-fibronectin, anti-vimentin [Abcam, Cambridge, UK]; and anti-GAPDH [Proteintech, Wuhan, People’s Republic of China]) overnight at 4°C. The membranes were washed with tris-buffered saline containing 0.1% Tween 20, and then incubated with appropriate horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit, 1:2,000; goat anti-mouse, 1:5,000; Boster, Wuhan, People’s Republic of China) for 1 hour at 37°C. Enhanced chemiluminescence reagent was used to detect the signal on the membrane. The data were analyzed and normalized to the expression of the internal control (glyceraldehyde 3-phosphate dehydrogenase).
5
Protein Isolation and Western Blot Analysis
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Cellular protein was isolated by lysis in RIPA buffer (Boster, Wuhan). Proteins in all samples were quantified with Bicinchoninic acid protein assay. Proteins of equal amounts from all samples were separated with SDS/PAGE gel (Bio-Rad, United States) and transferred onto PVDF membrane (Bio-Rad, United States). Bands were sealed with 5% skim milk, incubated in primary antibodies at 4°C overnight, then in secondary antibodies at room temperature for 1 h, and then examined and analyzed by using ChemiDoc™ XRS+ with Image Lab™ Software (Bio-Rad, United States). Following antibodies were purchased: anti-caspase-3 (#9662), anti-PARP (#9532), anti-Phospho-SAPK/JNK (Thr183/Tyr185) (#4688), anti-Phospho-p38 MAPK (Thr180/Tyr182) (#4511), anti-HDAC1 (#34589), anti- HDAC2 (#57156), and anti-Histone H3 (#4499) were purchased from Cell Signaling Technology (Beverly, MA, United States); anti-GAPDH (60004-1-Ig) was purchased from Proteintech Group (Chicago, IL, United States); anti-Phospho-ERK (AP0472), anti-Phospho-MEK (AP1021) and anti-CDK6 (A0705) were purchased from ABclonal Technology (Wuhan, China); anti-cyclin D1 (YT1172) was purchased from Immunoway Biotechnology Company (TX, United States); goat anti-rabbit and goat anti-mouse were obtained from Boster Biological Technology (Wuhan, China).
6
Western Blot Analysis for Protein Expression
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Total proteins were extracted from the cells and tissues using radio immunoprecipitation assay buffer (BIOSS) mixed with phenylmethylsulfonyl fluoride (BIOSS) and quantified using bicinchoninic acid assay (Beyotime Institute of Biotechnology). Protein extractions (30ug per well) were separated using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (MilliporeSigma). The membranes were blocked with 5% fat-free dried milk for 1 h at room temperature. After incubation with high-affinity anti-FTO (1:1000, Abcam, USA), anti-STAT3 (1:1000, Cell Signaling Technology, USA), anti- phosphorylation-STAT3 (1:1000, Cell Signaling Technology, USA), anti-Bcl-2 (1:1000, Cell Signaling Technology, USA), anti-c-Myc (1:1000, Abways Technology, China), anti-CyclinD-1 (1:1000, Abways Technology, China), anti-β-actin (1:5 000, Bioss, China), or anti-GAPDH (1:5000, Bioss, China) antibodies at 4°C overnight, the membranes were incubated with the HRP-conjugated secondary antibodies goat anti-rabbit (1:10,000; Boster, China) or goat anti-mouse (1:5000; Boster, China) for 1 h at room temperature. Proteins were detected using BeyoECL chemiluminescence kit (Biyuntian, China) and detected using the Amersham ImageQuant 800 system (Cytiva). The density of bands were measured using ImageJ (v1.51, National Institutes of Health).
7
Western Blot Analysis of p-AKT and AKT
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Total cell proteins were extracted at 4 °C using RIPA lysis buffer (Solabio, Beijing, China) containing protease and phosphatase inhibitors (Roche, Basel, Switzerland). Proteins were resolved using 8%-12% SDS polyacrylamide electrophoresis and electro-transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). Then we blocked the blots with 5% non-fat milk for 1 h at room temperature. Western blots were probed with antibodies against p-AKT (Ser473, 1:1000, CST, USA) and total AKT (1:1000, CST, USA), both from Cell Signaling Technology), GAPDH (1:1000, CST, USA) at 4°C overnight. After washing, the blots were then incubated with the secondary antibody, goat anti-mouse (1:2000) and goat anti-rabbit (1:2000) (Boster Biological Technology co., ltd, USA) for 2 h at room temperature. Western blotting was performed at least three biological replicates.
8
Profiling of Inflammasome Pathway in HUVEC Cells
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Total protein was extracted from HUVECs using Cell lysis buffer for Western and IP [20mM Tris(pH7.5), 150mM NaCl, 1% Triton X-100, Beyotime, China]. The concentrations of protein were determined by a BCA protein assay kit (Vazime, China). 20 μg proteins were separated by BeyoGel™ Plus Precast PAGE Gel (Beyotime, China) and transferred onto PVDF membranes (Millipore, USA). After blocking with 5% dry milk, the membrane was incubated with primary antibodies against NLRP3 (1:1000, AdipoGen, USA), ASC (1:1000, AdipoGen, USA), Caspase-1 (1:1000, Abcam, UK), GSDMD (1:500, Santa, USA), and IL-1β (1:500, Santa Cruz, USA), AMPKα (1:1000, Cell Signaling Technology, USA), and Phospho-AMPKα (Thr172) (1:1000, Cell Signaling Technology, USA), β-actin (1:2000, Proteintech, China) overnight at 4°C. Following incubation, the membrane was washed thrice with TBST (0.05% Tween 20). After incubation with the corresponding secondary antibody [goat anti-rabbit (1:5000, BOSTER Biological Technology co. ltd, China) and goat anti-mouse (1:5000, BOSTER Biological Technology co. ltd, China)], the membrane was exposed to an enhanced chemiluminescence kit (Vazime, China), and observed using a Clinx ChemiScope 3500 (Clinx Science instrument Co. Ltd, China).
9
Investigating FAK and Src Signaling
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1-CP-U, generously provided by Ms. Qizhi Ning, was diluted to the required concentrations in conditional medium and then stored at 4°C according to the reported procedure [19 ]. Matrigel was purchased from Corning, USA (356234). The antibody to FAK (ab131435) and p-FAK Tyr397 (ab81298) were from Abcam, UK. p-Src Tyr416 antibody (6943) and paxillin antibody (12065) were purchased from Cell Signaling Technology, MA, USA. Anti-β-actin, goat anti-mouse and anti-rabbit secondary antibodies were from Boster Biological Technology (CA, USA).
10
Protein Expression Analysis in Cell Lysates
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After 1 week induction, cells were rinsed with pre-cooled PBS and lysed using radioimmunoprecipitation assay lysis buffer (RIPA, Boster, China) containing 1% phosphatase and protease inhibitors. The protein concentration of the lysate was determined using a BCA protein assay kit. Cell lysates (20 μg protein) were resolved onto SDS-polyacrylamide gels (8–10%) and transferred onto 0.45-μm polyvinylidene difluoride (PVDF) membranes (Millipore, USA). After blocked with 5% bovine serum albumin (BSA) in Tris-buffered Saline-Tween solution (TBST) for 1 h at room temperature, the blots were incubated with the specific primary antibodies (OPN, RUNX2, COLI, VEGFR2, vWF, CD31, WNT1, and LRP-6 at 1:1000 dilution, β-catenin at 1:2000 dilution, Abcam, UK; GAPDH at 1:500 dilution, Boster, China) at 4 °C overnight. The membranes were next incubated with the corresponding horseradish peroxidase-conjugated goat anti-rabbit (1:5000 dilution) or goat anti-mouse (1:5000 dilution) antibodies (Boster, China) at 37 °C for 1 h after rinsing with TBST. The blots were then performed with an enhanced chemiluminescence (ECL, Thermo Fisher Scientific, USA). Relative expression was quantified using the Image Lab system version 5.1 (Bio-Rad Laboratories, USA) and normalized to GAPDH.
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