GSCs were subjected to lysis in radio-immunoprecipitation assay lysis buffer (Cell Signaling Technology) containing proteinase (Sigma-Aldrich) and a phosphatase cocktail (Thermo Fisher Scientific). The protein concentration in each supernatant was determined by a bicinchoninic acid protein assay (Bio-Rad). Samples were subjected to sodium dodecyl sulfate–polyacrylamide gel separation, and the separated proteins were electrophoretically transferred to polyvinylidene fluoride membranes. Blots were incubated with the primary antibody overnight at 4°C and incubated with horseradish peroxidase–linked secondary anti-rabbit or anti-mouse antibody (Bio-Rad). Antibodies against signal transducer and activator of transcription 3 (STAT3; cat. #9139), phosphorylated STAT3 (Tyr705; cat. #9135), caveolin-1 (cat. #3238), N-cadherin (cat. #4061), SMAD2 (cat. #5339), SMAD3 (cat. #9523), phosphorylated SMAD2 (cat. #3108), and phosphorylated SMAD3 (cat. #9520) were purchased from Cell Signaling Technology. Other antibodies used for Western blotting were POSTN (cat. #AP11962b; Abgent); HIF1 alpha (cat. #610958; BD Biosciences); integrin β1 (cat. #18887), integrin β3 (cat. #6627), and GAPDH (cat. #32233; all, Santa Cruz Biotechnology); and α-tubulin (cat. #T9026; Sigma-Aldrich).
Horseradish peroxidase linked secondary anti rabbit or anti mouse antibody
Manufactured by Bio-Rad
Horseradish peroxidase–linked secondary anti-rabbit or anti-mouse antibody is a laboratory reagent used in immunoassays and immunoblotting techniques. It functions as a detection system by binding to primary antibodies raised against rabbit or mouse antigens, enabling the visualization of target proteins or molecules.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence, by Cytiva (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Bicinchoninic acid protein assay, by Bio-Rad (1 mentions)
β1 integrin, by BD (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Cell Signaling Technology (1 mentions)
α tubulin, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Laminin b, by Abcam (1 mentions)
Bca protein assay, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Cell Signaling Technology (1 mentions)
Icam 1, by Cell Signaling Technology (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
6 protocols using horseradish peroxidase linked secondary anti rabbit or anti mouse antibody
1
Western Blot Analysis of Cell Signaling Proteins
2
Western Blot Analysis of NDC80 Protein
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Cells were lysed, and the protein in the supernatant extracts was quantified using a BCA protein assay kit (Beyotime Institute of Biotechnology). Fifty micrograms per lane of total cell lysates were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, United States). Membranes were incubated with the primary antibody overnight at 4°C. The next day, the membranes were incubated with a horseradish peroxidase–linked secondary anti-rabbit or anti-mouse antibody (Bio-Rad). Immunoreactivity was detected using enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ, United States) with a Chemidoc imaging system and Quantity One software (Bio-Rad). A densitometric analysis was performed by using the Quantity One software. β-actin was used as a loading control. NDC80 antibodies were purchased from Abcam, United States.
3
Western Blot Analysis of NDC80 Protein
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Cells were lysed and protein in supernatant extracts was quanti ed using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology). Fifty micrograms per lane of total cell lysates were resolved on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gels and transferred onto polyvinylidene uoride (PVDF) membranes (Millipore, Billerica, MA, USA). Blots were incubated with the primary antibody overnight at 4°C. Furthermore, the blots were incubated with horseradish peroxidaselinked secondary anti-rabbit or anti-mouse antibody (Bio-Rad). Immunoreactivity was detected using the enhanced chemiluminescence (Amersham Biosciences, Piscat-away, NJ, USA), the Chemidoc and Quantity One software (Bio-Rad). Densitometric analysis was performed by using Quantity One software.
β-actin was used as a loading control. NDC80 antibodies were purchased from the US Abcam company.
β-actin was used as a loading control. NDC80 antibodies were purchased from the US Abcam company.
4
Western Blot Analysis of Cellular Proteins
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Cells were lysed in an ice-cold lysis buffer containing 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1% TritonX-100, 1 mM PMSF, 1 μg/ml leupeptin, and 1 μg/ml pepstain A. The protein concentration in the supernatant was determined using a BCA protein assay (Pierce, Rockford, IL, USA). Samples were subjected to 8-12% SDS-polyacrylamide gel electrophoresis, and the separated proteins were electrophoretically transferred to nitrocellulose membranes. Blots were incubated with the primary antibody against IRF1 (1: 1000, Santa Cruz Biotechnology), LC3B (1:1000, CST), Bnip3 (1:1000, CST), Tubulin (1:3000, Sigma), AIF (1: 1000, CST), coxIV (1: 1000, Calbiocam), Laminin B (1: 1000, Abcam). The membranes were then incubated with horseradish peroxidase-linked secondary anti-rabbit or anti-mouse antibodies (Bio-Rad).
5
Protein Expression Analysis in GSCs
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GSCs were lysed using RIPA lysis buffer (Cell Signaling Technology) with proteinase (Sigma-Aldrich) and a phosphatase cocktail (Thermo Fisher Scientific). The protein concentration in the supernatant was measured using a BCA protein assay (Bio-Rad). Samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the separated proteins were electrophoretically transferred to polyvinylidene fluoride membranes. Blots were incubated with a primary antibody overnight at 4°C and incubated with horseradish peroxidase-linked secondary anti-rabbit or anti-mouse antibodies (Bio-Rad). Antibodies against phosphorylated AKT (cat. #4060), AKT (cat. #9272), phosphorylated p38 (cat. #4511), p38 (cat. #9212), phosphorylated ERK (cat. #9101), ERK (cat. #9102), phosphorylated pyruvate dehydrogenase lipoamide kinase isozyme 1 (PDK1; cat. #3061), PDK1 (cat. #3062), p27 (cat. #13715), phosphorylated FOXO3a (cat. #9465), and FOXO3a (cat. #2497) were purchased from Cell Signaling Technology. CLK2 (cat.#HPA055366) and α-Tubulin (cat. #T9026) were purchased from Sigma-Aldrich. The results of three independent Western blot experiments performed in triplicate were reported.
6
Western Blot Analysis of Cell Signaling
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Cells were lysed in an ice-cold lysis buffer containing 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1% TritonX-100, 1 mM PMSF, 1 μg/ml leupeptin, and 1 μg/ml pepstain A. The protein concentration in the supernatant was determined using a BCA protein assay (Pierce, Rockford, IL, USA). Samples were subjected to 8–12% SDS-polyacrylamide gel electrophoresis, and the separated proteins were electrophoretically transferred to nitrocellulose membranes. Blots were incubated with the primary antibody against ICAM-1 (1:1000; Cell signaling Technology, Danvers, MA), GFP (1:1000, CST), p-STAT3 (1:1000, Cell signaling), Tubulin (1:3000; Sigma), p-AKT (1: 1000, Cell signaling Technology, Danvers, MA), AKT (1: 1000, Cell signaling). The membranes were then incubated with horseradish peroxidase-linked secondary anti-rabbit or anti-mouse antibodies (Bio-Rad).
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