Proteins were isolated from the right lung tissues by lung homogenate, followed by incubation with RIPA buffer. Aliquots of 35 μg of protein were loaded onto 10% sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). After blocking in 5% skim milk (Sigma Aldrich) in Tris buffered saline containing 0.05% Tween 20 (TBS-T) for 1 at room temperature, membranes were incubated with primary antibodies against OPN (R&D Systems), Smad3, phosphorylated Smad3 (pSmad3) (Abcam, Cambridge, UK), and TGM2 overnight at 4 °C with gentle shaking. Then, membranes were washed three times with TBS-T for 10 min each and incubated with horseradish peroxidase conjugated with anti-goat or anti-rabbit antibody for 1 h at room temperature. Anti-β-actin antibody (Thermo Fisher Scientific) was used as an internal control. Signals were detected using ECL Plus Western Blotting Detection Reagents (GE Healthcare, Little Chalfont, UK). The intensity of bands was analyzed using a gel doc system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Phosphorylated smad3 psmad3
Manufactured by Abcam
Sourced in United Kingdom, China
Phosphorylated Smad3 (pSmad3) is a protein that has been modified by the addition of a phosphate group. Smad3 is a key intracellular signaling molecule that mediates the transforming growth factor beta (TGF-β) signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Smad3, by Abcam (2 mentions)
Anti β actin antibody, by Thermo Fisher Scientific (1 mentions)
Ecl plus western blotting detection reagent, by GE Healthcare (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Skim milk, by Merck Group (1 mentions)
Gel doc system, by Bio-Rad (1 mentions)
α sma, by Abcam (1 mentions)
Activated pvdf membrane, by Merck Group (1 mentions)
Sds page, by Ipsen (1 mentions)
Collagen 1, by Abcam (1 mentions)
Protein free rapid blocking buffer, by Ipsen (1 mentions)
Ripa lysis buffer, by Wuhan Servicebio Technology (1 mentions)
β actin, by Proteintech (1 mentions)
Bca protein kit, by Thermo Fisher Scientific (1 mentions)
Fibronectin, by Proteintech (1 mentions)
Gapdh, by Proteintech (1 mentions)
Tgf β1, by Proteintech (1 mentions)
α smooth muscle actin α sma, by Merck Group (1 mentions)
Gapdh, by Bioworld Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
3 protocols using phosphorylated smad3 psmad3
1
Lung Tissue Protein Analysis
2
Western Blot Analysis of Lung Proteins
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As reported previously [55 (link)], proteins in the lung or cells were extracted by RIPA lysis buffer containing protease and phosphatase inhibitors (Servicebio, China), and the protein concentration was determined using the BCA protein kit (Thermo, United States). After denaturation, proteins were separated by SDS-PAGE (Epizyme, China) and blotted onto activated PVDF membranes (Millipore, United States). After blocking with protein free rapid blocking buffer (Epizyme, China) for 10 min, the membranes were incubated with primary antibodies against GAPDH (1:50000, Proteintech), β-actin (1:50000, Proteintech), CDA1 (1:2000, Proteintech), α-SMA (1:1000, Abcam), collagen I (1:1000, Abcam), fibronectin (1:1000, Proteintech), TGF-β1 (1:1000, Proteintech), Smad3 (1:2000, Abcam), phosphorylated Smad3 (P-Smad3) (1:2000, Abcam), GFP (1:2000, Proteintech) at 4 °C overnight. After washing, the membranes were incubated with the appropriate secondary antibodies and visualized using an ECL Assay Kit (Epizyme, China). GAPDH/β-actin served as an internal control. The intensities of the target bands were quantified using ImageJ software.
3
Western Blot Analysis of Kidney Tissue
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Kidney tissues were lysed and homogenized using the radioimmunoprecipitation assay lysis buffer (50 mM Tris-HCl, pH 8.0, 1% Triton-X 100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 1 M NaF plus protease inhibitor cocktail [Sigma-Aldrich] and phosphatase inhibitor cocktail [Sigma-Aldrich]). Extracted protein samples were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (GVS S.p.A.). After blocking with 5% bovine serum albumin or skim milk for 30 minutes, the membranes were incubated with antibodies against α-smooth muscle actin (α-SMA, 1:20,000; Sigma-Aldrich), phosphorylated SMAD3 (p-SMAD3, 1:2,000; Abcam), p-SMAD2 (1:2,000; Abcam), t-SMAD 2/3 (1:2,000; Abcam), 4-hydroxynonenal (4-HNE, 1:2,000; Abcam), Ly6G (1:2,000; Fisher Scientific), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:10,000; Bioworld Technology) overnight at 4 °C. The membranes were then treated for 1 hour at room temperature with horseradish peroxidase (HRP)-labeled goat anti-mouse immunoglobulin G (IgG, 1:3,000; Bethyl-Laboratories) or HRP-labeled goat anti-rabbit IgG (1:3,000; Bethyl-Laboratories). Total protein expression levels were normalized to GAPDH. Band intensities were analyzed using ImageJ software.
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