Anti smad4

Manufactured by Proteintech

Sourced in United States, China

Anti‐SMAD4 is a primary antibody that recognizes the SMAD4 protein. SMAD4 is a critical mediator of TGF-beta signaling and plays a role in regulating gene expression. This antibody can be used to detect and study the SMAD4 protein in various experimental applications.

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SMAD4 (13 citations) Anti-TM (2 citations)

3 protocols using anti smad4

Western Blot Analysis of Protein Signaling

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Cells or tissues were collected and lysed to prepare protein samples, which were separated by SDS‐PAGE and transferred to nitrocellulose (NC) membranes. NC membranes were blocked with 5% skimmed milk in Tween 20 (TBST) tris‐buffered saline for 1.5 h at room temperature and shaken overnight at 4°C with primary antibody. Subsequently, the NC membrane was incubated with the corresponding secondary antibody for 1 h at room temperature. Finally, chemiluminescence reagents (Cat. No: BL520B, Biosharp) were added to the NC membrane, which was developed in a gel imager (FluorChem M, Proteinsimple). Signals were analyzed by Image J software (National Institutes of Health).
The primary antibodies were anti‐SMAD2/3 (1:1000, Cell Signaling Technology), anti‐Phospho‐SMAD2/3 (1:1000, Cell Signaling Technology), anti‐α‐SMA(1:1000, Proteintech), anti‐Phospho‐AKT (1:1000, Cell Signaling Technology), anti‐AKT (1:1000, Cell Signaling Technology), anti‐Phospho‐p44/42 MAPK (ERK1/2) (1:1000, Cell Signaling Technology), anti‐p44/42 MAPK (ERK1/2) (1:1000, Cell Signaling Technology), anti‐SMAD4 (1:1000, Proteintech), anti‐SMAD7 (1:1000, Santa Cruz), anti‐FN (1:1000, Proteintech), and anti‐GAPDH, (1:5000, Proteintech).

Jin Z., Tian L., Zhang Y., Zhang X., Kang J., Dong H., Huang N., Pan L, & Ning B. (2022). Apigenin inhibits fibrous scar formation after acute spinal cord injury through TGFβ/SMADs signaling pathway. CNS Neuroscience & Therapeutics, 28(11), 1883-1894.

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Protein Extraction and Western Blot Analysis

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Total proteins were isolated by Total Extraction Kit (KGP2100; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) following the manufacturer's protocols. Protein concentration was determined by the BCA reagent kit (KGPBCA; Nanjing KeyGen Biotech Co., Ltd.). Equal amounts of protein (60 μg) were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes, which were blocked in 5% non-fat milk, and then incubated with the primary antibodies against anti-HIF-1α (1:1,000; ProteinTech Group, Inc.), anti-TGF-β (1:500; Abcam, USA), anti-Smad4 (1:1,000; ProteinTech Group, Inc.), anti-LOX (1:2,000; Abcam, USA), anti-E-cadherin (1:200; Cell Signaling Technology, Danvers, MA, USA) and anti-β-actin (1:1,000; Elabscience Biotechnology Co., Ltd.). The samples were incubated with the antibodies overnight at 4°C. Subsequently, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (1:6,000; OriGene, Beijing, China) at room temperature for 1 h. An enhanced chemiluminescence reagent (ECL kit, KGP1121; Nanjing KeyGen Biotech Co., Ltd.) was applied as a chromogenic substrate for 1 min, and then visualized with an Amersham Imager 600 instrument (GE Healthcare Life Sciences). Grayscale analysis was performed with ImageJ software.

Xu Y., Wang X., Huang Y., Ma Y., Jin X., Wang H, & Wang J. (2018). Inhibition of lysyl oxidase expression by dextran sulfate affects invasion and migration of gastric cancer cells. International Journal of Molecular Medicine, 42(5), 2737-2749.

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Porcine Granulosa Cell Protein Expression

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After treatment for the indicated time, porcine GCs were collected and western blotting assays were performed as we previously described [24] . In brief, the total protein from porcine GCs were extracted by ice-cold RIPA lysis buffer with 1% PMSF, and the concentration of total protein were measured by BCA method. Then, equal amount (~15 μg) of total protein was separated on an 4-20% SDS-PAGE gel and subsequently transferred into PVDF membrane (Merck Millipore, Germany) after electrophoresis. After incubation with 5% non-fat milk at room termperature for 90 min, the membranes were incubated with primary antibodies at 4°C overnight and then with corresponding secondary antibodies for 1 h. The images were obtained after incubation with ECL reagent. The primary antibodies used here were as follows: anti-SMAD4 (1:1000, #10231-1-AP, Proteintech, Jiangsu, China), anti-ACVR1B (1:1000, #D120045, Sangon Biotech, Shanghai, China), anti-BMPR2 (1:1000, #D221406, Sangon Biotech, Shanghai, China), anti-TGFBR2 (1:800, #sc-400, Santa Cruz, USA), anti-CREB1 (1:1000, #9197, Cell Signaling Technology, USA), anti-c-JUN (1:2000, #9165, Cell Signaling Technology, USA), anti-SP1 (1:1000, #2250, Cell Signaling Technology, USA) and anti-GAPDH (1:3000, #TA802519, ORIGENE, Jiangsu, China).

Du X., Li Q., Liu Y., & Li Q. (2021). SMAD4 Feedback Activates The Canonical TGF-β Family Signaling Pathways.

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