Total protein was extracted with RIPA buffer supplemented with protease inhibitors. Protein extracts were resolved on 10% SDS–PAGE gels, transferred onto PVDF membranes (Millipore, Billerica, MA, USA), as descripted previously [14 (link)]. The primary antibodies anti-rabbit Smad4 (Cell Signaling, Danvers, MA, USA) or GAPDH (Kang Cheng Biotechnology, Shanghai, China) were applied overnight. The dilutions of the primary antibodies were 1:1000 for anti-SMAD4 and 1:10000 for anti-GAPDH. The following day, the membranes were incubated with HRP-conjugated anti-rabbit secondary antibodies (Kang Cheng Biotechnology, Shanghai, China) at room temperature for 60 minutes. A chemiluminescence detection system (Millipore, Billerica, MA, USA) was used for visualization of the results.
Rabbit anti smad4
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, China
Rabbit anti-SMAD4 is a primary antibody that specifically recognizes the SMAD4 protein. SMAD4 is a key mediator in the transforming growth factor-beta (TGF-β) signaling pathway, which plays a crucial role in various cellular processes such as cell growth, differentiation, and apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
Chemiluminescence detection system, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti mouse igg hrp, by R&D Systems (1 mentions)
Mouse anti α tubulin, by Merck Group (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Western lightning plus ecl, by PerkinElmer (1 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Rabbit anti erk, by Cell Signaling Technology (1 mentions)
Rabbit anti p p38, by Cell Signaling Technology (1 mentions)
Rabbit anti p38, by Cell Signaling Technology (1 mentions)
Rabbit anti p erk, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase coupled anti rabbit immunoglobulin, by Agilent Technologies (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Bca assay, by Bio-Rad (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Gapdh, by Abcam (1 mentions)
Phostop, by Roche (1 mentions)
Rabbit anti p smad2, by Cell Signaling Technology (1 mentions)
Mouse anti fibronectin, by Merck Group (1 mentions)
6 protocols using rabbit anti smad4
1
Western Blot Analysis of Smad4 and GAPDH
2
Western Blot Analysis of Smad Proteins
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Cells were lysed with RIPA buffer supplemented with protease inhibitor cocktail (Sigma). Denaturing sample buffer was added to protein samples, and samples were boiled and resolved on Tris–HCl polyacrylamide gels. Resolved proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes (GE Healthcare) and probed with the following primary antibodies: anti-rabbit Smad2 and anti-rabbit Smad4 (Cell Signaling Technology, Inc.), and anti-mouse α-Tubulin (Sigma). The following secondary antibodies were used: goat anti-rabbit IgG-HRP (Santa Cruz) and anti-mouse IgG-HRP (R&D Systems). The western blot was visualized by enhanced chemiluminescence (Western Lightning-ECL Plus, PerkinElmer) and exposed to film. Densitometric measurements were performed with ImageJ software (http://rsb.info.nih.gov/ij/ ).
3
Western Blot Analysis of Macrophages and DRGs
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Macrophages (1 × 106 cells) and L3-L4-L5 DRGs were lysed in RIPA buffer (Sigma-Aldrich) supplemented with antiphosphatase (Phostop, Roche) and protease inhibitor (Roche). Protein concentration was determined by BCA assay (Bio-Rad) prior to denaturation. Samples were loaded into 10% SDS-PAGE gels and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were probed with the following primary antibodies: rabbit anti-SMAD4 (1:1000; Cell Signaling Technology, 46535), rabbit anti–p-ERK (1:1000; Cell Signaling Technology, 9101), rabbit anti–p-p38 (1:1000; Cell Signaling Technology, 9211), rabbit anti-ERK (1:1000; Cell Signaling Technology, 4695), rabbit anti-p38 (1:1000; Cell Signaling Technology, 9212), and rabbit anti-CCL2 (1:1000; Invitrogen, MAS-17040). GAPDH (1:2000; Abcam, ab8245) and β-actin (1:1000; Cell Signaling Technology, 4967) were used as loading controls. Results were visualized with horseradish peroxidase–coupled anti–rabbit immunoglobulin (Dako, Agilent) using enhanced chemiluminescence detection reagents. Protein abundances were analyzed by densitometry scanning using Fiji (ImageJ 1.52i, NIH)
4
Immunoblotting for Protein Detection
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Standard immunoblotting procedures51 (link) were used to detect LMP1 (with mouse CS1-4 at 1:5), activin A (with mouse anti-inhibin βA at 1:50 [Serotec, Oxfordshire, UK]), β-actin (with mouse anti-β-actin at 1:20,000 [Sigma]), fibronectin (with mouse anti-fibronectin at 1:100 [Sigma]), phosphorylated Smad2 Ser465/Ser467 (with rabbit anti-pSmad2 at 1:250 [Cell Signaling Technology, Hitchin, UK]), Smad2 (with rabbit anti-Smad2 at 1:250 [Cell Signaling Technology]), Smad4 (with rabbit anti-Smad4 at 1:250 [Cell Signaling Technology]), phosphorylated JNK/SAPK (with rabbit anti-pJNK/SAPK at 1:500 [Cell Signaling Technology]), JNK/SAPK (with rabbit anti-JNK/SAPK at 1:500 [Cell Signaling Technology]).
5
Quantitative Western Blot Analysis
6
Protein Extraction and Western Blot Analysis
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Total proteins were extracted using an SDS Lysis Buffer (Beyotime Biotechnology) supplemented with 1% phenylmethanesulfonylfluoride fluoride (Beyotime Biotechnology) and 1% phosphatase inhibitor (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). To separate nuclear and cytoplasmic proteins, the Minute™ SC-003 kit (Invent Biotechnologies, Inc., Beijing, China) was used according to the manufacturer’s instruction. Proteins were run on 10% SDS-PAGE and transferred into a PVDF membrane, followed by incubation with a primary antibody overnight. The following primary antibodies were used: rabbit anti-CROT (1:1000 dilution), rabbit anti-LaminB1 (1:2000 dilution), rabbit anti-Bcl-2 (1:2000 dilution), and mouse anti-GAPDH (1:5000 dilution) from Proteintech Group, Inc (Wuhan, China) and rabbit anti-Bax (1:2000 dilution), rabbit anti-Smad4 (1:2000 dilution), mouse anti-Smad2 (1:2000 dilution), rabbit anti-phospho-Smad2 (1:2000 dilution), and mouse anti-β-actin (1:5000 dilution) from Cell Signaling Technology, Inc. (Danvers, MA, USA). Secondary antibodies were horseradish peroxidase-conjugated goat anti-rabbit IgG and anti-mouse IgG (1:10,000 dilution, Proteintech). Signals were detected using BeyoECL Moon (Beyotime) and quantified using ImageJ software.
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