Ab94483

Manufactured by Abcam

Sourced in United Kingdom

Ab94483 is a laboratory equipment product. It is a core product designed to perform a specific function in the laboratory setting. No further details are provided to maintain an unbiased and factual description.

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8 protocols using ab94483

Immunostaining for Angiogenesis and Cellular Markers

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For IF staining, sections were deparaffinized and hydrated, followed by antigen retrieval in citrate buffer. After quenching with 3% H₂O₂/H₂O and blocking in goat serum, the sections were incubated with the primary antibodies against CD105 (ab231774; Abcam, Cambridge, UK), CD31 (3528; CST), GFP (2956; CST), Smurf2 (ab94483; Abcam), and PTX3 (373951; Santa Cruz Bio) overnight at 4°C and then incubated with the respective secondary antibodies (4413, 4408, 4409, 4412; CST) at room temperature for 1 hour in the dark. Images of IF staining were acquired with a fluorescence microscope (Leica DM6B, Germany). The vessel density was quantified from at least three random visual fields per section using the Image‐Pro version 6.0 software.
Both HDMSCs and ASMSCs were fixed with 4% paraformaldehyde, blocked with 5% normal goat serum, incubated with primary antibodies against Smurf2 (ab94483; Abcam) and PTX3 (373951; Santa Cruz Bio) overnight at 4°C, and then incubated with the appropriate secondary antibodies and stained with DAPI to detect the colocalization of Smurf2 and PTX3. Images were obtained using an LSM 5 Exciter confocal imaging system (Carl Zeiss, Germany). Positively stained cells were quantified from at least three random visual fields per section using the Image‐Pro version 6.0 software.

Ma M., Yang W., Cai Z., Wang P., Li H., Mi R., Jiang Y., Xie Z., Sui P., Wu Y, & Shen H. (2021). SMAD‐specific E3 ubiquitin ligase 2 promotes angiogenesis by facilitating PTX3 degradation in MSCs from patients with ankylosing spondylitis. Stem Cells (Dayton, Ohio), 39(5), 581-599.

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Immunohistochemical Analysis of Mouse Brain

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Paraffin sections of mouse brain tissues were routinely dewaxed with gradient alcohol and hydrated, followed by antigen microwave thermal retrieval and blocking of endogenous peroxidase with 3% hydrogen peroxide. Then, overnight section incubation was implemented with rabbit polyclonal antibodies (Abcam) to Smurf2 (ab94483; 1:200), YY1 (ab109237; 1:100), HIF1α (ab228649; 1:100), and DDIT4 (ab106356; 1:200) at 4 °C, followed by 30-min incubation with horseradish peroxidase–labeled secondary goat anti-rabbit immunoglobulin G (IgG) antibody (ab6721: 1:10,000; Abcam) at room temperature. Subsequent to development with diaminobenzidine, hematoxylin counterstaining, and mounting, the number of positive cells was counted in five random high-power fields (×400) from each group. Positive cell rate = number of brown positive cells/total number of cells × 100%.

Liu H., Sun S, & Liu B. (2021). Smurf2 exerts neuroprotective effects on cerebral ischemic injury. The Journal of Biological Chemistry, 297(2), 100537.

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Western Blotting and Immunofluorescence of Cell Markers

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Western blot analysis was performed as previously described.⁵⁹ (link) The primary antibodies used were anti-GFP (50430-2-AP, 1:1,000, Proteintech), anti-FLAG (AF519, 1:1,000, Beyotime), anti-MYOD (ABP53067, 1:500, Abbkine), anti-MyHC (B103, 0.5 μg/mL, DHSB), anti-SMURF2 (ab94483, 1 μg/mL, Abcam), anti-SMAD2/3 (70R-51804, 1:500, Fitzgerald), anti-phosphorylated (phospho-)SMAD2 (bs-3419R, 1:500, Bioss), anti-phospho-SMAD3 (bs-3425R, 1:500, Bioss), anti-β-actin (bsm-33036M, 1:1,000, Bioss), and anti-GAPDH (60004-1-Ig, 1:5,000, Proteintech). ProteinFind goat anti-mouse immunoglobulin G (IgG) (H+L), horseradish peroxidase (HRP) conjugate (HS201-01, 1:1,000, TransGen Biotech, Beijing, China) and ProteinFind goat anti-rabbit IgG (H+L), HRP conjugate (HS101-01, 1:500, TransGen Biotech) were used as a secondary antibody.
The immunofluorescence was performed using anti-FLAG (AF519, 1:1,000, Beyotime) or anti-MyHC (B103, 2.5 μg/mL, DHSB). Images were obtained with a fluorescence microscope (DMi8; Leica, Germany). The area of cells labeled with anti-MyHC was measured and calculated as previously described.⁵⁹ (link)

Cai B., Li Z., Ma M., Zhang J., Kong S., Abdalla B.A., Xu H., Jebessa E., Zhang X., Lawal R.A, & Nie Q. (2020). Long noncoding RNA SMUL suppresses SMURF2 production-mediated muscle atrophy via nonsense-mediated mRNA decay. Molecular Therapy. Nucleic Acids, 23, 512-526.

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Immunofluorescence Staining of TRAF4 and Smurf2

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MSCs were seeded on sterile glass coverslips. When cells reached appropriate confluence, the growth medium was aspirated. Cells were washed 3 times with PBS before fixing in 4% paraformaldehyde for 30 min. To permeabilize the cells, 0.1% Triton X-100 was added for 15 min at room temperature, and 5% normal goat serum was used to block cells for 30 min. A primary antibody against TRAF4 (1:50, Abcam, ab88612) or Smurf2 (1:200, Abcam, ab94483) was added, and the cells were incubated overnight at 4 °C. After the cells were washed 3 times with PBS, anti-rabbit IgG (1:500, Cell Signaling Technology, 4413) and anti-mouse IgG (1:500, Cell Signaling Technology, 4408) were added, and the cells were incubated for another 1 h at room temperature. 4′,6-diamidino-2-phenylindole (DAPI) was used to counterstain the nuclei. Thereafter, the coverslips were mounted on glass slides with anti-fade mounting medium (Beyotime, P0126). Thereafter, the samples were viewed under a laser scanning confocal microscope at wavelengths of 488 nm (green, TRAF4), 555 nm (red, Smurf2), and 405 nm (blue, DAPI). Images were obtained using an LSM 5 Exciter confocal imaging system (Carl Zeiss).

Li J., Wang P., Xie Z., Wang S., Cen S., Li M., Liu W., Tang S., Ye G., Zheng G., Su H., Ma M., Wu X., Wu Y, & Shen H. (2019). TRAF4 positively regulates the osteogenic differentiation of mesenchymal stem cells by acting as an E3 ubiquitin ligase to degrade Smurf2. Cell Death and Differentiation, 26(12), 2652-2666.

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SMURF2 Protein Expression Analysis

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RIPA lysis buffer (Thermo Fisher Scientific) was used for isolating total protein. After measurement of protein concentration, approximately 40 μg of extracted protein was separated by SDS-PAGE, and then transferred (semi-dry method) onto PVDF membrane. The membranes were blocked and then probed with primary antibodies against SMURF2 (ab94483, 1:1000, Abcam, Cambridge, UK) or GAPDH (ab37168, 1:1000, Abcam) at 4°Cfor 12–14 h. After incubation with secondary antibody (D110058, 1:4000, Sangon Biotech, Shanghai, China), the combined signals were visualized using ECL reagent (Tanon, Shanghai, China). The band density was assessed by ImageJ software.

Wang X, & Chen J. (2021). Long non-coding RNA TUG1 promotes proliferation and migration in PDGF-BB-stimulated HASMCs by regulating miR-216a-3p/SMURF2 axis. BMC Molecular and Cell Biology, 22, 56.

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Immunofluorescence Assay for Smurf2 Protein

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Neurons were prepared into cell slides, which were fixed with 4% paraformaldehyde for 30 min at room temperature. Cells were treated with 0.2% TritonX-100 for 15 min at room temperature and blocked with 3% bovine serum albumin for 30 min at 4 °C, followed by overnight culture with fluorescent primary rabbit anti-Smurf2 antibody (ab94483; 1:200; Abcam) in a wet box at 4 °C. Then, a 2-h cell slide culture was conducted with fluorescent goat anti-rabbit secondary antibody (ab150080; 1:500) or goat antimouse secondary antibody (ab150113; 1:200) at room temperature in the dark. The cell slides were cultured with 4'-6-diamidino-2-phenylindole (ab104139; 1:100; Abcam) in dark at room temperature for 10 min and mounted, followed by observing the results under an inverted microscope.

Liu H., Sun S, & Liu B. (2021). Smurf2 exerts neuroprotective effects on cerebral ischemic injury. The Journal of Biological Chemistry, 297(2), 100537.

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Detecting SMAD2 Ubiquitination in HFSCs

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IP assay was performed for detecting SMAD2 ubiquitination level. In brief, HFSCs were lysed with lysis buffer (P0013, Beyotime) on ice for 30 min. The cell lysate was collected into a 1.5 mL microtube and centrifuged at 12,000 g at 4 °C for 15 min. Protein A and protein G beads (50 µL each) were then mixed in a 1.5 mL micro-tube. Protein A + G agarose beads (10 µL) and Smurf antibody (ab94483, Abcam) were incubated with the cell lysate overnight at 4 °C. After IP reaction, the mixture was centrifuged at 3000 rpm at 4 °C for 3 min. Agarose beads were carefully rinsed with 1 mL of lysis buffer. The immunoprecipitants were subsequently boiled with 2× SDS loading buffer (15 µL) for 5 min. Changes in SMAD2 ubiquitination levels were then determined using western blot assay.

Lin B., Huang D., Lin G., Miao Y., Wang J., Fan Z, & Hu Z. (2022). Smurf2-induced degradation of SMAD2 causes inhibition of hair follicle stem cell differentiation. Cell Death Discovery, 8, 160.

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Investigating SIX3 Protein Regulation via Co-Immunoprecipitation

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The details of these procedures were described previously [37 (link)]. Anti-SIX3 (Santa Cruz Biotechnology, Dallas, TX, USA; sc-81985), anti-FLAG (Abcam, Cambridge, MA, USA; ab205606), anti-HA (Abcam; ab9110), and anti-IgG (Santa Cruz Biotechnology; sc-2027) antibodies were used for co-immunoprecipitation (Co-IP). Immunoprecipitates were washed at least five times and subjected to western blot analysis using anti-TRIM27 (Abcam; ab78393), anti-NEDD4 (Abcam; ab236512), anti-SMURF2 (Abcam; ab94483), anti-RNF6 (Abcam; ab204506), anti-SYVN1 (Abcam; ab170901), anti-MDM2 (Abcam; ab16895), and Anti-SIX3 (Abcam; ab172131) antibodies. For ubiquitination assays, the lysates of A549 cells transfected with siTRIM27-1 or siNC were used for IP with an anti-IgG (Santa Cruz Biotechnology; sc-2027) or Anti-SIX3 antibody (Santa Cruz Biotechnology; sc-81985) and Protein A/G PLUS-Agarose (Novex, Oslo, Norway), which was performed at 4° C overnight. The eluted proteins were then detected by western blot analysis using an anti-ubiquitin (Ub) antibody (Abcam, ab7780).

Liu S., Tian Y., Zheng Y., Cheng Y., Zhang D., Jiang J, & Li S. (2020). TRIM27 acts as an oncogene and regulates cell proliferation and metastasis in non-small cell lung cancer through SIX3-β-catenin signaling. Aging (Albany NY), 12(24), 25564-25580.

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