Ab272332

Manufactured by Abcam

Ab272332 is a laboratory equipment product. It is designed for use in scientific research and experiments. The core function of this product is to facilitate specific tasks or processes within a laboratory setting. Further details about the intended use or capabilities of this product are not available.

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Lab products found in correlation

6 protocols using ab272332

Western Blot Analysis of Cardiac Fibrosis Markers

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Total proteins were extracted from frozen LV tissue and cultured PVFs using RIPA lysis buffer. Proteins were quantified using a BCA Protein Assay Kit (Thermo Fisher Scientific, USA). All sample proteins loaded equally (20 μg) were separated by SDS-PAGE and transferred to PVDF membranes. Subsequently, the PVDF membrane was blocked with TBST containing 5% BSA for 1 h and incubated at 4°C overnight with a primary antibody. Then, the membrane was washed with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h. The primary antibodies used were as follows: GAPDH (1 : 5000, 60004-1-Ig, Proteintech), CTGF (1 : 1000, sc-365970, Santa Cruz), α-SMA (1 : 1000, ab7817, Abcam), Col 1 (1 : 1000, ab34710, Abcam), Col 3 (1 : 1000, ab7778, Abcam), TGF-β1 (1 : 1000, ab215715, Abcam), TGFR-2 (1 : 1000, ab269279, Abcam), Smad2 (1 : 1000, ab33875, Abcam), Smad3 (1 : 1000, ab40854, Abcam), and phospho-Smad2/3 (1 : 1000, ab272332, Abcam). Immunoblots were quantified using a Tanon 5200 Multi Chemiluminescent Imaging System (Tanon Science & Technology Co., Ltd., Shanghai, China).

Shao S., Zhang Y., Gong M., Yang Q., Yuan M., Yuan M., Suo Y., Wang X., Li Y., Bao Q, & Li G. (2021). Ivabradine Ameliorates Cardiac Function in Heart Failure with Preserved and Reduced Ejection Fraction via Upregulation of miR-133a. Oxidative Medicine and Cellular Longevity, 2021, 1257283.

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Whole-Cell Lysis and Protein Analysis

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Whole-cell lysis assay (KeyGEN) was applied for extracting protein from SMSCs and BCA protein assay kit (Biyuntime, China) assessed protein concentration. Protein (20 μg) is transferred to a PVDF membrane by SDS-polyacrylamide gel electrophoresis. Primer antibody including anti-Runx2 (Abcam, 1:1 000, ab23981), anti-PTH1R (Abcam, 1:1 000, ab75150), anti-pCREB (Abcam, 1:1 000, ab32096), anti-CREB (Abcam, 1:1 000, ab32515), anti-pSmad2/3 (Abcam, 1:1 000, ab272332), anti-Smad2/3 (Abcam, 1:1 000, ab217553), and anti-β-actin (Santa Cruz Biotechnology, 1:1 000, CA) antibodies were incubated overnight at 4 °C. Next day, the secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, 1/5 000) was incubated in room temperature 1 h. ChemiDoc XRS + system (BD, Franklin Lakes, NJ) was used to detect the immunoreactive bands.

Zhang J., Pi C., Cui C., Zhou Y., Liu B., Liu J., Xu X., Zhou X, & Zheng L. (2022). PTHrP promotes subchondral bone formation in TMJ-OA. International Journal of Oral Science, 14, 37.

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Western Blot Analysis of Neurological Markers

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Total protein was extracted from tissues and cells using RIPA lysis buffer (R0010, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) supplemented with 1% phosphorylase inhibitor and 1% protease inhibitor, with the concentration determined using a BCA protein assay kit (GBCBIO Technologies Inc., Guangzhou, China). Next, the protein was separated with 10% SDS‐PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA). The membrane was blocked with TBST with 5% BSA at room temperature and probed overnight at 4°C with primary rabbit antibodies to TREM2 (1:1000, #61788, Cell Signaling Technologies, Beverly, MA), TGF‐β1 (1:800, ab215715, Abcam), Smad2/3 (1:500, ab236030, Abcam), p‐Smad2/3 (1:1500, ab272332, Abcam), DAT (1:200, ab128848, Abcam), TH (1:5000, ab137869, Abcam), and GAPDH (ab9485, 1:2500, Abcam) and then with secondary antibody goat anti‐rabbit IgG (1:10,000, ab97051, Abcam) at room temperature. ECL reagent was used to visualize the results by Image Quant LAS 4000C Gel Imager (GE Healthcare, Waukesha, WI). ImageJ software (National Institutes of Health) was applied for band intensity quantification, with GAPDH as an internal reference.

Liang H., Liu P., Wang Z., Xiong H., Yin C., Zhao D., Wu C, & Chen L. (2024). TREM2 gene induces differentiation of induced pluripotent stem cells into dopaminergic neurons and promotes neuronal repair via TGF‐β activation in 6‐OHDA‐lesioned mouse model of Parkinson's disease. CNS Neuroscience & Therapeutics, 30(2), e14630.

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Western Blot Analysis of Protein Expression

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The extraction of total protein was finished through the usage of RIPA buffer (Beyotime) and quantified through the usage of BCA Protein Quantification Kit (Vazyme). Then the proteins were subjected to sodium dodecyl sulfonatepolyacrylamide gel (Solarbio) electrophoresis and blotted onto polyvinylidene difluoride membranes (Amersham Biosciences, Chicago, IL, USA). Next, the membranes were blocked in 5% defatted milk for 1 h at indoor temperature and then cultivated with primary antibodies against RNF38 (ab121487; Abcam), Ki67 (ab16667; Abcam), Snail (ab53519; Abcam), E-cadherin (ab231303; Abcam), Smad2/3 (ab202445; Abcam), phospho-Smad2/3 (p-Smad2/3; ab272332; Abcam) and GAPDH (ab9485; Abcam) overnight at 4°C and secondary antibody (ab6789; Abcam) for 1.5 h at indoor temperature. The proteins were visualized with ECL reagent (Vazyme).

Hu X., Zhou K., Cao S., Zhu H, & Xie K. (2021). circ_LRIG3 contributes to the progression of hepatocellular carcinoma by elevating RNF38 via sponging miR-449a. General physiology and biophysics, 40(2).

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Quantifying TGF-β1 Signaling Pathway

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Totally 1 mM PMSF (P0100, Solarbio) was added to RIPA protein lysate (P0013C, Beyotime), and 100 μl mixed solution was added to each well of six-well plate for lysis. After 30 min, cell lysate was collected and centrifuged at 15000 g at 4 °C for 10 min to extract total protein. The total protein concentration was detected by BCA protein quantitative kit (P0012, Beyotime). After SDS-PAGE gel electrophoresis, the protein was transferred to PVDF membrane (88585, Thermo Scientific) by semi-wet transfer method. The 5% skimmed milk powder was incubated for 1 h, then TGF-β1 (1:1000, ab92486, Abcam), Smad7 (1: 1000, ab216428, Abcam), Smad2/3 (1:1000, ab217553, Abcam), p-Smad2/3 (1:1000, ab272332, Abcam) and GAPDH (1:10000, ab181602, Abcam) Primary Antibody Dilution Buffer was added and cultivated at 4 °C all night. The sample was washed three times with 0.1% PBST, and then the corresponding secondary antibody diluent (1:4000, ab205718, abcam) was added and incubated for 1 h at indoor temperature. Subsequently, it was cleaned three times with 0.1% PBST again, developed with ECL chemiluminescence reagent (P0018FS, Beyotime), fixed and photographed by chemiluminescence imaging system (BioSpectrum 510, UVP), and analyzed by ImageJ 1.52.

Kang Y., Bai M., Deng L., Fan L, & Wang X. (2021). MiRNA-21 Regulates Bronchial Epithelial Cell Proliferation by Activating Tgfβ1/Smad Signaling Pathway and Its Correlation with Asthma Severity in Children. Iranian Journal of Public Health, 50(10), 1973-1982.

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TGF-β Signaling Modulation in 3T3 Cells

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3T3 cells were incubated in a 6-well plate at a density of 1 × 10⁵ cells per well and treated overnight with TGF-β (20 ng/ml), TGF-β (20 ng/ml) plus SB-431542 (MCE, USA), a kind of TGF-β inhibitor, different 4T1 cultured medium after treatment with PBS (4T1-CM), DTX (4T1@DTX), C-Lp/DTX (4T1@C-Lp/DTX), Nanoxel-PM (4T1@Nanoxel-PM), Rg3 (4T1@Rg3), Rg3-Lp (4T1@Rg3-Lp), Rg3/DTX (4T1@Rg3/DTX) or Rg3-Lp/DTX (4T1@Rg3-Lp/DTX) (DTX 0.5 μg/mL). The protein of the cells was then harvested according to the procedures described previously [32 (link)]. 50 mg of protein per lane were loaded on the polyacrylamide gel and then transferred onto a PVDF membrane. The PVDF membrane was incubated with anti-α-SMA (ab124964, Abcam), anti-phospho-Smad2/3 (ab272332, Abcam) and anti-GAPDH (30202ES40, Yeasen) overnight at 4℃ respectively. The following procedure was performed as previously described [32 (link)].

Xia J., Zhang S., Zhang R., Wang A., Zhu Y., Dong M., Ma S., Hong C., Liu S., Wang D, & Wang J. (2022). Targeting therapy and tumor microenvironment remodeling of triple-negative breast cancer by ginsenoside Rg3 based liposomes. Journal of Nanobiotechnology, 20, 414.

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