Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris–HCl pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with 1 × protease inhibitor cocktail (Complete Mini, Roche), 0.2 mM sodium orthovanadate, 20 mM sodium fluoride, and 1 mM phenylmethylsulfonyl fluoride (SIGMA); 25 μg of protein lysate was loaded onto 8% SDS PAGE gels. Immunoblots were performed with antibodies against ITG11A1 (Abnova, 1:700), COL11A1 (Abcam 1:1,000), MMP13 (Thermo Scientific, 1:500), INHBA (Abcam, 1:500), SULF1 (Abcam, 1:500), EPYC (SIGMA, 1:2,000), TNFSF4 (BioLegend, 1:300) and ERK2 (Santa Cruz Biotechnology, 1:2,000) overnight at 4°C.
Inhba
Manufactured by Abcam
Sourced in United States
INHBA is a protein-coding gene that provides instructions for the production of the inhibin beta A subunit. This subunit is a component of the inhibin and activin protein complexes, which play important roles in regulating the reproductive system and other physiological processes.
Automatically generated - may contain errors
Lab products found in correlation
Complete mini, by Roche (1 mentions)
Mmp 13, by Thermo Fisher Scientific (1 mentions)
Col11a1, by Abcam (1 mentions)
Sulf1, by Abcam (1 mentions)
Bca kit, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
E cadherin, by Abcam (1 mentions)
Snail, by Abcam (1 mentions)
N cadherin, by Abcam (1 mentions)
Odyssey fc, by LI COR (1 mentions)
Snai1, by Cell Signaling Technology (1 mentions)
Fibronectin, by Cell Signaling Technology (1 mentions)
Abcg2, by Cell Signaling Technology (1 mentions)
Smad3, by Cell Signaling Technology (1 mentions)
Itgb4, by Cell Signaling Technology (1 mentions)
Smad4, by Santa Cruz Biotechnology (1 mentions)
3 protocols using inhba
1
Quantitative Proteomics Analysis of Extracellular Matrix Proteins
2
Western Blot Analysis of EMT Markers
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RIPA buffer (R0278, Sigma) was used for cells lysis and the cell lysate was centrifuged for 15 min at 13,000 r/min, the supernatant was collected for protein measurements. The concentration of total protein was measured by Bicinchoninic Acid (BCA) Kit (71,285 M, Sigma), and 50 µg of protein was separated on 10% SDS-PAGE gel for electrophoresis and then transferred to PVDF membranes. The membranes were blocked using nonfat milk at room temperature (RT) for 1 hours, followed by incubation with primary antibodies at 4°C overnight. After three washes with TBST buffer, the membrane was further incubated with the HRP-conjugated secondary antibody at RT for 2 hours. All antibodies (INHBA, E-cadherin, ZO-1, CK-8, CK-18, CK-19, ZEB1, Snail, Slug, N-cadherin, Vimentin and Fibronectin, and HDRP-conjugated secondary antibodies) were purchased from Abcam (Cambridge, MA, USA).
3
Quantifying Protein Abundance with Western Blot
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Abundance of specific proteins was quantified using Western blot analysis as described (76 (link)) with the following primary antibodies: ABCG2 (Cell Signaling, 4477S, 1:250), SMAD4 (Santa Cruz, sc-7966, 1:500), phosphorylated SMAD3 (Cell Signaling, 9520S, 1:500), SMAD3 (Cell Signaling, 9523S, 1:500), fibronectin (Cell Signaling, 26836S, 1:500), SNAI1 (Cell Signaling, C15D3, 1:500), ITGB4 (Cell Signaling, 14803S, 1:500), INHBA (Abcam, ab56057, 1:500), and FST (Santa Cruz, sc365003, 1:500). The following secondary antibodies were utilized to detect bound primary antibodies: IRDye 680RD or 800CW goat anti-rabbit or mouse IgM, 1:5000, LI-COR, 926-32211, 926-68070, 926-68071. Images of Western blots probed with the fluorescent secondary antibody were collected with a LI-COR Odyssey Fc or CLx, and target proteins were quantified and normalized to total protein present. Total protein was ascertained utilizing REVERT staining. Total protein staining was used for normalization of Western blots aligning with reports demonstrating that total protein staining is a more accurate loading control than the use of housekeeping proteins (83 , 84 (link), 85 (link)). Western blots were quantified using Image studio v5.2.
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