Western blot assay was performed as previously described [16 (link)]. In brief, the treated A498 cells were lysed with RIPA lysis buffer containing proteinase inhibitors and phosphatase inhibitors (Roche). Then, the obtained cell lysates were used for western blot analysis. The MTA1 (BA2749) was from BOSTER, and β-actin (10230-1-AP) antibodie was purchased from Proteintech, and p65 (AF5006) and p-p65 (AF2006) antibodies were obtained from Affinity Biosciences.
P p65 af2006
Manufactured by Affinity Biosciences
Sourced in China
P-p65 (AF2006) is a laboratory equipment product manufactured by Affinity Biosciences. It is used for the detection and quantification of phosphorylated p65, a subunit of the NF-kB transcription factor, in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
P65 af5006, by Affinity Biosciences (2 mentions)
Proteinase inhibitors and phosphatase inhibitors, by Roche (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Iκb 4814s, by Cell Signaling Technology (1 mentions)
Imagej software, by Media Cybernetics (1 mentions)
Myd88, by ABclonal (1 mentions)
2 protocols using p p65 af2006
1
Western Blot Analysis of MTA1, p65, and p-p65
2
Ruminal Epithelial Protein Extraction and Western Blot Analysis
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Total protein from ruminal epithelial cells was extracted using RIPA lysis buffer and quantified using a BCA Protein Assay kit (P0012, Beyotime Biotechnology, China). Protein was separated by SDS-PAGE on 10% gels and transferred to PVDF membranes. Then the membrane was Blocking with 5% skimmed milk or BSA in TBST buffer for 2 h, and followed incubated with TLR4 (ab217274, abcom, Cambridge, the United Kingdom), MyD88 (A0980, Abclonal, Wuhan, Chian), p-p65 (AF2006, Affinity, Jiangsu, China), p65(AF5006, Affinity, Jiangsu, China), p-IκB (9246S, Cell Signaling Technology, Danvers, MA, USA), IκB(4814S, Cell Signaling Technology, Danvers, MA, USA ). antibodies overnight at 4°C, subsequently incubated for 2 h at room temperature with secondary antibodies. The target bands were detected using the chemiluminescence (ECL) system and analyzed using the ImageJ software (Media Cybernetics, Bethesda, MD, United States).
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