Chromatin immunoprecipitation (ChIP) was operated according to the guidelines by the SimpleChIP Plus Enzymatic Chromatin IP Kit (Magnetic Beads) (Cell Signaling Technology, United States, 9005). Chromatin was crosslinked and immunoprecipitated with 2 μg of anti-SMAD2+SMAD3 (Abcam, United States, ab202445) and 30 μl of protein G beads at 4°C overnight. The negative control was normal rabbit IgG. Lastly, the purified immunoprecipitated chromatin was analyzed by quantitative real-time PCR. The sequences of the primers are provided in Supplementary Table S1 .
Anti smad2 smad3
Manufactured by Abcam
Sourced in United States
Anti-SMAD2+SMAD3 is a lab equipment product that detects the presence of SMAD2 and SMAD3 proteins. SMAD2 and SMAD3 are intracellular signal transducers and transcriptional modulators activated by transforming growth factor-beta (TGF-beta) and activin type 1 receptor kinases.
Automatically generated - may contain errors
Lab products found in correlation
Normal rabbit igg, by Abcam (1 mentions)
Ez magna rip rna binding protein immunoprecipitation kit, by Merck Group (1 mentions)
Pierce magnetic chip kit, by Thermo Fisher Scientific (1 mentions)
Chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Anti pd l1, by Thermo Fisher Scientific (1 mentions)
Anti bub1, by Abcam (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
4 protocols using anti smad2 smad3
1
Chromatin Immunoprecipitation (ChIP) Assay
2
RNA-Binding Protein Immunoprecipitation of SMAD2/3 and DROSHA
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Primary HSCs were isolated from uninfected and S. japonicum-infected mouse livers and assayed with the EZ-Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Cat. 17-701; Millipore). To obtain concentrated cell lysates, at least 2 × 107 cells are required for 100 μL of RIP Lysis Buffer. The assay was performed following the manufacturer’s protocol with anti-SMAD2+SMAD3 (Cat. no. ab65847; Abcam), anti-DROSHA antibodies (Cat. no. ab12286; Abcam), and normal rabbit IgG as a negative control antibody (Cat. no. PP64B; Millipore).
3
Chromatin Immunoprecipitation Protocol
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The ChIP was performed according to the Pierce Magnetic ChIP Kit (26157, Thermo Fisher, Waltham, MA, USA) guidelines. The chromatin was cross-linked and immunoprecipitated with 2 μg of anti-Smad2 + Smad3 (Abcam, Cambridge, USA, ab202445) and 30 μL of protein G beads overnight at 4 °C. The negative control was normal rabbit IgG. Finally, the purified immunoprecipitated chromatin was analyzed by a quantitative real-time PCR. According to previous research methods, the results of the ChIP-qPCR were normalized and presented as % input [74 (link)] The primer sequences are shown in Supplementary Table S5 .
4
Western Blot Analysis of Cell Signaling Proteins
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Radioimmunoprecipitation assay buffer supplemented with phenyl methane sulfonyl uoride was used to lyse tumour cells. After the lysate was centrifuged, the supernatant was collected for further analysis. After the protein concentration was measured, equal amounts of protein were loaded onto 10% SDS-PAGE gels and transferred onto polyvinylidene uoride membranes. Then, the membranes were blocked using 5% skimmed milk at 37 ºC for 2 h and were incubated with the primary antibodies (anti-SMAD2/SMAD3, anti-BUB1, anti-PSMAD2/PSMAD3, 1:1,000, Abcam; anti-PDL1, 1:1,000, eBioscience, California; anti-GAPDH, 1:1,000, Proteintech, Chicago) at 4 ºC overnight. Then, the anti-mouse/rabbit antibodies (1:3,000, Beyotime, Shanghai) were used to incubate the membranes at 37 ºC for 1h. The protein bands were detected by the chemiluminescence kit (Thermo Fisher, Shanghai).
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