Cells were lysed on ice using RIPA buffer. The lysate was centrifuged at 10,000 × g for 10 min, and the protein concentration in the supernatant was determined using the microBCA protocol (Pierce, Thermo Scientific, Rockford, IL). The protein lysate was separated by SDS-PAGE and then transferred to PVDF membranes (BioRad). The membranes were blocked with 5% BSA/TBS-0.1% Tween20 and then probed with rabbit anti-Musashi1 primary antibodies (Abcam ab52865, Cambridge, MA, 1/2,000) or rabbit anti-NF-YA primary antibodies (Abcam ab6558, Cambridge, MA, 1/1,000) for 1 h at room temperature. After washing, the blots were incubated with an anti-rabbit horseradish peroxidase-conjugated secondary antibody (SigmaAldrich, 1/50,000) and visualized by ECL plus Membrane Blotting Detection System (GE Healthcare) using a laser scanner (Typhoon 9410, GE Healthcare).
Ab6558
Manufactured by Abcam
Sourced in United States, Hong Kong
Ab6558 is a mouse monoclonal antibody that recognizes the protein kinase A regulatory subunit RIα. It can be used in various laboratory applications to detect and quantify the target protein.
Automatically generated - may contain errors
Lab products found in correlation
Ab9110, by Abcam (2 mentions)
Sc 2027, by Santa Cruz Biotechnology (2 mentions)
Ab52865, by Abcam (1 mentions)
Horseradish peroxidase conjugated anti rabbit secondary antibody, by Merck Group (1 mentions)
Typhoon 9410, by GE Healthcare (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Orange chip kit, by Diagenode (1 mentions)
4 protocols using ab6558
1
Musashi1 and NF-YA Protein Detection
2
Chromatin Immunoprecipitation Assay in HepG2 Cells
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ChIP assays was performed with HepG2 cells transfected with pcZHX2-HA or pcEGFP-HA as reported [16 (link)]. Briefly, fixed cells were sonicated to shear DNA to 200~1000 bp and immunoprecipitated using anti-HA antibody (ab9110; Abcam), anti-NF-YA antibody (ab6558; Abcam) or rabbit IgG (sc-2027, Santa Cruz, CA, USA). As controls, 1/20th of the starting chromatin (Input) was used. Analysis was performed using specific primers for the NF-YA binding region of MDR1 promoter.
3
ChIP Assay for Histone-DNA Interactions
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For ChIP assays, homogenized endometrial tissues were cross-linked with 1% formaldehyde and subjected to immunoprecipitation after sonication. The ChIP experiments were performed using the Orange ChIP kit (Diagenode, Belgium), according to the manufacturer’s instructions on three biological replicates. Real time PCR was used to amplify specific promoter DNA (ID1–4) bound to the immunoprecipitated histones (NF-Y) after reversing the histone-DNA cross-links (primers listed in Table 1 ). All the samples were amplified in triplicate. Data were represented as the percentage of input DNA associated with immunoprecipitated NF-Y relative to input chromatin. A Rabbit polyclonal antibody against NF-Y (Abcam, ab6558) was used.
4
HA-Tag Immunoprecipitation from HepG2 Cells
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Cell lysates from HepG2 cells transfected with pcZHX2 were precipitated with control IgG (sc-2027, Santa Cruz, CA, USA) or NF-YA antibody (ab6558; Abcam, Hong Kong). After incubation with protein A/G Plus-Agarose(sc-2003, Santa Cruz, CA, USA), the precipitates were immunobloted with anti-HA antibody (ab9110; Abcam).
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