The DARTS assay was performed as described25 to examine the effect of compounds on the stability of FOXM1 to proteolysis by exogenous pronase. Cell lysates were incubated without or with compound for 1 h at room temperature, and with varying concentrations of pronase (none; 1:103; 1:3 × 103; 1:5 × 103; 1:104; and 1:105 dilution of a 12.5 mg/ml pronase solution) for an additional 30 min at room temperature. Proteins were then separated on 4–12% SDS-PAGE gels and gels were exposed to FOXM1 antibody (Genetex GTX102170, 1:750) and β-actin antibody (1:500, mouse monoclonal).
Gtx102170
Manufactured by GeneTex
GTX102170 is a laboratory centrifuge that can be used to separate components of a liquid mixture based on their different densities. It is designed to provide consistent and reliable results for a variety of applications in life science research.
Automatically generated - may contain errors
Lab products found in correlation
β actin antibody, by GeneTex (1 mentions)
H3s10ph, by Cell Signaling Technology (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Hisprobe hrp conjugate, by Thermo Fisher Scientific (1 mentions)
Cyclin b1, by BD (1 mentions)
Aurora b, by BD (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Foxm1, by Santa Cruz Biotechnology (1 mentions)
2 protocols using gtx102170
1
DARTS Assay for FOXM1 Stability
2
Immunoblotting and Immunofluorescence Protocol
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Antibodies against ACA (HCT-0100, ImmunoVision, 1:5000), Aurora B (611082, BD Transduction Laboratories, WB: 1:1000, IF: 1:250), CDH1 (CC43, Millipore, 1:500), Cyclin B1 (554177, BD Pharmingen, 1:5000), EGFP (sc-9996, Santa Cruz, 1:2000), Flag (F1804, Sigma-Aldrich, 1:1000), FOXM1 (sc-500, Santa Cruz, 1:500 for WB; GTX-102170, GeneTex, 1:250 for ChIP), GAPDH (2118, Cell Signaling, 1:5000), HA (home-made, WB: 1:5000), ubiquitin-HRP (AUB01, Cytoskeleton, 1:1000), H3S10ph (9706, Cell Signaling for IF, 1:1000; 06-570, Upstate-Merck, for WB, 1:1000), and RepoMan (HPA030049, Sigma, WB: 1:1000, IF: 1:300) were obtained from the indicated sources. For detection of His-tagged ubiquitin in Supplemental Figures S4G and 5C, the PVDF membrane was incubated with HisProbe-HRP Conjugate (15165, Thermo Scientific, 1: 2500). Secondary HRP–conjugated antibodies were purchased from Dako (Heverlee, Belgium). Secondary Alexa 488, 555, and 633 antibodies were obtained from Invitrogen (Carlsbad, CA).
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