Human anti c myb antibody

RIP was performed according to the protocol for the EZ-Magna RIP kit (EMD Millipore, Billerica, MA, USA). Brie y, DU145 cells grown to 80-90% con uency were lysed in complete RIP lysis buffer, and 90% of 100 µL of whole cell extract was incubated with RIP buffer containing magnetic beads conjugated with 5 µg human anti-C-myb antibody (Abcam) or immunoglobulin G (IgG) control. Incubation with proteinase K with shaking was performed to digest the protein, and RNA was isolated by immunoprecipitation. Finally, the immunoprecipitated RNA was puri ed and analysed by qRT-PCR.

To identify the potential transcription factors that bind to the LEF1-AS1 promoter, a ChIP assay was performed using an EZ-Magna ChIP kit (Millipore) according to the manufacturer's protocol. In brief, cells were xed with 4% paraformaldehyde and incubated with glycine for 10 min to generate DNA-protein cross-links. Then, the cells were lysed with cell and nuclear lysis buffers and sonicated to generate 400-800 bp chromatin fragments. The chromatin fragments were immunoprecipitated with an anti-C-myb antibody (Cell Signaling Technology, Danvers, MA, USA) or control IgG, and the resulting DNA was puri ed for PCR analyses All primers sequence was shown as Table S1.
RNA binding protein immunoprecipitation (RIP) assay RIP was performed according using an EZ-Magna RIP kit (EMD Millipore, Billerica, MA, USA) following the manufacturer's instructions. Brie y, DU145 cells grown to 80-90% con uency were lysed in complete RIP lysis buffer, and 90 μL of whole cell extract was incubated with RIP buffer containing magnetic beads conjugated with 5 μg of a human anti-C-myb antibody (Abcam) or immunoglobulin G (IgG) control. The samples were then incubated with proteinase K with shaking to digest the protein, and RNA was isolated by immunoprecipitation. Finally, the immunoprecipitated RNA was puri ed and analysed by RT-qPCR.