Ab2410

LINC00586 binding with LSD1 was evaluated using the Magna RIP™RNA-Binding Protein Immunoprecipitation Kit (Millipore, United States) following the manufacturer’s recommendation. In brief, two parts of cell extracts were incubated with either anti-LSD1 antibody (ab17721, Abcam) or nonspecific rabbit IgG (ab2410, Abcam) and immunoprecipitated with magnetic Protein A/G beads. Following proteinase k incubation, the immunoprecipitated RNA and total RNA from the whole cell lysates (input controls) were extracted for RT-qPCR analysis.

Enrichment of LSD1 and H3K27me3 in the ASXL1 promoter region was evaluated by the EZ ChIP™Chromatin Immunoprecipitation Kit (Millipore) following the manufacturer’s recommendation. In brief, HCT116 and LoVo cells were fixed with 10% formaldehyde for 10 min to generate DNA-protein cross-links. Cell lysates were then sonicated to generate chromatin fragments. The chromatin fragments were incubated with Protiein G Agarose (1 h) and then centrifuged (5,000 g, 1 min). Cell extracts were split into four parts of which one was used as “Input” and others were probed with anti-LSD1 antibody (ab17721, Abcam), anti-H3K27me3 antibody (ab6002) or nonspecific rabbit IgG (ab2410, Abcam) at 4°C overnight. The DNA-protein complexes were precipitated by using protein G Agarose. The immunoprecipitation was de-crosslinked, and the DNA samples were extracted for real-time qPCR analysis.

The EZ-Magna ChIP A/G kit (17–371; Millipore, Billerica, MA, USA) was applied in accordance with manufacturer’s instructions. After being sonicated, the cells were centrifuged at 12,000×g at 4 °C for 10 min to remove the insoluble components. The cells were then added with Protein G Agarose, incubated at 4 °C for 1 h and centrifuged at 5000×g for 1 min to remove the supernatant. Then 10 µL of supernatant was used as “Input” and the remaining supernatant was divided into two parts, which were further treated with H3K4me3 antibody (ab185637; 1:20, Abcam) and NC rabbit anti-human IgG (ab2410; 1:25, Abcam) at 4 °C using overnight incubation. Protein G Agarose was inverted and incubated at 4 °C for 1 h to precipitate the protein-DNA complexes. Following centrifugation at 5000×g for 1 min, the supernatant was discarded. The non-specific complex (protein-DNA complex) was eluted, and de-crosslinked at 65 °C. The recovered and purified DNA fragments were used as amplification templates for RT-qPCR experiments, and the enrichment of H3K4me3 in the LSD1 promoter region was detected by ChIP assay [10 (link), 12 (link)].

An amount of 2 × 10⁷ cells were lysed with 0.5% NP40 and the supernatant was collected as a lysate by centrifugation. An amount of 50 μL of Protein-A magnetic beads was equilibrated with 5% BSA-NT2 buffer and 3–5 μg of antibody was added, then incubated overnight at 4 °C. The beads were washed 4–5 times with pre-chilled NT2 buffer, and cell lysate added and mixed well. An amount of 100 μL was taken as Input, and the remaining samples were incubated at 4 °C for 4 h. The magnetic beads were washed 4 times with pre-chilled NT2 buffer. An amount of 500 μL Trizol was added to extract RNA for qRT-PCR analysis. The following antibodies were used: anti-AR (Abcam, ab108341), anti-NONO (Proteintech, 11058-1-AP) and anti-IgG (Abcam, ab2410).

ChIP assays were implemented via ChIP assay kit (Millipore) in tune with the supplier’s requirements. Briefly, HEK-293 T cells were fixed with 1% formaldehyde for 10 min at 37 °C and then sonicated on ice. The sonicated chromatin was subsequently immunoprecipitated with antibodies against c-JUN and immunoglobulin G (IgG) (ab2410, Abcam). The qRT-PCR was applied to detect the precipitated DNA fragments.