Protein a g magnetic beads

The whole protein extracts of SMCs were prepared using the special lysis buffer (Cell Signaling Technology no. #9803, Danvers, MA, USA) for immunoprecipitation. The lysates were centrifuged, and protein concentrations were measured (BCA kit) and immunoprecipated at 4°C by gently rocking overnight with the protein A/G magnetic beads (Cell Signaling Technology) prebound to anti‐TWIST1 or anti‐p68 antibodies prior to bind with the immunoprecipitates. Precipitated proteins were washed 5 times with PBS, boiled in 2 × sample loading buffer, and separated by 12% SDS/PAGE. The separated protein was then transferred to PVDF membrane and immunoblotted with anti‐TWIST1 or anti‐p68 antibody, respectively. Rabbit normal IgG (Cell Signaling Technology) served as negative control.

To investigate the interactions between Oip5-as1 and proteins, RNA immunoprecipitation (RIP) assays were carried out using an EZ-Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Merck Millipore). HL-1 cell lysates was prepared with the supplied RIP lysis buffer, enriched with the Halt protease inhibitor cocktail and RNase inhibitor. The lysate was subsequently incubated with protein A/G magnetic beads that were conjugated with 5 µg of specific antibodies (anti-rabbit-IgG, AKAP1, or CaN; Cell Signaling Technology). Following an overnight incubation at 4 °C with gentle rotation, the RNA–protein complexes bound to the beads were washed with the supplied washing buffers. The complexes were then exposed to proteinase K to degrade the proteins, and the accompanying RNA was purified as per the manufacturer’s guidelines. The purified RNA was subsequently subjected to downstream RT-qPCR analysis to quantify the enrichment of Oip5-as1. Additionally, the EZ-Magna RIP Kit contains antibodies against SNRNP70 (small nuclear ribonucleoprotein 70 kDa) and primers for U1, which serve as positive controls for the experiments.

H1299 and H1650 cells were lysed in nondenaturing lysis buffer. Next, the supernatant of the cell lysate was precleaned by protein A/G magnetic beads (Thermo Fisher Scientific) for 2 h at 4 °C. Subsequently, approximately 300 μg of protein sample was incubated overnight at 4 °C with 1 μg of anti-NIPBL antibody (#ab245539, Abcam) or anti-RAD21 antibody (#4321, Cell Signaling Technology) and 25 μL of protein A/G magnetic beads. The next day, the protein A/G magnetic beads were collected using a magnetic separation device (Thermo Fisher Scientific), and the precipitated complexes were cleansed with washing buffer (Thermo Fisher Scientific). Finally, the bound proteins were analyzed by Western blotting using KDM6B antibody (#ab38113, Abcam) or EZH2 antibody (#21800-1-AP, Proteintech). Rabbit IgG was used as the negative control.
For competitive Co-IP, different dosages of NIPBL expression vector (0.5 μg, 1 μg, and 2 μg) was transfected into control H1299 or H1650 cells and then the interaction RAD21 and NIPBL or EZH2 was evaluated by Co-IP using anti-RAD21 antibody (#4321, Cell Signaling Technology) as above described.

To detect PGC-1α acetylation in cells, cultured cardiomyocytes were collected and lysed in lysis buffer (P0013C; Beyotime, Shanghai, China) for 30 min at 4 °C. After centrifugation at 14,000 g for 20 min at 4 °C, the supernatants were incubated with 1 μg of anti-PGC-1α antibody overnight at 4 °C. Rabbit IgG (Santa Cruz Biotechnology) was used as control. Each sample was incubated with protein A/G magnetic bead slurry (Selleckchem, Houston, TX, USA) for 2 h at 4 °C to avoid nonspecific binding. A total of 500 μg of protein from each sample was incubated with 2 μg of specific antibody to acetylated lysine (#9441, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C according to the instructions of the Protein A/G Magnetic Beads for IP. The blot was probed with rabbit anti-PGC-1α (Abcam) and anti-acetylated lysine antibodies and then treated with appropriate secondary antibodies conjugated to HRP (Santa Cruz Biotechnology, Dallas, TX, USA). Then, western blotting was performed as described above.

We used the ChIp Assay Kit (Biyotime) to perform the ChIp Assay in accordance with the manufacturer’s instructions. Cells were cross-linked with 1% formaldehyde for 10 min at 37 °C and quenched with 125 mM glycine for 5 min. The DNA fragments with 200–1000 bp were prepared and then immunoprecipitated with Protein A + G Magnetic beads coupled with anti-HSPB1 (Cell Signaling Technology, Boston, MA, USA) antibodies. After reverse crosslinking, ChIP and input DNA fragments were end-repaired and A-tailed using the NEBNext End Repair/dA-Tailing Module (E7442, NEB) followed by adapter ligation with the NEBNext Ultra Ligation Module (E7445, NEB). The DNA library was amplified in 15 cycles and sequenced using the Illumina HiSeq (Illumina) with 2 × 150 pairs.

All of Myc‐Synr, HA‐Debcl, and Flag‐Buffy were expressed in HEK293T cells. Their lysates were incubated with the monoclonal mouse ANTI‐FLAG M2 antibody (F1804, Sigma) and Protein A/G Magnetic Beads (#88802, Thermo Fisher) for 4 h. After washing beads, proteins were eluted with 200 μg/ml 3XFlag peptide and then elution was again incubated with new Protein A/G Magnetic Beads and Myc‐tag mouse antibody (9B11, #2276, Cell Signaling Technology) O/N. After washing the beads with the cell lysis buffer, proteins were eluted by heating at 95°C for 5 min in the 2× sample buffer and they were separated by SDS–PAGE (8% SDS–PAGE gels).

Co-immunoprecipitation of AGO2-bound RNAs was conducted as reported earlier [21 (link)]. In brief, the 293T cells were first cross-linked with 0.3% formaldehyde. Approximately 1.0 × 10⁷ cells were collected and lysed. Then, the protein A/G magnetic beads (Cell Signaling Technology) coupled with AGO2 (Cell Signaling Technology) or IgG control antibody were added into the cell lysates, and incubated at 4°C overnight. After extensive washing, AGO2-bound RNAs were eluted using elution buffer containing proteinase K, followed by reverse cross-linking. The eluted RNA was purified and reverse transcribed to cDNA according to the manuals.