Protein a g plus agarose

Immunoprecipitation of NSUN2 binding small RNA was performed using an anti-NSUN2 antibody (Cat. No.20854-1-AP; Proteintech) at 4 ℃ for 2 h. The m6A-containing small RNA–anti-m6A-antibody complexes were mixed with Protein A/G PLUS-Agarose (Cat. No. sc-2033, Santa Cruz Biotechnology, inc.) at 4 °C for 2 h. The mixture was isolated by centrifugation at 12,000 rpm for 20 s. The NSUN2 binding RNA was reverse cross-linked by incubating tubes in a 67 °C water bath, mixing occasionally over two hours. Remove beads by centrifugation and continue incubating supernatant at 67 °C overnight. The RNA not recovered by centrifugation was designated as small RNA not binding to NSUN2.

The lysate of IPEC-J2 cells infected with TGEV Miller for 36 h was prepared with RIPA lysis buffer (Proteintech) containing the protease inhibitor phenylmethanesulfonyl fluoride (PMSF) (1 mM). After centrifugation at 10 000 × g for 20 min and measurement of the protein concentration using the BCA method, the lysate supernatant was pretreated with protein A/G PLUS-Agarose (Proteintech) for 60 min at 4 °C to purify the protein. The lysate supernatant (700 μg) was incubated with 3 μg of a rabbit pAb against UBXN1 overnight at 4 °C. Next, 10 μL of protein A/G PLUS-Agarose was added to this mixture and incubated with shaking at 4 °C for 4 h. After washing four times with lysis buffer, the eluted proteins were analyzed by SDS-PAGE and Western blotting using pAbs recognizing the S1 protein of TGEV and rabbit pAbs recognizing UBXN1. The lysate of IPEC-J2 cells uninfected with TGEV was used as the control.

After the above treatment, ESCs were lysed with IP lysis buffer (25 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 1% protease inhibitor). The cell lysates were precleared with Protein A/G PLUS-Agarose (Thermo, USA) and then mixed with Bcl-2 (4ug, Proteintech) or control IgG (Proteintech, USA) for 1 h at 4 °C. The immunoprecipitates were captured on Protein A/G PLUS-Agarose and analyzed using a western blot with antibodies against Bcl-2 or Beclin1 (1:1000, Proteintech).