Anti myc beads

WDR4-based Cul4 ligase complex was purified with anti-Myc beads (Sigma-Aldrich) from HEK293T cells cotransfected with Myc-Cul4A or Myc-Cul4B, together with other complex subunits. His-PTPN23 was purified from baculovirus by incubating lysates with Ni-Sepharose beads (Cytiva) and eluted with imidazole. The E3 ligase complex bound on Myc beads was incubated at 37 °C for 2 h in 20 μl reaction mixture containing 40 ng yeast E1, 500 ng E2 (UbcH5a), 300 ng His-PTPN23 and other components as described previously [57 (link)]. The E1, E2, and His-ubiquitin were purchased from R&D systems.

Co-IP assays were carried out as described previously^{47 (link),48 (link)}. Briefly, PAC1 cells were co-transfected with expression plasmids encoding myc-tagged myocardin and YY1 as indicated. 48 h after transfection, nuclear protein was harvested and Co-IP assays were performed using the nuclear complex Co-IP kit as described by the manufacturer (Active Motif, cat. #: 54,001). One hundred micrograms of nuclear protein extracts were incubated with 50 μl of anti-Myc beads (Sigma, cat. #: E6654) or 3 μg control rabbit IgG in 500 μl low salt IP buffer (Active Motif) overnight at 4 °C. Fifty microliters of anti-rabbit IgG beads (True Blot, cat. #: 00–8800-25) were added to control IgG sample for an additional 1 h with rocking and then immobilized complexes from both anti-myc and anti-rabbit IgG beads were washed six times with the low salt IP buffer with or without BSA. The immunoprecipitated proteins were then mixed with 45 μl 2 × SDS sample buffer and analyzed by Western blot as indicated. Images were acquired by ImageQuan LAS4000 Imaging Station (GE).

After 48 h, insect cells were infected with pVL-TRPC5-Myc, pVL-Tlr9-Myc, and pVL-Eno1-Myc, pVL-Eno2-Myc, pVL-Eno2(Δ405–431)-Myc, and pVL-Eno2(Δ432–434)-Myc. The recombinant protein of Pgk1-Flag, Pgk1(1–145)-Flag, Pgk1(146–417)-Flag, Pgk1(225–417)-Flag or Pgk1(325–417)-Flag was then added into the insect cell culture medium and incubated for 2 h, followed by mixing with bis[sulfosuccinimidyl] suberate (BS3) to perform crosslinking-IP according to the procedures described for BS3 Crosslinkers (Thermo Fisher Scientific). Cell lysates were then subjected to IP with either anti-Flag (Sigma-Aldrich) or anti-Myc beads (Sigma-Aldrich). The IP products were then prepared for western blot analysis with anti-Flag (RRID:AB_446355; 1:10,000) and anti-Myc (RRID:AB_439694; 1:1000). To carry out the antibody-blocking experiment, we added antibody against Eno2 (NSE-P1) (RRID: AB_627513) into the insect cell culture medium at a concentration of 2 μg/ml and incubated for 1 h before performing Crosslinking-IP.

Plasmids were constructed as follows: the SAMD12-AS1 and SAMD12-AS1-S1, SAMD12-AS1(1-350)-S1 and SAMD12-AS1(351-701)-S1 tagged genes were cloned into a lentiviral plasmid, pLentiLox3.7; the SAMD12-AS1, SAMD12-AS1-T, SAMD12-AS1-TT, and HBx genes were cloned into pcDNA3.1 with a C-terminal FLAG tag.; and pGL2-SAMD12-AS1-luc was constructed by cloning the predicted SAMD12-AS1 promoter (−425 to 1, genome location: nt118620575-nt118621000) into the pGL2-Basic vector.
Rabbit anti-NPM1 antibody was produced by immunizing rabbits with E. coli-expressed NPM1. Mouse anti-FLAG antibody (M2), rabbit anti-Myc antibody (9E10) and anti-Myc beads were purchased from Sigma Aldrich, USA. Mouse anti-GAPDH antibody, goat anti-human lamin B antibody, and anti-p53 antibody were purchased from Santa Cruz Biotechnology, USA. Rabbit anti-human tubulin antibody and rabbit anti-human poly(ADP-ribose) polymerase (PARP) antibody were purchased from Cell Signaling Technology, USA. Rabbit anti-HDM2 antibody was purchased from Bioworld, USA. HRP-conjugated secondary antibodies were purchased from Jackson Laboratory, USA. Anti-Mouse IgG (Heavy Chain)-HRP, anti-Mouse IgG (Light Chain)-HRP, anti-Rabbit IgG (Light Chain)-HRP, and anti-Rabbit IgG (Heavy Chain)-HRP were purchased from EASYBIO, China. The anti-HA-Tag beads were purchased from Abmart, and Ni-NTA beads were purchased from Qiagen, Germany.

Cells were lysed in lysis buffer containing 0.5% NP40, 150 mM NaCl, 20 mM HEPES (pH 7.4), 10% glycerol, 1 mM EDTA, and protease inhibitor cocktail. After centrifugation, the supernatants were incubated with anti-FLAG, anti-Myc beads (Sigma), or Protein A/G PLUS-Agarose beads (Santa Cruz) for 4 hr at 4°C. After five washes in washing buffer (0.5% NP40, 300 mM NaCl, 20 mM HEPES (pH 7.4), 10% glycerol, and 1 mM EDTA), the immunoprecipitates were analyzed by immunoblot analysis. Native PAGE was performed with an 8% acrylamide gel without SDS. The gel was pre-run for 30 min at 40 mA on ice with 25 mM Tris-HCl (pH 8.4) and 192 mM glycine with or without 0.5% deoxycholate in the cathode chamber and anode chamber, respectively. Samples in the native sample buffer (50 mM Tris-HCl, pH 6.8, and 15% glycerol) were applied on the gel and underwent electrophoresis for 60 min at 35 mA on ice followed by immunoblot analysis.