Supersignal pico substrate

The cells treated as described were centrifuged at 2,500 g for 5 min, and the pellets were washed twice in PBS, and in 0.5% Triton X buffer, for isolation of nuclear fraction, before being resuspended in RIPA buffer (Thermofisher), added 1 mini tablet Complete (Roche protease inhibitors) for protein extraction. Western blots for detection of TBP and TAF9 were performed by loading 20 μg of whole cell lysates and 5 μl of PageRuler Plus Prestained Protein Ladder (Thermofisher) per well, onto a Mini-Protean TGX 4–20% pre-cast gel (Biorad). Proteins were transferred to a nitrocellulose membrane using a G2 Blotter, and blocked with 5% Casein in TBST for 1 h at room temperature. The N-terminus of the TATA binding protein (TBP) was detected at 36–38 kD using a TBP monoclonal antibody (1TBP18/ MA1-189) at a dilution of 1:1,000. TATA box associated factor 9 (TAF9) was detected at ~20 kD using a polyclonal antibody (PA5-40919), at 1:1,000. Both antibodies were incubated overnight at 4oC on a rocking platform, followed by a Goat anti-Mouse IgG (H+L) HRP conjugate (Cell Signaling) for 1 h at room temperature. Chemiluminescent detection was performed using SuperSignal Pico substrate (Thermofisher).

Cells were lysed in 2% SDS lysis buffer and sonicated. A total of 15 μg of protein was loaded after quantification (Pierce 23225). Then, the proteins were transferred to a 0.45 μm PVDF membrane. After 1 hour of blocking with 5% BSA, the membrane was incubated with the primary antibody overnight at 4°C and then the secondary antibody at room temperature for 1 hour on the next day. Antibody information can be found in Tables [Link], [Link], [Link]. After washing, the blots were developed with the Super Signal Pico substrate (Pierce Biotechnology).

Total cellular or tissue lysates were prepared with 1% SDS and sonicated to break up DNA. Antibody data can be found in the Supplementary Table. Each immunoblot was repeated three times containing samples coming from different cell cultures. The relative intensity of protein bands was measured with NIH image J software. The blots were developed with Super Signal Pico substrate (Pierce Biotechnology, Shanghai, China).

Cells were washed in PBS buffer, then lysed with EBC buffer (50 mM Tris, pH 8.0; 120 mM NaCl; 0.5% NP-40) supplemented with a protease inhibitor cocktail. In some cases 1% SDS solution was added to the cells in the plates instead of EBC buffer, then the solution was sonicated at 20 A for 3 × 15 s pulses and mixed with sample buffer. The same total amount of total protein was loaded and resolved by SDS–PAGE and analyzed by immunoblotting. The blots were developed with Super Signal Pico substrate (Pierce Biotechnology, Rockford, IL) or Immobilon Western substrate (Millipore, Billerica, MA). Immunoprecipitations were performed with EBC lysates as previously described (Niu et al., 2012 (link)).
The antibodies used in this manuscript are: HA (Covance MMS-101P-1000 HA.11 (16B12 for IB, Santa Cruz Biotech sc-805 for IP), Flag (Sigma F3165-1mg (M2)), KDM5C (Bethyl A301-034A and A301-035A), BAP1 (Santa Cruz Biotech sc-28383), γ-Actin (Santa Cruz Biotech sc-8432), PBRM1 (Bethyl A301-591A and A301-590A), STAT1 (Cell signaling 9172S), phospho-STAT1 (Cell signaling 9171L), STAT2 (Bethyl A303-512A-T), phospho-STAT2 (Cell signaling 88410), IRF9 (Cell signaling 76684S), MX1 (R and D AF7946), PLSCR1 (Santa Cruz Biotech sc-59645), Vinculin (Santa Cruz Biotech sc-73614), GAPDH (Santa Cruz Biotech sc-59540), HIF2α (Novus NB100-122), OAS1 (Cell signaling 14498S).

Cells were washed in PBS buffer before being lysed with EBC buffer (50 mM Tris, pH 8.0; 120 mM NaCl; 0.5% NP-40) supplemented with a protease inhibitor cocktail. The same total amount of each lysate was loaded and resolved by SDS–PAGE and analyzed by immunoblotting. The blots were developed with Super Signal Pico substrate (Pierce Biotechnology, Rockford, IL) or Immobilon Western substrate (Millipore, Billerica, MA). The antibodies were the same as the ones used for IHC.