Sc 59

Co-immunoprecipitation (Co-IP) of proteins as an indication of protein-protein interactions was carried out as described earlier [34 (link), 35 (link)]. Briefly, cells were washed with 1X PBS and harvested in NP-40 Buffer (50mM Tris pH 7.5, 150mM NaCl, 2mM EDTA, 0.5% NP-40 supplemented with PMSF and protease inhibitors). Cells were lysed for 30min on ice and passaged through a 27G needle three times. Lysates were centrifuged and protein concentrations were determined using the BCA Protein Assay Kit (Thermo Scientific; Waltham, MA). Equal protein amounts were used for IP. Protein extracts were precleared with protein A agarose rocking at 4°C for one hour. The extract/bead mix was centrifuged and the supernatant was transferred to new tubes. Extracts were then incubated with an antibody against p53 (PAb 421), CBP (sc-369, Santa Cruz), Sp1 (sc-59, Santa Cruz), or Ets1 (sc-350, Santa Cruz) and protein A agarose beads while rocking at 4°C overnight. The following morning the extract/bead/antibody mix was centrifuged and the beads were washed three times with NP-40 Buffer. The buffer was removed and equal volume 2X Laemmli loading buffer was added and boiled for ten minutes. Extracts were then resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Additionally, a small aliquot of the IP supernatant was set aside and co-electrophoresed as a loading control.

Immunoblotting of whole Jurkat T cell lysates or nuclear extracts (Sutcliffe et al., 2012 (link)) was performed with: anti-PKC-θ (sc-212, Santa Cruz Biotechnology), anti-PKC-θ S676p (ab47774, Abcam), p65 (ab7970, Abcam), H3 (ab1791), Sp-1 (sc-59, Santa Cruz Biotechnology), p50 (sc-1191, Santa Cruz Biotechnology), p65 Ser486p (3039, Cell Signaling Technology), p65 Ser536p (3031, Cell Signaling Technology), IκB-α (Ser32/36; 9246, Cell Signaling Technology), H2B Ser32p (ab10476, Abcam), and H2B Ser36p (ECM Biosciences HP4331) antibodies with a dilution range of 1:200 to 1:1000. Signals were detected by enhanced chemiluminescence on a LAS4000 Fluoimager.

Western blotting was performed according to a previously published study [12 (link)]. Anti-CD147 mAb was prepared in our lab [11 (link)]. Primary antibodies against Sp1 (sc-59), Krueppel-like factor 6 (KLF6, sc-365633), DNA methyltransferase 3A (DNMT3A, sc-373905), methyl-CpG-binding protein 2 (MeCP2, sc-137070), Tet1 (sc-293186), Tet2 (sc-398535), TDG (sc-376652), and β-actin (sc-8432) were purchased from Santa Cruz Biotechnology. Anti-SMAD2/3 mAb (ab63672) was purchased from Abcam (Abcam, Cambridge, UK) and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Thermo Fisher Scientific (31430 and 31460, Thermo Fisher Scientific, Waltham, MA, USA).