Ma1 827

Immunoprecipitation was carried out as previously described^{8 (link)} using an anti-C/EBPβ antibody (2 μg per 1 mg total protein, MA1–827, ThermoFisher).

Cell lysate was prepared and Western blotting was performed as described previously^{8 (link)}. The primary antibodies used for western blotting were as follows: anti-acetylated lysine (1:1000, 9814, Cell Signaling Technology), anti-p300 (1:500, sc584, Santa Cruz Biotechnology), anti-PCAF (1:1000, p3378, Cell Signaling Technology), anti-C/EBPβ (1:1000, MA1–827, ThermoFisher), anti-atrogin1/MAFbx (1:1000, AP2041, ECM Bioscience), anti-UBR2 (1:500, NBP1–45243, Novus Biologicals), anti-LC3 (1:2000, NB100–2220, Novus Biologicals), anti-p62 (1:1000, H00008878-M01, Novus Biologicals), anti-p38 MAPK (1:1000, 9212, Cell Signaling), anti-pp38 MAPK (1:1000, 4511, Cell Signaling), anti-p65 (1:250, 8008, Santa Cruz), anti-pp65 (1:500, 166748, Santa Cruz) and anti-MHC (1:1000, MAB4470, R&D Systems); whereas the custom-antibody targeting acetylated Lys39 of C/EBPβ (1:2000) were generated by Pocono Rabbit Farm & Laboratory from rabbits using a peptide (CLAYGAK(Ac)AARAAPRA) synthesized by Novoprotein Scientific Inc (Summit, NJ 07901). Data were normalized to α-Tubulin (antibody was from Development Studies Hybridoma Bank at the University of Iowa, Iowa City, IA).

All procedures were adhered to our previous publication (12 (link)). The following primary antibodies were used: anti-p300 (1:500, sc584, Santa Cruz), anti-C/EBPβ (1:1000, MA1–827, Thermo Fisher), anti-pT188-C/EBPβ (1:1000, 3084, Cell Signaling Technology), anti-TLR4 (1:500, sc16240, Santa Cruz), anti-p38α MAPK (1:500, sc271120, Santa Cruz), anti-p38β MAPK (1:500, 2339, Cell Signaling Technology), anti-p38 MAPK (1:1000, 9212, Cell Signaling Technology), anti-p-p38 MAPK (1:1000, 4511, Cell Signaling Technology), anti-MAFbx (1:1000, AP2041, ECM Bioscience), anti-UBR2 (1:500, NBP1–45243, Novus Biologicals), anti-LC3 (1:2000, NB100–2220, Novus Biologicals) and anti-MHC (1:1000, MAB4470, R&D Systems). Antibody against acetylated Lys-39 of C/EBPβ (1:2000) was generated as previously described (12 (link)). Antibody against phosphorylated Ser-12 of p300 (1:1500) were generated by Pocono Rabbit Farm & Laboratory (Canadensis, PA) from rabbit using the peptide PGPPS(P)AKRPKLSSPAC. The specificity of the antibody was validated using mutated p300 with point mutantions (Figure 1A). Data were normalized to α-Tubulin (Development Studies Hybridoma Bank at the University of Iowa, Iowa City, IA).