Ciita

Immunohistochemical staining was carried out as described in [25 (link)]. Primary antibodies used were as follows: HDAC2 (dilution 1:250, Santa Cruz Biotechnology, Dallas, TX, USA; #sc-55541), CIITA (dilution 1:300, Abcam, Cambridge, UK; #ab117598), and B2M (dilution 1:250, Cell Signaling, Danvers, MA, USA; #9899).

After removal of the culture medium, the cells were washed with cold 1X PBS and were lysed using a lysis buffer supplemented with protease and phosphatase inhibitors: 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP40, 10 mM sodium fluoride, 0.1 mM sodium orthovanadate, 40 mg/mL phenylmethylsulfonyl fluoride (PMSF), 20 g/mL aprotinin, 20 mg/ml leupeptin, 2 mg/mL antipain, 10 mM p-nitrophenyl phosphate, 10 mg/mL pepstatin A and 20 nM okadaic acid. Cells were then centrifuged at 13000 rpm for 15 minutes at 4°C, and protein content of supernatant was used to determine the protein concentration by colorimetric assay (Biorad, Italy). Cell extracts were diluted 1:1 in sample buffer 2X Laemmli (0.217 M Tris-HCl pH 8.0, 52.17% SDS, 17.4% glycerol, 0.026% bromo-phenol blue, 8.7% beta-mercapto-ethanol), and then boiled for 3 minutes. Equal amounts of protein (50 μg) were run and separated by SDS-PAGE gel (acrylamide gel). Primary antibodies used were HDAC1 (Santa Cruz), HDAC2 (Alexis), HDAC3 (Sigma), CIITA (Abcam), all diluted 1:500; 100mg/ml anti-ERK1 antibody (Santa Cruz Biotechnology) was used for normalization.