γ-Interferon induction of HLA class II mRNAs in dermal fibroblasts studied by RNAse protection analysis

Co-occupancy of the interferon regulatory element of the class II transactivator (CIITA) Type IV promoter by interferon regulatory factors 1 and 2

“…The GBP RPA probe, which protects a 138 bp mRNA fragment, was prepared by in vitro transcription of HindIII digested GBP plasmid, pGT7 (a generous gift from Thomas Decker, Fraunhofer Institute of Toxicology) by T7 polymerase. The g-actin RPA probe was generated as previously described (Blanck et al, 1990;Lu et al, 1996). RPA was performed using 10 mg of cytoplasmic RNA prepared from cells either untreated or treated with recombinant human IFN-g (Genzyme) at 400 U/ml for 48 h, as previously described (Blanck et al, 1990;Lu et al, 1996).…”

“…The g-actin RPA probe was generated as previously described (Blanck et al, 1990;Lu et al, 1996). RPA was performed using 10 mg of cytoplasmic RNA prepared from cells either untreated or treated with recombinant human IFN-g (Genzyme) at 400 U/ml for 48 h, as previously described (Blanck et al, 1990;Lu et al, 1996). Preparation of nuclear extracts…”

Impaired class II transactivator expression in mice lacking interferon regulatory factor-2

“…The PCR product was digested with EcoRI and gel puri®ed. The PCR product (100 ng) was used for in vitro synthesis of the RPA probe by T7 RNA polymerase as described previously (Blanck et al, 1990;Lu et al, 1996). The synthesized 32 P-labeled probe protected +1 to +59 region of the mouse CIITA type IV mRNA.…”

“…The 32 P-labeled b-actin antisense RNA probe was prepared by in vitro transcription of pTRI-actin-mouse template (Ambion) using T7 RNA polymerase in the presence of 50-fold unlabeled UTP. Twenty-®ve mg of total RNA isolated from kidneys and lungs was analysed by RPA as previously described (Blanck et al, 1990;Lu et al, 1996). For synthesis of the RPA probe speci®c for the mouse type IV CIITA mRNA, the RPA templates were generated by PCR using the following primer set: 5'-CGCGGAATTCAT AGCTGCCAGGAGAC (forward primer, an EcoRI site is indicated as the italic type); 5'-TAATACGACTCACTA-TAGGGAGGAAGCTTGCTCTGAGTG (reverse primer, the T7 promoter is underlined).…”

Different levels of control prevent interferon-γ-inducible HLA-class II expression in human neuroblastoma cells

“…MHC-II molecules expressed by DC play an essential role in the control of the immune response, since they present antigenic peptides to CD4 þ T lymphocytes (Steinman, 1991;Germain, 1994;Guermonprez et al, 2002). MHC-II molecule expression in most nonimmune cells can be induced by different stimuli, among which the most potent is IFN-g (Collins et al, 1984;Blanck et al, 1990;Glimcher and Kara, 1992;Steimle et al, 1994). In tumors, a noticeable heterogeneity of MHC-II constitutive and inducible expression has been reported (Brocker et al, 1984;Sheen-Chen et al, 1994;Blanck, 1999;Van der Stoep et al, 2002).…”