Fig. 2

Cellular and molecular characterization of U937 Plus cells stably expressing exogenous CIITA. a U937 Plus cells were stably transfected with pcfCIITA vector expressing flag-tagged CIITA, generating Plus-CIITA cells. Bulk population was analyzed for HLA-II DR expression by FACS analysis. HLA-II-positive clones were isolated by sorting and cloning. FITC-conjugated isotype-matched secondary antibody (IgG2a) (dotted line) represents the negative control. b HLA-II DR mRNA expression was assessed by qRT-PCR in U937 Minus cells, U937 Plus cells and in clones 1C11, 1F6, 4G2. The results of a representative experiment performed in triplicates are shown. mRNA values are expressed relatively to that of Minus cells set to 1. Unpaired two-tailed t test has been performed (****P < 0.0001; ***P < 0.005). Error bars represent the standard deviation. c Cell lysates obtained from U937 Plus cells (60 × 106 cells) (lane 1), U937 Minus cells (60 × 106 cells) (lane 2) and CIITA-transfected Plus clones (30 × 106 cells) (lanes 3–5) were immunoprecipitated with an anti-CIITA antibody (IP a CIITA) and analyzed for the presence of CIITA by western blotting. Raji cell lysate (30 × 106 cells) was used as a positive control of CIITA expression (lane 6). Eight percent of the pre-cleared cell lysates was analyzed for the expression of α-tubulin by western blotting (input)