Enhancement of CIITA transcriptional function by ubiquitin

Supplementary Fig. 1. (pdf 25K)
Flag-CIITA and Myc-CIITA have comparable half-lives. Pulse-chase analysis was used to measure the stability of transfected Flag-CIITA and transfected Myc-CIITA in COS7 cells. Cells were transfected with 1 g Flag-CIITA or 1 mg Myc-CIITA and 24 h post-transfection were labeled with ³⁵S-methionine and immunoprecipitated with anti-Flag (lanes 1-5) or anti-Myc (lanes 6-10). Both versions of CIITA have similar, extended half-lives. Identical data were obtained with a Hemagglutinin-tagged CIITA protein (not shown).

Supplementary Fig. 2 (pdf 42K)

Supplementary Fig. 3. (pdf 108K)
Ubiquitin enhances the association of CIITA with the endogenous MHC class II promoter. (a) Chromatin immunoprecipitation assays were performed on 293T cells stably expressing Fg-CIITA alone or in the presence of HA-ubiquitin. Chromatin immunoprecipitation was performed using anti-FLAG mAb M5 and MHC class II HLA-DRA promoter DNA was detected by quantitative real-time PCR. Real-time PCR values were determined by subtracting values obtained from bead-only immunoprecipitations. Input values demonstrate the total amount of MHC class II promoter DNA present in lysates (right top panel). As a negative control, Actb promoter DNA sequence was also assessed (right bottom panel). Input represents 1% of the total chromatin introduced into each immunoprecipitation reaction. Representative of four independent experiments. (b) Identical to (a) except mono-ubiquitin is shown. (c) The rabbit polyclonal anti-CIITA was generated against E coli derived 6xHis, FLAG tagged CIITA aa 1-333. Specificity was tested as follows: 1 x 10⁶ COS7 were transfected with 1 g vector containing Flag-CIITA aa 336-884 (lane 1), or HA-CIITA (lane 2). Eighteen hrs post-transfection cells were lysed in RIPA buffer and immunoblotted with anti-FLAG (panel 1), anti-CIITA (panel 2) or anti-preimmune serum (panel 3). Blots are representative of three independent experiments. (d) 1 x 10⁶ COS7 cells were transfected with 1 g Fg-CIITA. Eighteen hours post-transfection cells were lysed in RIPA buffer and the following samples were immunoblotted with the anti-FLAG mAb M2: total lysate (lane 1), lysate depleted of CIITA after immunoprecipitation with anti-CIITA (lane 2), anti-CIITA immunoprecipitation (lane 3) or anti-PKA control antibody immunoprecipitation (lane 4). Blot is representative of three independent experiments.

Top