GM-CSF gene constructions with or without ARE.

(A) Schematic representation of the CMV-GMTag AU⁺and AU⁻ DNA constructs. Gray box indicates CMV promoter; thick black lines, 5′ and 3′ UTRs; thin lines, introns; open boxes, coding region; gray thick line, ARE; and hatched box, c-Myc Tag. Primers used for PCR or RT-PCR and relevant restriction sites are indicated. (B) RT-PCR products derived from RNA of L-929 cells untransfected or transfected with both constructs. Lane 1, untransfected L-929 cells; lane 2, L929 cells treated with cycloheximide; lane 3, L929 cells stably transfected with pBK-CMV–GMTag AU⁺; lane 4, pBK-CMV–GMTag AU⁺transfected L-929 cells treated with cycloheximide; lane 5, L929 cells stably transfected with pBK-CMV–GMTag AU⁻. (C) Detection of GMTag by Western blot; 15 μL 30 × concentrated supernatant of nontransfected (lane 1) or pBK-CMV–GMTag AU⁻ transfected HeLa cells was loaded on 15% SDS-PAGE and blotted on nylon membrane. The membrane was incubated with goat antimouse GM-CSF primary antibody and HPR-coupled rabbit antigoat secondary antibody and revealed by ECL. (D) Translation of in vitro transcribed GM-Tag AU⁺ and AU⁻ mRNAs in rabbit reticulocyte lysate. After translation, 10 μL of the reaction was immunoprecipitated with anti–GM-CSF or anti-Tag antibodies. The immunoprecipitated products were analyzed by SDS-PAGE and autoradiography. Lanes 1, 2, and 3, GMTag AU⁺ RNA translation product immunoprecipitated with antimouse GM-CSF antibody (lane 1), anti-Tag antibody (lane 3), or no antibody (lane 2); lanes 4, 5, and 6, GMTag AU⁻translation product immunoprecipitated with anti–GM-CSF (lane 4), anti-Tag (lane 6) antibodies, or no antibody (lane 5).