Cotransfection of Stat3 enhances Jak3 promoter activity in M1 cells, and Sp1 and Stat3 proteins present in nuclear lysates of M1 cells bind to a radiolabeled probe corresponding to theJak3pr sequence. (A) Either wild-type Stat3 or a constitutively active mutant of Stat3 (Stat3-C) were cotransfected with the -529 Jak3pr construct into either unstimulated M1 cells or M1 cells stimulated with IL-6. Following a 20-hour incubation period, cells were lysed and analyzed for luciferase activity. Relative expression refers to the fold increase in luciferase activity over vector alone. Values expressed are the average of 3 independent experiments plus or minus one standard deviation. (B) EMSA analysis using a radiolabeled probe corresponding to the -39 to -89 sequence of the Jak3 promoter and nuclear lysates from M1 cells is depicted. Lanes 1 to 3 demonstrate gel shifts obtained by using nuclear lysates from unstimulated M1 cells. A band supershift is seen with the addition of anti-Sp1 antibody (arrow, lane 2). The band shift was competed away with the addition of 100-fold excess of cold -39 to -89 probe (lane 3). Lanes 4 to 6 demonstrate gel shifts obtained by using nuclear lysates from M1 cells stimulated with IL-6. A band supershift is seen with the addition of anti-Sp1 antibody (arrow, lane 5). The band shift was competed away with the addition of 100-fold excess of cold -39 to -89 probe (lane 6). (C) Lanes 1 to 3 demonstrate band shifts obtained using a radiolabeled probe corresponding to the -38 to -58 sequence of the Jak3 promoter. A band supershift is seen with the addition of anti-STAT3 antibody (arrow, lane 2). The band shift was competed away with excess of cold -38 to -58 probe (lane 3). The band shift obtained using the m67 STAT3 consensus oligonucleotide19 is seen in lane 4, and a band supershift is seen with the addition of anti-STAT3 antibody (arrow, lane 5). A different complex is obtained by using a labeled probe containing -45 and -46 A to C mutations that disrupt the -44 to -53 GAS element (lane 6).
