Fig. 1
Cell model and Co-IP optimization. (a) PRA and PRB protein structure. PRB is a full-length isoform, PRA lacks 164 amino acids at the N-terminus domain. HA-tag was appended at the C-terminus domain of both isoforms. The ligand-binding domain (LBD), hinge region (H), the DNA-binding domain (DBD), and activation function domains (AFs) are indicated. (b) Western blot results of PRA and PRB expression in T47DC42-PRA and T47DC42-PRB cells treated (+) or untreated (−) with 1000 ng/mL of Dox or Dox with 10 nM R5020 for 1 h to induce the expression of PRA or PRB proteins. Cell lysates were prepared and immunoblotted with a 1294, PR-specific antibody. Thirty micrograms of protein were loaded in each lane. Ten micrograms of T47D cell lysate was loaded as a positive control. Actin was used as a loading control. (c) Co-IP optimization, 1.5 mg of proteins from T47DC42-PRA or T47DC42-PRB treated with 1000 ng/mL of Dox for 24 h were separately IP with HA ab (1:100) and blotted with PR specific 1294 ab. Input, unbound supernatant (sup) and rabbit IgG isotype (IgG) were used as a control.
