Fig. 1.

Knockdown of PRLR in T47D cells results in augmented mature GHR abundance. A, PRLR, GHR, STAT5, and JAK2 levels. Serum-starved T47D-SCR and T47D-ShPRLR cells were extracted in detergent-containing lysis buffer. Equal amounts of protein from each cell extract were evaluated without immunopreciptation or, in a separate experiment, immunoprecipitated with anti-JAK2. Precipitated eluates or extracts were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Actin blotting served as a loading control for cell extracts. B, Densitometric analysis of data from three separate experiments, performed such as in A, was used to estimate GHR abundance. The T47D-SCR GHR level in each experiment considered 100%. Data are expressed as mean ± se. C, EndoH treatment. Serum-starved T47D-SCR and T47D-ShPRLR cells were extracted in detergent-containing lysis buffer. Equal amounts of protein from each cell extract were immunoprecipitated with anti-GHR, and precipitates were treated with EndoH or vehicle, as indicated. Eluates or extracts were resolved by SDS-PAGE and immunoblotted with anti-GHR. The positions of the mature (M; endoH resistant) and precursor (P; EndoH sensitive) forms of GHR are indicated. D, GHR mRNA levels. Total RNA was extracted from serum-starved T47D-SCR and T47D-ShPRLR cells, and quantitative real-time PCR for GHR (normalized for 18S rRNA) was performed. Pooled data are represented as mean ± se. The GHR mRNA level in the T47D-SCR cells were arbitrarily set to 100%. NS, No significant difference; WB, Western blot; IP, immunoprecipitated.