Western blot detection of EpoR with C-20 anti-EpoR antibodies. Whole-cell extracts were prepared from unstimulated UT-7 cells (lane 1), Mo7E cells (lane 2), UT-7 cells stimulated for 10 minutes (lanes 3 and 10) or for 90 minutes (lane 4) with Epo, UT-7 cells incubated for 6 hours with cycloheximide (lane 5), and EpoR-transfected BaF3 cells stimulated for 10 minutes with Epo (lane 11). UT-7 cells stimulated for 10 minutes with Epo or biotinylated Epo (lane 6) were solubilized as previously described,⁴  and cell extracts were precipitated with streptavidin (lane 6), a laboratory-made anti-GST–EpoR antibody (lane 7), anti-GST antibodies (lane 8), and anti-Epo antibodies (lane 9). Portions corresponding to 250 × 10³ cells (whole-cell extracts) or to 10⁶ cells (precipitations) were separated on 8.5% (A-B,D) or 10% polyacrylamide gels (C) and transferred to nitrocellulose and analyzed by WB using Santa Cruz Biotechnology C-20 antibodies (catalog no. SC-695) batch K200 (A left, B-D) or batch B2105 (A right). Images were recorded on a LAS 3000 FujiFilm camera. (A-C) Chemiluminescence detection of the proteins recognized by C-20 antibody. Panel B is an enlarged view of panel A, lanes 1-3. (D) Molecular weight markers from Biolabs (Saint-Quentin en Yvelines, France) (catalog no. P7702S) were stained with Ponceau Red after transfer on nitrocellulose and pencil labeled (MW). The upper and lower limits of the BSA band are indicated. After processing for WB analysis, 2 images of the nitrocellulose sheet were successively recorded to detect chemiluminescence and pencil labels, respectively. The images were overlaid and analyzed using MultiGauge software (Fujifilm), giving the molecular masses indicated in the text (molecular mass of the EpoR mature band, 67.6 to 70.4 kDa; molecular mass of the EpoR maturing band, 64 kDa).