Detection of EpoR with anti-EpoR antibodies. Protein extracts were prepared from tumor cell lines and COS-7 cells expressing FLAG-EpoR and subjected to Western immunoblotting. Each antibody was used to probe blots that had been processed simultaneously: (A) Anti-FLAG (M-2) antibody (1.8 μg/mL); (B) H-194 (0.03 μg/mL); (C) 07-311 (0.4 μg/mL); (D) M-20 (0.2 μg/mL); and (E) blot D was stripped and then reprobed with anti–cyclophilin B antibody (0.25 μg/mL) as a loading control; (F) C-20 (1.32 μg/mL); (G) C-20 preincubated with a 50-fold mass excess of HSP70-2 peptide (note absence of 66-kDa band); (H) C-20 preincubated with 10-fold mass excess of C-20p. The arrows indicate the position of the 59-kDa proteins. The positions of the molecular weight markers are illustrated on the left (kDa). FLAG-EpoR Δ40 protein was detected by the H-194 and 07-311 antibodies that recognize the extracellular domain and the N-terminal 15 amino acids, respectively, of EpoR.