Immunodetection of RPK in the circulating erythroid fraction and peripheral mononuclear cells. (A) Western blot analysis using mouse antibodies against recombinant human RPK on PBMCs and red cells from a healthy control and the patient. The 11-bp insertion resulted in the lack of the wild-type subunit bands (61.7 and 57.2 kDa) from the red cell fraction of the patient. (B) Western blot analysis performed on the same set of samples using antibodies against recombinant human RPK raised in rabbits detecting the RPK (61.7 and 57.2 kDa) and the M2PK (58.1 kDa) wild-type subunit bands. M2PK was detected in PBMCs from the control and the patient, but not in the corresponding RBC fractions. The top extra band in all the tracks has a molecular weight not corresponding to any PK species and was interpreted as an unspecific reaction of this rabbit antibody. If the 32.2-kDa band detected in the PBMC was a degradation product of M2PK, the same band appearing in the erythroid fraction from the patient but absent in the healthy control could be attributable to the presence of M2PK relics from the abundant erythroblasts in the patient's erythroid fraction. It should be noted that reticulocytes/erythroblasts, mainly in the patient, could have been present in the mononuclear cells and mature red cell fractions when separation was performed by centrifugation. Molecular weights for the RPK monomers were calculated according to published data.³⁰