Figure 2. Cell lysates from both control and patient lymphocytes show evidence of MCM4 protein.

(A) Lysates from patient and control human lymphocytes were immunoblotted with an anti- MCM4 antibody. While control subjects have two main protein species, of approximately 96 and 85 kDa, the predicted full-length 96-kDa product is missing in the patients. (B) HEK293 cells were transfected with full-length MCM4 or MCM4 with the first methionine mutated, both of which were tagged with a C-terminal HA tag. The lysates were then blotted with both MCM4 antibody (left panel) and HA antibody (right panel). Abolition of the initiating methionine of MCM4 leads to expression of a smaller MCM4 species, of approximately 85 kDa. (C) FLAG-tagged full-length MCM4 and constructs beginning at the second and third in-frame ATGs were expressed in HEK293 cells. Lysates from each were run individually (lanes 1–3), all together (lane 4), or in combination with the full-length and third ATG (lane 5) and immunoblotted using an anti-FLAG antibody. The relative mobility of the different species indicates that the smaller MCM4 species may be produced by translation from the third in-frame ATG. (D) Different cell lysates show evidence of both the major full-length and smaller MCM4 isoforms when immunoblotted with the MCM4 C-terminal antibody. However, when the same lysates are immunoblotted with the N-terminal antibody, the smaller MCM4 isoform is no longer seen, suggesting that the smaller isoform is missing the N terminus of MCM4.