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. 2009 Aug 14;85(2):290–295. doi: 10.1016/j.ajhg.2009.07.009

Figure 3.

Figure 3

Gene Sequence Analysis of RRM2B and Protein Analysis of p53R2

(A) Partial DNA sequence of normal RRM2B gene.

(B) Partial DNA sequence of patient's RRM2B gene showing the heterozygous c.979C→T mutation.

(C) RRM2B cDNA of patient (family 1, IV:7) myoblasts also contains the heterozygous c.979C→T mutation. We extracted total RNA from the cells with an RNeasy kit (QIAGEN). The RNA was used to generate cDNA with random primers and M-MLV Reverse Transcriptase (Promega) followed by amplification of part of the RRM2B cDNA with primers listed in Table S1 and Phusion polymerase (Finnzymes).

(D) Western analysis of the p53R2 protein in myoblasts shows a truncated p53R2 protein present only in the patient sample (Pt) and not in the control samples (1–3). Ten micrograms of total cellular protein lysate was analyzed by western blot with p53R2 antibody (ab8105 rabbit polyclonal, Abcam). Actin was used as a loading control (sc-1616 goat polyclonal, Santa Cruz Biotechnology).

(E) The C-terminal amino acids of the p53R2 protein are highly conserved. The R327 residue is indicated by an arrow, and the heptapeptide is underlined.