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. 2013 Jun 21;8(6):e67313. doi: 10.1371/journal.pone.0067313

Figure 1. BRCA1 is a ribosome-associated protein.

Figure 1

A/MCF7 cells were lysed in 25 mM KCl buffer and the post-mitochondrial cytoplasmic lysate was layered onto a 1 M sucrose cushion and centrifuged as described in the “Material and methods” section. Immunoblotting for BRCA1 using the MS110 antibody was performed on the following samples: initial total cell lysate (L), nuclear fraction (N), cytoplasmic fraction (C) and ribosome pellet (R). PABP1 and eIF4G were used as markers for pellet fraction containing ribosome-associated proteins. The analyzed L, N and C fractions represent 5% of the total cell lysate. B/MCF7 cells were lysed in 25 mM KCl buffer and the cytoplasmic fraction was separated onto a 10–40% sucrose gradient. (Top) A characteristic ribosome profile. (Middle) Extracts of total RNA from half of each fraction were subjected to gel analysis to determine the presence of 18S and 28S rRNAs. rRNAs were detected by Gel Red staining. (Bottom) The remaining half of each fraction was precipitated with TCA. BRCA1 protein was identified with immunoblot analysis using D9 antibody. PABP1 and eIF4G served as controls.