Figure 2. Validation of specific RNA–protein interactions from yeast and mammalian cells isolated by 2C.

(A) Evaluation of 2C performance examining known yeast RBPs by Western blotting. 2C eluates equivalent to 12.5 μg of RNA were treated or not with RNase I, boiled in loading buffer, and separated through SDS–PAGE. Specific RBPs were visualized with antibodies against Pab1 and GAPDH. The DNA-binding histone H3 was probed as a negative control. (B) Validation of additional non-canonical yeast RBPs by Western blot after 2C. The samples were treated as in (A), and hexokinase, triose phosphate isomerase, and tubulin, as a negative control, were analyzed with specific primary antibodies. Note that S. cerevisiae hexokinase B is known to aggregate under denaturing conditions in vitro into amyloid-like fibrils (Ramshini et al, 2011). The denaturing conditions during 2C capture, thus, may have promoted the formation of hexokinase aggregates resistant to SDS–PAGE separation. (C) Analysis of mammalian 2C eluates by Western blot. The samples were treated as above (A), and the proteins hnRNPC1/C2, GAPDH, FASTKD4, and histone H3 were detected with specific primary antibodies. R−, non–RNase-treated samples; R+, RNase I–treated samples.