Figure 4.

RNA addition makes coilin more vulnerable to calpain digestion. (A) Coomassie blue stained SDS-polyacrylamide gel shows untreated and calpain digested coilin levels. DNA and RNA free bacterial expressed dialyzed coilin was digested with increasing concentrations of calpain in the presence of 1 mM CaCl₂ at 30°C for 15 min, following pre-incubation (5 min) of dialyzed coilin with or without HeLa total RNA. A portion of the reactions was subjected to SDS-PAGE and stained with Coomassie. (B) The remainder of the reactions was subjected to SDS-PAGE then Western transfer, and blots probed with anti-coilin H300 antibodies (left panel) and anti-coilin pδ antibodies (right panel). Note the lane assignments in (B) are the same as in (A). The letters a, b and c indicate other fragments released from full length coilin upon calpain digestion in addition to the cleaved fragment.