Figure 5.

Purification and identification of coilp1. (a) A flow chart depicting the overall process used to purify and identify the peptide sequence of the coilin derivative coilp1. (b) Western blot showing that coilp1 can be enriched in the ammonium sulfate supernatant fraction (lane 1) compared to coilin, which is enriched in the ammonium sulfate precipitant (lane 2). (c) The ammonium sulfate supernatant fraction shown in lane 1 of (b) was subjected to HPLC. Western blotting of some of the fractions from the HPLC run was conducted and the membrane probed with anti-coilin antibodies. The location of coilp1 is indicated and found to be enriched in fraction 8. Fraction 8 was subjected to tryptic digestion and LC/MS to identify peptide sequences. (d) Alignment of coilin and coilp1 amino acid sequence. Peptides identified by mass spectrometry are highlighted in yellow or pink. Regions of overlap between peptides is indicated by an orange color. Note that 7 out of the 8 peptides unambiguously show that the COILP1 pseudogene can encode the coilp1 protein. One peptide contains sequence (NTTADK) that is shared between coilp1 and coilin (Fig. S2 and Tables S1 and S2). Peptide SLIWNQKLSQIRLPAKK has low confidence (Table S1), but complementary MRM evidence (Table S2) support the existence of this and other peptides. (e) Reduction of coilp1 protein via RNAi. Cells were transfected for 48 hrs with control or coilp1 (13.3 and 13.11) siRNAs, followed by lysate generation, SDS-PAGE, Western transfer and probing with anti-coilin antibodies.