FIG 8.

Identification of proteins associated with C9. (A) Coomassie blue-stained protein bands. Control and A549 2×Myc-C9 cells were pretreated with 2,000 IU/ml of IFN-β for 24 h. The cells were lysed, and proteins were affinity purified using Myc-Trap agarose beads. The bound proteins were eluted and resolved by SDS-PAGE, and the gel was stained with Coomassie blue. Gel bands 1 to 4 were excised individually from the 2×Myc-C9 cells and the corresponding positions of control cells and analyzed by mass spectrometry. A ladder of protein markers (in kilodaltons) is on the left. (B) Protein interaction network. Proteins identified by mass spectrometry and listed in Table 1 were analyzed using the String Protein-Protein Interaction Network. Connecting lines indicate known and predicted protein interactions. Node colors (Gene Ontology terms) are as follows: red, proteolysis (GO: 0006508); blue, cullin deneddylation (GO: 0010388) and COP9 signalosome (GO: 0008180); green, SCF ubiquitin ligase complex (GO: 0019005); white, unassociated proteins. (C) Verification of the mass spectrometry results. Proteins from lysates of control or A549 2×Myc-C9 cells with (lanes +) or without (lanes −) IFN-β treatment were captured with Myc-Trap beads and analyzed by Western blotting using antibodies to the Myc epitope, SKP1, CUL1, and COPS7A.