FIGURE 2.

Isolation and analysis of proteins that interact with Gadd45a TAP in mouse skeletal muscle fibers. TA skeletal muscle fibers of 48 mice were transfected with 20 μg of empty TAP plasmid (one TA per mouse) or 20 μg of Gadd45a TAP plasmid (the contralateral TA in each mouse). Ten days post-transfection, bilateral TA muscles were harvested and used to prepare pooled protein extracts from each of the two groups of skeletal muscles (control and Gadd45a). The pooled protein extracts were then subjected to sequential purification steps with anti-FLAG magnetic beads and S-protein affinity gel. An aliquot of each final pulldown sample was visualized by SDS-PAGE and silver staining, as shown. The remaining portions of control and Gadd45a pulldown samples were subjected to mass spectrometry and a data analysis workflow that is summarized here and fully detailed under “Experimental Procedures” and the supplemental Tables.