Fig. 8.

Multiple polyUb linkages were detected in TDP-S6 and TDP-43 insoluble proteome. Transfected cells were harvested and mixed with metabolically labeled cells as internal standards followed by differential extraction. The Sarkosyl-insoluble samples were used for SRM-based analysis to quantify all polyUb linkages and two other proteins (E1 and Rpn2 as loading control). A, the monitored product ion pair of Lys-48 linkage-specific peptide. B, the co-elution of Lys-48 linkage peptide pair during an LC/SRM run. C, the comparison of measured linkages and proteins in three samples: mock-, HA-TDP-43- or HA-TDP-S6-transfected cells. The data were normalized to values in the mock sample. Error bars indicate S.E., and significance (*) was determined by performing an unpaired two-tailed t test (p < 0.01). WT, wild type (Full length TDP-43).