Extended Data Fig. 6: PSME4 is incorporated into multiple species of proteasome complex in multiple localizations.
a,b, Cytosolic and nuclear cellular fractions of A549 were stained with Coomassie (a) and immunoblotted for PSME4 (b). GAPDH and Histone H3 were used as cytosolic and nuclear markers respectively. c, Proteasome complexes were immunoprecipitated (IP) with PSMA1 from A549 cells (with PSME4 depletion or control) that were either treated with TNFα and IFNγ or left untreated and blotted for the indicated proteasome subunits. This experiment was repeated twice with similar results. d,e, Abundance of the proteasome subunits following mass spectrometry analysis of the αPSMA1 immunoprecipitate from nuclear and cytosolic fractions. A heatmap of abundances where values are scaled by row (d). A volcano plot showing the log2 transformed fold change between median abundance of each subunit in the nuclear and cytosolic proteasomes is plotted against the significance of the difference (two-sided student’s T.test; e). Colors indicate the identity of the proteasome subunits. f, The carboxy terminal residue of peptides was used to classify these peptides based on the proteasome activity attributed to their cleavage. The abundance of PSME4 in the samples based on whole cell proteomics correlated with the chymotryptic-like signature (spearman rho = 0.33, n = 8 tumor and 8 adjacent samples). g, Workflow of the size exclusion separation. A549 cells were treated with TNFα and IFNγ (TI) for 24 hours or left untreated, lysed and then recombinant PSME4 was added to the TI treated A549 lysates. h, Lysates were separated by size using Superose6 gel filtration column and blotted against different proteasome subunits as indicated. i, Input lysates of A549 cells treated with TNFα and IFNγ (TI) or untreated used for immunoprecipitation shown in Fig. 3f blotted with the indicated antibody. j,k, Input lysates of A549 cells treated with TNFα and IFNγ (TI) or untreated (j) and the immunoprecipitation with the indicated antibody (k) blotted for the indicated proteasome subunit. Experiments in h-k were repeated twice with similar results.
