Figure 4.

ASK2 protein is degraded in response to LPS stimulation of macrophages and is shown to interact with proteins from the blue down module of Database #3. Western blots visualizing the presence of recombinant ASK2-HA-V5 protein in the fractions that were combined to make the HMW samples (0.5 mL fractions from the 8 to 10 mL elution range) analyzed in Database #4 are shown in (A), along with the changes in relative quantification as compared to the total intensity from all fractions; unpaired t-test results are shown. Full elution profiles and quantification of the LMW range are shown in Figure S14, and full blots are shown in Figure S15. SILAC quantification of ASK2 protein levels following PARPi treatment, varying amounts of LPS stimulation, lysis, and immunoprecipitation are shown in (B), revealing that the global ASK2 protein level decreases in response to LPS, and this response is unaffected by PARP inhibition. These SILAC ratios are based on data from four independent replicates that were analyzed with a one-way ANOVA with Bonferroni post-testing. A PCA-based comparison of the ASK2 interactome with the top 10 proteins from each of the known down modules from Database #3 reveals similarity between the ASK2 interactome and the MAPKKK regulatory [blue down] module, suggesting that the ASK interactome may be represented by this module (C). The SEC elution profiles of four proteins that are included in both the MAPKKK regulatory module and the ASK interactome are shown in (D), both before (black) and after (red) a 30 min LPS stimulation. A STRINGdb³⁷ diagram of the overall ASK2 interactome is shown in (E); inclusion in the interactome was based on the proteins being identified by at least two peptides in at least three of eight biological replicates, in the bait (αV5) but not the isotype control pulldown, and appearing in the CRAPome⁴² 25 times or less. *p = <0.05, **p = <0.01, and ***p = <0.001.