Figure 7: Identification of HACE1 interacting proteins by MS analysis of HA Co-IP.

(a,b) Hace1^−/− MEF cells were stably transfected with HA-tagged WT HACE1 (HA-HACE1) and empty vector MSCV. (a) Immunoblot for HA shows stable expression of HA-tagged HACE1 in HA-HACE1 MEF cell lysate. (b) HA-HACE1 MEF and control MSCV MEF cell lysates were subjected to Co-IP with anti-HA antibody and coomassie blue staining shows a couple of distinct bands only detected in HA-HACE1 MEF IP pull-down as indicated by red arrows. (c–f) Identification of HACE1 interacting proteins by MS analysis of Co-IP products. (c) Hierarchical clustering (heat) map demonstrating the clustering of proteins based on their expression intensities in HA-HACE1 MEF and control MSCV MEF HA-IP pull-down products. To the left of the heat map, selected annotations found to be enriched in the protein clusters are shown. n=5 for HA-HACE1 group and n=4 for control MSCV group. (d) Pie chart shows enriched representative HACE1-interacting proteins according to molecular function by GO term. (e) Selected HACE1-interacting proteins identified by HA-IP MS were evaluated by western blot with the indicated antibodies (IB). HA-HACE1 denotes HA-IP pull-down from HA-HACE1 MEF; MSCV denotes HA-IP pull-down from control MSCV MEF; and input indicates HA-HACE1 MEF lysate before IP. (f) HA-HACE1 MEF and control MSCV MEF lysates were subjected to co-IP with an anti-Unc-45b antibody and the pull-down were analysed by western blot with an anti-HA antibody. (g,h) HA-IP pull-down products from HA-HACE1 MEF (HA-HACE1) and Hace1 KO MSCV MEF (MSCV) immortalized in HA beads were further subjected to Co-Co-IP with WT mouse heart lysates, and the pull-down products were analysed by western blot using the indicated antibodies. HACE1 and those proteins with which it interacts were still detectable (g). There were also increased ubiquitinated proteins in the Co-Co-IP heart tissues (h).