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. Author manuscript; available in PMC: 2021 Aug 3.
Published in final edited form as: FASEB J. 2020 Oct 23;34(12):16476–16488. doi: 10.1096/fj.202001875R

FIGURE 1.

FIGURE 1

USP7 and USP10 are NHE3 binding DUBs. A, The HA-UbVME probe was used to identify DUBs interacting with NHE3. Post-nuclear supernatant (PNS) of Caco-2bbe/NHE3 and SK-CO15 cells were incubated HA-UbVME with or without NEM, which inhibits the covalent linkage between DUBs and HA-UbVME. Immunoblotting using anti-HA antibody showed the presence of NEM-sensitive 110 and 130 kDa bands (arrows) in both cell lines. B, PNS of Caco-2bbe/NHE3 bound to HA-UbVME was immunoblotted with anti-HA, anti-USP10 or anti-USP7 antibody. C, USP7 and USP10 co-immunoprecipitate with NHE3. Caco-2bbe/NHE3 lysate was immunoprecipitated with anti-VSVG antibody P5D4 or IgG as a control, followed by immunoblotting using anti-USP7, anti-USP10, or anti-NHE3 antibody. Lower four panels show immunoblotting of cell lysates. D, USP7, USP10 and NHE3 are present in early endosomes of Caco-2bbe cells. Early endosomal and cytosomal fractions were immunoblotted using antibodies to the proteins indicated. Rab5a and HSP70 were used as a marker for early endosome and cytoplasm, respectively. Representative blots from three or more independent experiments are shown