Fig. 2: GP73-mediated PKM2 secretion depends on the interaction of GP73 with PKM2. | Cell Death & Disease

Fig. 2: GP73-mediated PKM2 secretion depends on the interaction of GP73 with PKM2.

From: GP73-mediated secretion of PKM2 and GP73 promotes angiogenesis and M2-like macrophage polarization in hepatocellular carcinoma

Fig. 2

A–C GP73 interacts with PKM2 in vivo. A The interaction between exogenous GP73 and PKM2 was confirmed in HEK293T cells. HEK293T cells co-transfected with FLAG-PKM2 and HA-GP73 were lysed with IP lysis buffer and Co-IP assay was carried out by using anti-FLAG/HA antibody and followed by western blotting with anti-FLAG/HA antibody. B The interaction between endogenous GP73 and PKM2 was confirmed in HepG2 cells. HepG2 cells were lysed with IP lysis buffer and subjected to immunoprecipitation with anti-GP73 or anti-PKM2 antibody followed by western blotting with anti-GP73 or anti-PKM2 antibody. C IF images reveal the colocalization of GP73 and PKM2 in the cytoplasm. HCC cells were stained with GP73 antibody (red) and PKM2 antibody (green). DAPI (blue), nucleus. Scale bar, 5 μm. D GP73 interacts with PKM2 in vitro. GST pull-down assay was performed by using HA-GP73/His-PKM2 with GST or GST-PKM2/GST-GP73 followed by western blotting with anti-HA/His antibody. E Schematic diagram of the truncated regions of GP73 and the interaction of GP73 with PKM2. PKM2 interacts with domain III of GP73. GST pull-down assay was performed by using His-PKM2 and GST or GST-tagged various domains of GP73 protein and followed by western blotting with anti-His antibody. Arrows indicate the location of target proteins. F Schematic diagram of the GP73 functional region and the interactions between PKM2 and deletion mutant GP73. PKM2 interacts with 146-205 aa in domain III of GP73. HEK293T cells co-transfected with FLAG-PKM2 and HA-tagged GP73, Δ36-100, Δ91-150, or Δ146-205 were lysed by IP lysis buffer and subjected to immunoprecipitation with anti-FLAG/HA antibody followed by western blotting with anti-FLAG/HA antibody. Arrows indicate the location of the target proteins. Δ represents the deletion of amino acid residues. G Top, IF images show that HCC cells co-transfected with HA-Δ146-205 and PKM2 are less co-localized than those co-transfected with HA-GP73 and PKM2. HCC cells co-transfected with PKM2 and HA-tagged GP73 or Δ146-205 were stained with HA antibody (red) and PKM2 antibody (green). DAPI (blue), nucleus. Scale bar, 2 μm. Bottom, Pearson’s correlation analysis of the correlation between PKM2 and GP73 or Δ146-205 in HCC cells. H Deletion of 146-205 aa in domain III of GP73 reduces PKM2 secretion. The protein levels of GP73 and PKM2 in cell lysate and supernatant were measured by western blotting in HCC cells transfected with vector, GP73 or Δ146-205 plasmid. β-actin was used as an intracellular loading control, and GST as an extracellular loading control. Arrows indicate the location of the target proteins. Δ represents the deletion of amino acid residues. Error bars represent mean ± SD (n = 3). *p <0.05**, p <0.01.

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