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. 2021 May 20;12(6):517. doi: 10.1038/s41419-021-03782-w

Fig. 4. TSP50 binds with PKM2 and depends on PKM2 for its metabolic effects.

Fig. 4

A Identification of proteins binding to endogenous TSP50 by LC-MS/MS in Huh7 cells. Candidate binding proteins to TSP50 were obtained after non-specific proteins were removed. B, C Huh7 and Bel7402 cells were harvested and subjected to immunoprecipitation with anti-TSP50 antibody, followed by Western blot analysis with anti-PKM2 antibody. D, E Huh7 and Bel7402 cells were harvested and subjected to immunoprecipitation with anti-TSP50 antibody, followed by Western blot analysis with anti-PKM1 or anti-PKLR antibody. F, G GST pull-down of Flag-PKM2 by GST-TSP50 using proteins purified in B21 bacteria, followed by Western blot analysis with anti-PKM2 and anti-GST antibody. H Huh7 and Bel7402 cells were fixed and subjected to immunofluorescence analysis. TSP50 co-localized with PKM2 in Huh7 and Bel7402 cells. Scale bar, 10 μm. I–P Glucose consumption, lactate production and ATP levels were analyzed after silencing of PKM2 abrogates the metabolic effects of TSP50. All aerobic glycolysis values were normalized to protein levels. N = 3 biologically independent replicates. t-Test statistical analysis was used. Data were presented as means ± s.d.