Fig 1: Anx A5 inhibited PKM2 tyrosine 105 (Y105) phosphorylation and enhanced its enzymatic pyruvate kinase activity (A) BMDMs were incubated with various concentrations of Anx A5 (60, 120 and 240 nM) in LPS&IFN-γ or IL-4 as described above, the expression of p-PKM (Y105), p-PKM2 (Ser37), PKM2 and β-Actin were determined by western blot. (B) Cellular localization of PKM2 was analyzed by immunofluorescence staining. The nuclei were stained with DAPI. Pictures are representative of five different visual fields. (C) Cells were incubated with Anx A5 (240 nM) with LPS&IFN-γ or IL-4, proteins of nuclear and cytoplasmic fractions were isolated respectively, and PKM2, Lamin B, β-Actin expression were determined by western blot and analyzed in histogram. (D) Cells were treated with Anx A5 (240 nM) with LPS&IFN-γ or IL-4, then collected and cross-linked with DSS. Tetramer, dimer and monomer form of PKM2 were analyzed by western blot. (E) Enzymatic pyrunate kinase activity was measured. (F) Association of PKM2 and Hif1α was detected by co-immunoprecipitation. Data of western blot are the representatives of three independent experiments. Date of histograms are represented as mean ± SEM of three independent experiments. #P < 0.05 vs. normal group; *P < 0.05, **P < 0.01 vs. M1 group.
Fig 2: Anx A5 attenuated M1 macrophage glycolysis and enhanced OXPHOS and fatty oxidation. BMDMs were seeded in a Seahorse XF96 analyzer culture plates and treated with Anx A5 (240 nM) in the presence of 10 ng/ml LPS and 10 ng/ml IFN-γ or 20 ng/ml IL-4 for 6 h. Real-time rate changes in extracellular acidification rate (ECAR) (A) and oxygen consumption (OCR) (C) were analyzed under basal conditions and then with sequential additions of various agonist and inhibitors at the indicated times. Basic and maximum capacity of ECAR (B) and OCR (D) were shown. (E) OCR/ECAR was analyzed. (F) Cells were incubated with various dose of Anx A5 in the presence of 10 ng/ml LPS and 10 ng/ml IFN-γ or 20 ng/ml IL-4 for 6 h. Expression of HK2, HIF1α, PKM2, LDH, GAPDH, p-AKT, AKT, β-Actin were determined by western blot. (G) Lactate dehydrogenase (LDH) enzymatic activity was assessed. (H) Lactic acid production was measured. (I) Glucose uptake was analyzed. (J) Protein expression of some transcriptional factors about lipid metabolism (PGC1β, CTP1, PPARα, PPARγ) were determined by western blot. (K) BMDMs were incubated with JC-1 at working stock and mitochondrial membrane potential was measured by flow cytometry. Data of western blot and flow cytometry are the representatives of three independent experiments. Date of histograms are represented as mean ± SEM of three independent experiments. #P < 0.05 vs. normal group; *P < 0.05, **P < 0.01, ***P < 0.001 vs. M1 group.
Fig 3: Anx A5 directly interacted with PKM2 at ASP101, LEU104 and ARG106 (A) BMDMs were incubated with FITV-Anx A5 (240 nM) or FITC (as negative control) in the presence of 10 ng/ml LPS&IFN-γ for 6 h, and washed with PBS for 3 times. Cellular location FITC-Anx A5 were observed by ultrahigh resolution laser confocal. (B) Anx A5-interacted proteins were explored by co-immunoprecipitation and mass spectrometry, and some target proteins with higher score were about glycolysis and OXPHOS, as shown. (C) Cells were treated with Anx A5 (240 nM) in the presence of 10 ng/ml LPS & IFN-γ, the interation of Anx A5 and PKM2 was determined by co-immunoprecipitation. (D) The cellular location of Anx A5 and PKM2 complex was observed by ultrahigh resolution laser confocal. (E–F) Cells were incubated with PBS or Anx A5 for 6 h, and CETSA analyzed the thermal stabilization of PKM2 protein at different temperatures and concentrations. (G) Molecular docking showed binding domain of Anx A5 and PKM2. (H) The interaction of Anx A5 with PKM2, or Anx A5 with PKM2-mutant (D101A, L104A, R106A) was measured by microscale thermophoresis. The Kd value of Anx A5 and PKM2 interaction was determined with MO. Affinity Analysis Software. The graphs displayed data are represented as mean ± SEM of three independent experiments. (I) HA-Anx A5, Flag-PKM2 or Flag-PKM2 mutant (D101A, L104A, R106A) plasmid was transfected into HET293T cells, and protein-protein interaction was determined by co-immunoprecipitation. Immunofluorescence pictures are the representatives of five different visual fields.
Fig 4: Anx A5 inhibited p-PKM2(Y105) expression in hepatic macrophages. C57BL/6 mice were treated as described in the legend of Fig. 6. Anx A5, CD163 and p-PKM2(Y105) expression in liver sections was detected by immunofluorescence. Scale bar 50 μm. Pictures are the representatives of five different fields for per mouse in every experimental group.
Fig 5: Anx A5 ameliorated HFD-induced NASH in mice C57BL/6 mice were fed HFD for 16 weeks to induce NASH. After 8 weeks of HFD, mice were administrated with 10, 30, 100 μg/kg Anx A5 once every 3 days by tail intravenous injection for 8 weeks, and fasted for 12 h before harvest (n = 9 mice per group). (A) HE staining with liver paraffin sections, scale bar 20 μm. (B) Immunohistochemistry for CD11c expression. (C) Sirius red stain for fibrosis. (D) mRNA level of α-Sma, Tgf-β, Col3a1 in liver was measured by quantitative PCR. (E) TG, TC in serum and liver tissue was analyzed by commercial kit. (F) Liver Hyp concentration. (G) Liver fibrosis index analysis, including hyaluronic acid, laminin, Type III procollagen. (H) Protein level of Fasn, Scd1, p-STAT1/STAT1, p-P65/P65, LDH, p-PKM2 (Y105), p-STAT6, PPARγ and β-actin was determined by western blot. (I) Serum inflammatory cytokines of IL-1β, IL-6 and TNFα were determined by ELISA. (J–K) The mRNA level of Fasn, Scd1, Acc, Il-1β, Il-6, Tnf-α, Cd206, Fizzl, Il-10 was determined by quantitative PCR. Date are represented as mean ± SD, n = 9 mice for each experimental group. #P < 0.05 vs. normal group; *P < 0.05, **P < 0.01 vs. HFD group. Pictures (A–C) were the representative of five field for per mice in every group. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Supplier Page from R&D Systems, a Bio-Techne Brand for PKM2 Protein
Available conjugates: Sizes Available: 20 ug