Skip to main content
. 2022 Jan 20;9(8):2104055. doi: 10.1002/advs.202104055

Figure 6.

Figure 6

AKT1‐dependent PKM2 S222 phosphorylation is necessary for PKM2 and FACT interaction upon DNA damage. IP and IB analyses were performed with indicated antibodies. Data are representative of at least three independent experiments. A,B) Co‐IP was performed with anti‐Flag antibody in HEK293T cells transfected with A) SFB‐SSRP1 or B) Flag‐SPT16 and HA‐PKM2 after etoposide treatment (200 × 10–6 m, 1 h). The precipitated complex of SSRP1 or SPT16 was treated with or without CIP. C) HEK293T cells were transfected with Flag‐PKM2 WT or S222A and then treated with or without etoposide (200 × 10–6 m, 1 h). D) U251, U87 cells, or normal human astrocyte cells (HA1800) stably expressing Flag‐PKM2 were treated with or without etoposide (200 × 10–6 m, 1 h). E) HEK293T cells were transfected with SFB‐SSRP1 and HA‐PKM2 WT or S222A and then treated with or without etoposide (200 × 10–6 m, 1 h). F) HEK293T cells were transfected with HA‐SPT16 and Flag‐PKM2 WT or S222A and then treated with or without etoposide (200 × 10–6 m, 1 h). G,H) PKM2‐depleted HEK293T cells were rescued with rPKM2 WT or S222A. The cells were transfected with G) SFB‐SSRP1 or H) Flag‐SPT16 and then treated with or without etoposide (200 × 10–6 m, 1 h). Co‐IP experiment was performed with anti‐Flag antibody in the nonchromatin‐bound protein fractions. I) PKM2‐depleted U251 cells were rescued with rPKM2 WT or S222A and then treated with or without etoposide (200 × 10–6 m, 1 h). Chromatin fraction was prepared. J) U251 cells stably expressing Flag‐PKM2 were treated with or without ATM/DNA‐PK inhibitor Torin2 (100 × 10–9 m), EGFR inhibitor afatinib (5 × 10–6 m), PI3K inhibitor LY294002 (1 × 10–6 m), AKT1/2/3 inhibitor MK‐2206 (1 × 10–6 m), MEK1/2 inhibitor U0126 (5 × 10–6 m), JAK1/2 inhibitor ruxolitinib (50 × 10–9 m) or NF‐κB inhibitor BAY11‐7082 (50 × 10–6 m) for 12 h and then treated with or without etoposide (200 × 10–6 m) for 1 h. K) Co‐IP was performed with anti‐Flag antibody in HEK293T cells transfected with HA‐AKT1 and Flag‐PKM2 after etoposide treatment (200 × 10–6 m, 1 h). L) In vitro kinase assays were carried out with purified recombinant His‐PKM2 and commercially purchased recombinant GST‐AKT1.