All lanes: Western blot analysis of PKM with anti-PKM antibody (ER1901-90) at 1:500 dilution. Lane 1: Wild-type MDA-MB-231 whole cell lysate. Lane 2: PKM knockout MDA-MB-231 whole cell lysate. ER1901-90 was shown to specifically react with PKM in wild-type MDA-MB-231 cells. No band was observed when PKM knockout samples were tested. Wild-type and PKM knockout samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary Anti-PKM antibody (ER1901-90, 1/500) and Anti-GAPDH antibody (ET1601-4, 1/10000) were used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG H&L (HRP) Secondary Antibody (HA1001) at 1:200,000 dilution was used for 1 hour at room temperature. Cell lysate was provided by Ubigene Biosciences (Ubigene Biosciences Co., Ltd., Guangzhou, China).

Flow cytometric analysis of PKM2 was done on F9 cells. The cells were fixed, permeabilized and stained with the primary antibody (ER1901-90, 1/50) (red). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor 488-conjugated Goat anti-Rabbit IgG Secondary antibody at 1/1000 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; black).