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. 2014 Mar 21;9(3):e92949. doi: 10.1371/journal.pone.0092949

Figure 2. HSP40 impairs PKM2 stability and functions in HeLa cells.

Figure 2

(A, B and C) HeLa, A549 and HepG2 cells were transfected with Flag-HSP40, after 48 h incubation, endogenous PKM2 protein levesl were detected by anti-PKM2 antibody. (D) HeLa cells were transfected with HSP40 siRNA, after 48 h incubation, PKM2 protein level was analyzed using anti-PKM2 antibody. (E) Cell lysates of HEK293T cells transfected with HA-HSC70 and Flag-HSP40/DNAJB1 were immunoprecipitated with anti-Flag antibody, the bound proteins were detected by anti-HA antibody. (F) HeLa cells transfected with HA-HSC70 or both HA-HSC70 and Flag-HSP40 were lysed, then western blot was analyzed using anti-PKM2, anti-HA and anti-Flag antibodies. (G) Pyruvate kinase activity of extracts prepared from HeLa cells transfected with HSP40 siRNA. The pyruvate kinase activity is detected by PK assay kit and normalized by protein measurement (mean ± S.D., n = 3). (H and I) HeLa cells were transfected with HSP40 siRNA or Flag-HSP40, after 24 h normal incubation followed with 24 hr anaerobic incubation, RT-PCR was performed to analyze PDK1 mRNA level (mean ± S.D., n = 3).