Anti pkm2

Consecutive 3-μm sections were cut from each block and subjected to immunohistochemical staining with the EnVision+DualLink system (Dako, Carpinteria, CA, USA). An immunoperoxidase technique was applied following antigen retrieval with microwave treatment (95 °C) in citrate buffer (pH 6.0) for 45 min. Anti-PKM1 (Abgent, San Diego, CA, USA), anti-PKM2 (Cell Signaling Technology, Danvers, MA, USA), HIF1α (Thermo Fischer Scientific, Waltham, MA, USA), and anti-Ki-67 antibody (DAKO) diluted at 0.5 μg/mL were used as primary antibodies. After 2 h of incubation at room temperature, the sections were incubated with a secondary antibody for 30 min. The specimens were color-developed with diaminobenzidine (DAB) solution (Dako) and counterstained with Meyer’s hematoxylin (Sakura Finetek, Tokyo, Japan). Immunostaining of all samples was performed using the same conditions of the antibody reaction and DAB exposure. Immunoreactivities of PKM1 and PKM2 were classified according to the Allred’s score (AS) [50 (link)]: grade 1 for AS = 0, 2 for AS = 2–4, and 3 for AS = 5–8. Grade 2 and 3 cases were regarded as immunopositive [5 (link)]. We also observed 20 microscopic fields per case at 200× magnification and counted the tumor cells per case; the results were expressed as the percentage of tumor cells, with positive nuclei defined by Ki-67 and HIF1α LI [10 (link)].

Cell lines were lysed in RIPA buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and sodium orthovanadate, sodium fluoride, EDTA, leupeptin, etc.] with protease inhibitors and the total protein concentration was quantified with BCA assay (Thermo Fisher Scientific, Waltham, MA). The normalized samples were analyzed by SDS-PAGE and western blot using standard protocols and the following primary antibodies: anti-PKM1 (1:1,000 dilution, sigma, SAB4200094), anti-PKM2 (1:1,000 dilution, Cell Signaling, 4,053), and anti-actin (1:2,000 dilution, Cell Signaling, 4,970), the secondary antibodies: Anti-rabbit IgG, HRP-linked antibody (1:3,000 dilution, Cell Signaling, 7,074). Results are represented as mean and s.e.m. of at least three independent experiments.