Sc 377092

Paraffin-embedded sections from primary tumor of 102 patients with lymph node metastasis were deparaffinized in xylene and hydrated in decreasing concentrations of ethyl alcohol. The sections were incubated with 3% hydrogen peroxide to block endogenous peroxidase activity, and heated for 10 min at 105 °C by autoclave in Target Retrieval Solution (DAKO, Carpinteria, CA, USA). Nonspecific binding was blocked by incubating with 10% normal rabbit serum for 10 min. The specimens were incubated with anti-GLUT1 antibodies (sc-377228; 1:150; Santa Cruz, Dallas, TX, USA; RRID: AB_2716767) for 30 min at room temperature, anti-PKM2 antibodies (sc-365684; 1:200; Santa Cruz; RRID: AB_10844484) at 4 °C overnight, anti-PDH-E1α antibodies (sc-377092; 1:100; Santa Cruz; RRID: AB_2716767)^{35 (link)} at 4 °C overnight, and with CA9 antibodies (NB100–417; 1:1000; Novus Biologicals, Centennial, CO, USA; RRID: AB_10003398) for 30 min at room temperature. These sections were incubated with a mouse linker for 10 min and peroxidase-labeled polymer (Histofine SAB-PO(M) kit, Nichirei Biosciences Inc., Tokyo, Japan) for 5 min, followed by counterstaining with Mayer’s hematoxylin.

Purified PDP1-His₆ was immobilized on HisPur resin columns (Pierce Pull-down polyHis protein: protein interaction Kit, Thermo Scientific). WT and TAZ-KO myoblasts (2 × 10⁶) were lysed in a buffer containing 25 mM Tris, 75 mM NaCl, and 0.1% Tween 20 (pH 7.4) and washed once with PBS. After lysing on ice for ∼30 min, the cells were briefly sonicated and centrifuged to obtain a clarified supernatant that was then incubated overnight at 4 °C in a column with immobilized PDP1-His. The next day, the column was washed 5 times before elution. The lysates were then used for immunoprecipitation by using specific antibodies. PDH-E1, 1:500 (sc-377092, SantaCruz), PDH-E2, 1:2000 (13426-1-AP, Proteintech), ACTIN, 1:1000 (sc47778, Santa Cruz); PDP1, 1:1000 (65,575, Cell Signaling).