Ap1062

Eluted PDP1 from the PDP1 pull-down assay was further incubated with purified PDH complex in PDP1 buffer containing 10 mM HEPES, pH 7.5, 100 μM EDTA, 0.5% BSA, and 1 mM DTT at room temperature (41 (link)). The dephosphorylation of Ser²⁹³ of PDH was tested using p-PDH (Ser293), 1:1000 (AP1062; Millipore).

Protein was extracted in RIPA buffer with proteinase and phosphatase inhibitor cocktail (ChemCruz). 20 μg of denatured total protein for each sample was loaded into an SDS-PAGE gel and separated by electrophoresis. The gel was transferred onto PVDF membranes using a wet transfer method. After transfer, the membranes were probed with the following primary antibodies against NDUFB6, 1:10,000 (ab110244, Abcam); p-PDH (Ser293), 1:1000 (AP1062, Millipore); IP3R 1:500 (sc-271197, Santa Cruz), GRP75 1:500 (sc-133137, Santa Cruz), and VDAC1 1:500 (sc-390996, Santa Cruz). Total protein was quantified from each sample using a No-Stain labeling reagent (Invitrogen) and subsequently used for total protein normalization (TPN). Anti-mouse or anti-rabbit secondary antibodies (1:10,000, Invitrogen) were incubated for 1 h at room temperature, and membranes were developed using SuperSignal West Pico PLUS or West Atto ECL (Thermo Scientific).

Protein was extracted in RIPA buffer with proteinase and phosphatase inhibitor cocktail (ChemCruz). 20 μg of denatured total protein for each sample was loaded into an 8–12% SDS-PAGE gel and separated by electrophoresis. The gel was transferred onto polyvinylidene fluoride (PVDF) membranes using a wet transfer method. After transfer, the membranes were probed with the following primary antibodies against PDK4, 1:1000 (ab214938; Abcam); NDUFB6, 1:10,000 (ab110244; Abcam); FOXO1, 1:1000 (14,952; Cell Signaling); FOXO1, 1:1000 (2880; Cell Signaling); ACTIN, 1:1000 (sc47778; Santa Cruz); p-AMPK (Thr172), 1:2000 (2535S; Cell Signaling); AMPKα 1:1000 (2793S; Cell Signaling); TUBULIN, 1:10,000 (ab184970; Abcam); H4, 1:1000 (ab16483; Abcam); H3, 1:500 (sc517576; Santa Cruz) GLUT4, 1:2000 (66,846; Proteintech); PDH-E1, 1:1000 (sc377092; Santa Cruz); and p-PDH (Ser293), 1:1000 (AP1062; Millipore). Anti-mouse or anti-rabbit secondary antibodies (1:10,000) were incubated for 1 h at room temperature, and membranes were developed using SuperSignal™ West Pico PLUS or West Atto ECL (Thermo Scientific).