Sab4200094

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.

Cell lysis was performed with 1% Triton lysis buffer as previously^{37 (link)}. Cell lysates were clarified by centrifugation (20,000 × g for 15 min at 4 °C), and protein concentrations were measured with Bradford assay (Biorad, 500-0006). Samples (20–30 µg of protein lysate/sample) were subjected to SDS-PAGE followed by immunoblotting using the indicated antibodies primary antibodies: GART (Proteintech, 13659-1-AP, 1:1000), S6 Kinase (CST, 2708, Lot:8, 1:1000), p-S6 Kinase -T389 (CST, 9234, Lot:12, 1:1000), PKM1 (Sigma, SAB4200094, Lot:117M4754V, 1:1000), PKM2 (Cell Signaling, 4053 S, Lot:6, 1:1000), PHGDH (Proteintech, 14719-1-AP, 1:1000), PSAT1 (Proteintech, 10501-1-AP, 1:1000) DHODH (Proteintech, 14877-1-AP, 1:1000), APRT (Abcam, ab196558, 1:1000), HPRT1 (Santa Cruz, sc-393901, 1:1000), MTHFD1L (Proteintech, 16113-1-AP, 1:1000), β-actin (Sigma, A5316, Lot: 059M4770V, 1:5000), and HRP-conjugated anti-mouse (CST, 7076, 1:5000) and anti-rabbit (CST, 7074, 1:5000) secondary antibodies were used.