Pcad ser1859

After desired treatments as specified as indicated, cells were washed twice with PBS and lysed in buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycer-ophosphate, 1 mM sodium vanadate, 1 mg ml⁻¹ leupeptin, 1 mM phenylmethyl-sulfonylfluoride). Equal amounts of protein (30 μg) were loaded onto 10% SDS–PAGE gels. Western detection was carried out using a Li-Cor Odyssey image reader (Li-Cor, USA) (Supplementary Fig. 17). The goat anti-rabbit IgG (Cat#C30502-01) and goat anti-mouse IgG (Cat#C30509-01) secondary antibodies were obtained from Li-Cor (USA). The final concentration of the secondary antibodies used was 0.1 μg ml⁻¹ (1:10000 dilution). The primary antibodies against β-Actin (Cat#60008-1, 1:5000 dilution), GOT1 (Cat#14886-1-AP, 1:1000 dilution), DHODH (Cat#14877-1-AP, 1:1000 dilution), HIF1α (Cat#20960-1-AP, 1:1000), and UMPS (Cat#14830-1-AP, 1:1000) were purchased from Proteintech (USA). Antibodies against CAD (Cat#sc-376072 from Santa Cruz, USA) and pCAD (Ser1859) (Cat#70307 from Cell Signaling Technology) were used with a dilution of 1:1000.

After desired treatments as specified as indicated, cells were washed twice with PBS and lysed in buffer on ice (20 mM Tris–HCl, pH 7.5, 150mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1mM β-glycer-ophosphate, 1mM sodium vanadate, 1mgml-1 leupeptin, 1mM phenylmethyl-sulfonylfluoride). Equal amounts of protein (30 μg) were loaded onto 10% SDS–PAGE gels. Western detection was carried out using a Li-Cor Odyssey image reader (Li-Cor, United States). The goat anti-rabbit IgG (Cat#C30502-01) and goat anti-mouse IgG (Cat#C30509-01) secondary antibodies were obtained from Li-Cor (United States). The final concentration of the secondary antibodies used was 0.1 μg/ml (1:10000 dilution). The primary antibodies against β-Actin (Cat#60008-1, 1:5000 dilution), GAPDH (Cat#60004-1-Ig, 1:5000 dilution), FLAG Tag (Cat#80010-1-RR, 1:3000 dilution), α-Tubulin (Cat#11224-1-AP, 1:5000 dilution), CPS1 (Cat#18703-1-AP, 1:1000 dilution), OTC (Cat#26470-1-AP, 1:1000 dilution), ARG1 (Cat#16001-1-AP, 1:1000), GDH1 (Cat#14299-1-AP, 1:1000 dilution), and GS (Cat#11037-2-AP, 1:1000 dilution) were purchased from Proteintech (United States), The primary antibodies against CAD (Cat#sc-376072, Santa Cruz, United States) and p-CAD (Ser1859) (Cat#70307, Cell Signaling Technology, United States) were used with a dilution of 1:1000.