Western blot of HIF1α and SULT1E1
Western blot was conducted as in Maiti et al. 2007 with a slight modification. A 12 % denaturing gel was loaded with 25 μg of protein (obtained from the rat liver tissue of animals treated with a single dose of DAS, Chalcone and DAS + Chal for 24, 48and 72 hours) and electrophoresis carried out at 100v for 3 hours, transfer to nitrocellulose membrane was done at 100v for 2 hours. The membrane was washed and incubated with primary antibodies (anti rHIF1α and anti rSULT1E1) and secondary antibodies as mentioned in the referenced protocol (20). The membrane were treated with Diaminobenzidine (DAB) until the brown colored bands were developed.