Immunodetection was performed using antibodies against pAKT (Ser473), total AKT, pRPS6 (Ser240/244), total RPS6, hexokinase II (HK2, Cell Signaling, Beverly, MA, USA), CHKA (Sigma), p21Cip1 (Cell Signaling), Cdc25A (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and GAPDH (Chemicon, Billerica, MA, USA). Blots were revealed with peroxidase-conjugated secondary anti-rabbit antibody (GE Healthcare, Chalfont St. Giles, UK) followed by ECL chemiluminescence solution (Amersham Biosciences, St. Giles, UK). The western blot procedure used for the tumor extracts was the same used for the cell lines.
An MSD assay, more sensitive than immunoblots with tissue extracts, was used to identify target inhibition ex vivo in tissue samples. It was based on a previously described protocol with minor modifications [58 (link)]. The protein concentration was determined using a Direct Detect spectrophotometer according to the manufacturer's instructions.
An MSD assay, more sensitive than immunoblots with tissue extracts, was used to identify target inhibition ex vivo in tissue samples. It was based on a previously described protocol with minor modifications [58 (link)]. The protein concentration was determined using a Direct Detect spectrophotometer according to the manufacturer's instructions.