2.6. Effect of metformin treatment on the activation of AMPK pathway in xenograft tumours

At the end of the treatment period, mice were killed, and ~10 mg of tumour tissues was isolated, washed, and lysed in RIPA buffer (Santa Cruz Biotechnology, Santa Cruz, USA). Protein in tissue lysates was measured by the bicinchoninic acid assay (Santa Cruz Biotechnology) and subjected to Western blot analyses (40 μg) as previously described (Cai et al., 2016; Han et al., 2015) using a rabbit monoclonal IgG primary antibody against phospho‐AMPK (Cell Signaling Technology, Danvers, USA, RRID:AB_2799368). GAPDH rabbit monoclonal antibody (Santa Cruz Biotechnology, RRID:AB_629536) was used as a loading control. An anti‐rabbit IgG, HRP‐linked antibody was used as secondary antibody (Cell Signaling Technology, RRID:AB_2099233). The intensity of the phospho‐AMPK band was normalized to the GAPDH band to reduce variation in loading. The immuno‐related procedures used comply with the recommendations made by the British Journal of Pharmacology.