4.8. Western Blotting (WB) Analysis

After treatment of human pancreatic cancer cells for 24–72 h with NAB, GEM and their combination, both in presence and in absence of CM-HMC-1, the protein extracts were obtained by homogenization in RIPA buffer and treated with 1 mM phenylmethylsulfonyl fluoride (PMSF). The protein level of the selected proteins (pErk1/2 Erk1/2, pSMAD3, pAkt (Ser473), Akt, was analyzed by WB as previously described [44]. The blot detection was performed with ChemiDoc™ Imaging Systems and analyzed with the ImageLab software (version, Bio-Rad-USA, Hercules, CA, USA). Antibodies: the monoclonal antibody anti Erk1/2, p-Erk1/2, anti Akt, anti p-Akt(Ser473) were provided by Cell Signalling-USA and anti-β-actin (AC-15) by Sigma-Aldrich. A mouse-HRP and a rabbit-HRP (Bio-Rad Laboratories, Hercules, CA, USA) were used as secondary antibody. All experiments were performed in triplicate.