Molecular imager fxtm system

Following treatment with drugs, cells were lysed and whole cell fractions were isolated as previously described [17 (link)]. Proteins (10–30 μg) were separated by 10%–12% (w/v) polyacrylamide gel electrophoresis and electroblotted to PVDF membranes. After blocking with non-fat milk for 1 h, membranes were incubated overnight with the following primary antibodies (1:1000 dilution) from Cell Signaling Technology (Danvers, MA, USA): E-Cadherin (Cat #3195), N-Cadherin (Cat #13116), TCF8/ZEB1 (Cat #3396), β-Catenin (Cat #8480), Vimentin (Cat #5741), phospho-Akt (Ser473) (Cat #4060), Akt (Cat #9272), phospho-IGF-I Receptor β (Tyr1135/1136) (Cat #3024), IGF-I Receptor β (Cat #9750), phospho-PI3 Kinase p85 (Tyr458)/p55 (Tyr199) (Cat 4228), PI3 Kinase p85 (Cat 4257), phospho-mTOR (Ser2448) (Cat #5536), mTOR (Cat #2983). β-Actin (Cat #8457) was used at the same time as a loading control. After incubation with the secondary antibody (HRP-conjugated; 1:5000 dilution) for 1 h, at room temperature, the conjugates were developed and visualized using a Molecular Imager FX^TM System (BioRad; Hercules, CA, USA).

Whole cell protein lysates were prepared, and electrophoresis and electroblotting were performed as previously described [31 (link)]. Membranes were probed overnight with the following primary antibodies (1:1000 dilution) from Cell Signaling Technology (Danvers, MA, USA): PFKP (Cat #8164), PKM2 (Cat #4053), HK2 (Cat #2867), LDHA (Cat #3582), phospho-Chk1 (Ser345) (Cat #2348), phospho-p53 (Ser15) (Cat #9286), p53 (Cat #2527), p21 Waf1/Cip1 (Cat #2947), cdc2 (Cat #28439), Cyclin B1 (Cat #12231), Caspase-3 (Cat #14220), Caspase-7 (Cat #12827), Caspase-9 (Cat #9508), and PARP (Cat #9542). β-Actin (Cat #8457) was used at the same time as a loading control. After incubation for 60 min at room temperature in the presence of the secondary antibody (HRP-conjugated; 1:5000 dilution), the conjugates were developed and visualized using a Molecular Imager FX^TM System (BioRad; Hercules, CA, USA).