Pierce ecl substrate chemiluminescence kit

NSCLC cells were cultured as described previously [38 (link)]. H2170-ER and H358-ER cells were then treated with TKIs (10 μM erlotinib in serum-free RPMI media with 0.5% BSA for 24 h) and subsequently with ligands (15 ng/mL EGF for 2.5 min or 40 ng/mL HGF for 7.5 min in serum-free media). Cell lysates were prepared, electrophoresed, and transferred onto nitrocellulose membranes, as described previously [38 (link)]. Membranes were probed with antibodies for VEGFR-2 and NP-1. Immunoblots were developed using a Pierce ECL Substrate Chemiluminescence Kit (Thermo Fisher Scientific, Waltham, MA, USA), and modulations in protein expression were calculated by densitometric analysis using NIH ImageJ software.

Following all cell pre-treatments (as described above), cells were lysed in buffer (20 mM Tris, 150 mM NaCl, 10% glycerol, 1% NP-40, 0.42% NaF, 1 mM phenylmethylsulfonyl fluoride, 1 nM sodium orthovanidate, and 10 mM protease inhibitor cocktail (Sigma-Aldrich). Cell lysates were then electrophoresed for separation on 7.5% or 10% SDS-PAGE. Separated proteins were then transferred on to nitrocellulose membranes (Bio-Rad Laboatories, Hercules, CA) and probed with antibodies for Wnt or mTOR pathway related proteins. Immunoblots were developed using Pierce ECL Substrate chemiluminescence kit (Thermo Fisher Scientific, Rockford, IL, 32109) and modulations of different proteins were calculated by densitometric analysis using NIH ImageJ software.