Western Blot Analysis of PLGA-CSNP Formulations

Western blot analyses were performed according to previously reported procedures^{6 ,9 (link)}. Briefly, protein extracts from cells treated with PTX- or CDDP- PLGA-CSNP-(RGD+/−) formulations and from control groups were run in sodium dodecyl sulfate-polyacrylamide gels and transferred onto Immobilon PVDF membrane (Millipore, Billerica, MA, USA). The membranes were then blocked using 5% milk in TBST buffer (pH 7.5), followed by blocking with primary antibodies (Cell Signaling Technology) in 1:1000 dilution in 5% milk in TBST buffer (pH 7.5) overnight at 4 °C. The next day, the membranes were washed and blocked with secondary antibodies (rabbit or mouse, depending on the primary antibodies; Sigma-Aldrich, USA) and imaged using Syngene GBOX after incubation with Clarity Western enhanced chemi-luminescence substrate (Biorad, USA). Beta actin was used as the internal control for all western blot experiments. Protein bands from western images were quantified using GelQuant.NET software.