3.8.3. Western Blotting

HeLa cells (1 × 106 cells per well in 6-well plate) treated with curcumin-loaded NPs (0.005 and 0.01 mg/mL of curcumin-loaded NPs) for 72 h were washed with PBS and then lysed in a buffer containing 50 mM Tris–HCl, pH 7.2, 0.1% sodium deoxycholate, 1% Triton X-100, 5 mM EDTA, 5 mM EGTA, 150 mM NaCl, 40 mM NaF, 2.175 mM NaVO4, 0.1% SDS, 0.1% aprotinin and 1 mM PMSF. Forty micrograms of protein underwent SDS–PAGE following transfer on PVDF membranes (Amersham™ Hybond® P, Dasser Germany). Bands were detected using Immobilon ECL Western Blotting Substrate (ThermoFisher Scientific, Waltham, MA, USA) on a Chemidoc digital imaging machine (Bio-Rad). An anti-Cleaved Caspase-3 primary antibody (1:1000 in 1% BSA, Cell Signaling Technology, Inc. MA, USA) and an anti-PARP-1 primary antibody (1:1000 in 1% BSA, Santa Cruz Biotechnology, Dallas, TX, USA), were used both followed by a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:10,000; Abcam Cambridge CB2 0AX UK). Internal loading control was performed using the anti-α-Tubulin mouse primary antibody (1:10,000 in 1% BSA; Sigma-Aldrich) followed by a goat anti-mouse horseradish peroxidaseconjugated secondary antibody (1:10,000, Sigma-Aldrich). Densitometry quantification was performed with ImageJ Software (NIH) and expressed as ratio of cleaved caspase-3 and PARP to α-tubulin.
