Caspase 3 d3r6y

Total proteins were extracted from human glioma cells using RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) and heated for 5 min. 4–15% Mini-PROTEAN® TGX™ Precast Gels (BIO-RAD, California, USA) were used to separate the proteins which were then transferred onto PVDF membranes. The membranes were blocked for 20 min at room temperature using StartingBlock™ (PBS) Blocking buffer (Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 4 °C overnight with primary antibodies including CD13 antibody (rabbit, Cat #ab108310, 1/500; Abcam, Cambridge, UK), BAX (D2E11) (rabbit, Cat#5023, 1/1000; Cell Signaling Technology, Danvers, MA, USA ), BCL-2 (mouse, Cat#15071, 1/1000; Cell Signaling Technology), NOXA (mouse, Cat#ab13654, 1/1000; Abcam), Caspase-3 (D3R6Y) (rabbit, Cat#14220, 1/500; Cell Signaling Technology) and GAPDH antibody (mouse, Cat #ab9484, 1/1500; Abcam). After washing several times with TBS-T (0.05% Tween20), the membranes were incubated with HRP-conjugated secondary antibodies (1/200; Dianova, Hamburg, Germany) for 2 h at room temperature. The blots were washed several times before the enhanced chemo-luminescence detection kit (ECL Advance) was applied and luminescence was measured.

Whole protein extraction and immunoblotting analysis were performed as previously described [32 (link)]. In detail, T98G cell pellets were resuspended in ice-cold RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 8.0, 1 mM Triton X-100, all from Sigma) supplemented with a Complete Mini protease inhibitor cocktail (Roche, Basel, Switzerland). Protein samples were quantified by Qubit fluorimeter, using a Protein Assay kit (Invitrogen) following the manufacturer’s instructions. Before loading in SDS-PAGE, protein extracts were boiled in Laemmli sample buffer (2% SDS, 6% glycerol, 150 mM B-mercaptoethanol, 0.02% bromophenol blue, and 62.5 mM Tris-HCl pH 6.8). After electrophoresis, proteins were transferred onto a nitro-cellulose membrane Hybond-C Extra (GE Healthcare, Milan, Italy). Membranes were blocked with 5% nonfat milk in PBS containing 0.1% Tween 20 (v/v) and incubated overnight at 4 °C with primary antibodies. The primary employed antibodies were caspase-3 (#D3R6Y) and PARP-1 (#46D11) (Cell Signaling, Danvers, MA, USA; diluted 1:2000); α-tubulin (Cell Signaling, #4967 diluted 1:6000) was used as the internal loading control. Species-specific peroxidase-labeled ECL secondary antibodies (Cell Signaling, diluted 1:4000) were employed. Protein signals were revealed by the Weststar Supernova Kit (Cyanagen, Bologna, Italy) and visualized using Chemidoc MP system (Biorad).