Ab8414

Platelets treated with TRAIL or PBS were washed and homogenized on ice using RIPA buffer (50 mM TRIS pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 1 mM phenylmethanesulfonyl fluoride). Lysates containing equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene difluoride membranes using a semi-dry transfer system (Bio-Rad, Hercules, CA, USA). Blots were blocked with 5% nonfat dry milk in TBST (10 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.1% Tween-20) for 2 hours at room temperature, and then probed with mouse monoclonal antibody against DR5 (1.5 µg/mL, ab16329, Abcam, Cambridge, MA, USA), mouse monoclonal antibody against DR4 (1.5 µg/mL, ab8414, Abcam), and rabbit polyclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:800; sc-32233; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight. The blots were then washed with TBST and incubated with fluorescence-conjugated goat anti-mouse/rabbit IgG (Rockland Immunochemicals Inc., Gilbertsville, PA, USA) for 1 hour at room temperature. The immunoreactive bands were visualized using an Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE, USA), and protein expression was quantified by densitometric analysis using Odyssey Imaging Software v3.0 (Li-Cor Biosciences).

The 22Rv1 cells (1×10⁶) were seeded in a 25-cm² flask and treated with linalool (2.5 mM) for 24 h at 37°C. After the treatment, the cells were collected and lysed with RIPA buffer on ice for 30 min. The protein concentration was determined by a BCA kit (Beyotime Institute of Biotechnology). The proteins (30 µg per lane) were separated on a 10% SDS-PAGE gel and electrotransferred onto a PVDF membrane. The membrane was blocked with 5% bovine serum albumin at room temperature for 1 h, and then immunoblotted with antibodies against caspase-3, cleaved caspase-3 (cat. nos. 9665 and 9664, respectively; both 1:1,000; Cell Signaling Technology, Inc.), caspase-8 (cat. no. SC56070; 1:500 dilution; Santa Cruz Biotechnology, Inc.), cleaved caspase-8 (cat. no. 9748; 1:1,000; Cell Signaling Technology, Inc.), caspase-9, cleaved caspase-9, DR4, DR5 (cat. nos. ab32539, ab2324, ab8414 and ab8416, respectively; all 1:1,000; Abcam), p53 (cat. no. AF0879; 1:1,000; Affinity Biosciences, Inc.), Bcl-2 (cat. no. ab59348; 1:1,000; Abcam), Bax, and β-actin (cat. nos. GB11007 and GB12001, respectively; 1:300 and 1:3,000, respectively; Wuhan Servicebio Technology, Co., Ltd.). The BeyoECL Plus kit (Beyotime Institute of Biotechnology) was used for visualization. The bands were analyzed by AlphaEaseFC 4.0 software (Genetic Technologies, Inc.).