Rabbit anti β actin monoclonal antibody

Cells were seeded in chamber slides and Dox for induction x 48 hours. Slides were fixed in cold methanol for 15min in −20°C and treated with cold acetone for 1 minute at room temperature. Mouse monoclonal anti-TMSB4Y antibody (Clone 6G4, SAB1403013, Sigma Aldrich) at 1:200 and rabbit monoclonal anti-β-actin antibody (Clone 13E5, 4970, Cell Signaling Technology) at 1:200 were incubated overnight. PLA was performed as per instructions of the DuoLink® In Situ Red Starter Kit Mouse/Rabbit (DUO92101, Sigma Aldrich).

According to relative gene expression results, HSP27 and Survivin (BIRC%) were selected for protein expression analyzes. Parental HSC-1 and resistant cells were lysed using a RIPA buffer (50 mM Tris, pH 7.2; 150 mM NaCl; 1% Triton X-100; and 0.1% SDS) containing protease and phosphatase inhibitor cocktail (1:100, Thermoscientific, Rockford, IL, USA). Protein concentrations were determined by a bicinchoninic acid assay (ThermoFischer, Waltham, MA, USA). Fifty micrograms of proteins were separated by SDS-PAGE on a 12% NuPAGE^® Bis-Tris Precast Gel (Invitrogen) and transferred to PVDF membranes (Millipore, Burlington, MA, USA). Protein expressions were detected through the use of rabbit monoclonal antibodies against HSP27 and survivin (1:1000, Cell Signaling Technology, Danvers, MA, USA). All antibodies were diluted in TBST-1% BSA solution (Cell Signaling Technology). The expressions of these proteins were standardized to human β-actin using a rabbit monoclonal anti-β-actin antibody (1:5000, Cell Signaling Technology, USA). Primary antibodies were detected using goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000, Santa Cruz Biotechnology, Dallas, TX, USA). Immunoreactive bands were visualized through chemiluminescence in a MyECL image platform (ThermoFischer).

Parental and resistant cellular pellet was obtained at 70% of confluence after three subcultures. Subsequently, protein lysates were harvested using RIPA buffer (50 mM Tris, pH 7.2; 150 mM NaCl; 1% Triton X-100; and 0.1% SDS) containing protease (1:100, Roche, USA) and phosphatase (1:100, Sigma-Aldrich, USA) inhibitors. The protein concentrations were determined using a bicinchoninic acid assay (Pierce, Thermo Scientific, USA). An amount of 40 μg of the proteins were separated by SDS-PAGE and transferred (Bio-Rad, USA) to PVDF membranes (Millipore, USA). Human SERPINA1, BTC and CCL5 protein expression levels were quantified using goat monoclonal antibody specific for human SERPINA1 and BTC (1:500, Santa Cruz Biotechnology, USA) and goat polyclonal antibody specific for CCL5 protein (1:500, R&D Systems, USA). The expression levels of these three proteins were standardized to human β-actin using a rabbit monoclonal anti- β-actin antibody (1:5000, Cell Signaling, USA). Primary antibodies were detected using donkey anti-goat or goat anti-mouse or mouse anti-rabbit- radish peroxidase (HRP)-conjugated secondary antibodies (1:5000, Santa Cruz Biotechnology, USA). Immunoreactive bands were visualized using myECL^™ Imager (Thermo Scientific, USA) according to the manufacturer’s instructions, and then quantified by densitometry using a ChemiGenius Gel Bio Imaging System (Syngene, USA).