Setdb1

The SETDB1 protein expression level was assessed by western blotting. The confluent siRNA SETDB1 and control cells were washed with 1 mL of Dulbecco’s phosphate-buffered saline (DPBS) at 4 °C three times and lysed in a radioimmunoprecipitation assay (Thermo Scientific, USA). Mammalian Protein Extraction Reagent (M-PER; Thermo Scientific, USA) was used to isolate the total proteins. The concentrations of these isolated proteins were measured using the Bradford method. For western blotting, 20 µg of each protein was used. The proteins were loaded onto SDS-PAGE gel and run in ProSieve EX running buffer (Lonza Bioscience, USA) by electrophoresis. Next, the protein was transferred to membranes (Lonza Bioscience, USA). The membranes along with the transferred proteins were blocked in 5% nonfat milk and incubated with primary rabbit monoclonal antibody-SETDB1 diluted to 1:1000 (Cell Signaling, USA) and subsequently with secondary anti-rabbit IgG HRP-linked antibody. β-Actin was used for normalization. Protein bands were observed with ECL solution (Bio-Rad, USA) and their relative densities were assessed with densitometric analysis by using Image Lab software (Bio-Rad, USA) (Sun et al., 2011; Taylor et al., 2013; Özdaş, 2018).