Radioimmunoprecipitation assay (ripa)

Immunoblot and immunoprecipitation analyses were carried out as previously described.30 Briefly, tissues and cells were lysed in radioimmunoprecipitation assay (Thermo) buffer with complete protease inhibitor cocktail (Roche) and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. For immunoprecipitation, precleared cell extracts were incubated with primary antibodies overnight at 4°C, followed by incubation with protein A‐agarose or G‐agarose beads (Roche Diagnostics) for 1 h and washing three times with cell extraction buffer. Precipitates were analyzed by western blotting. Surface biotinylation was performed as previously explained.34 Briefly, biotinylated surface proteins were precipitated with streptavidine‐agarose beads (Pierce) and analyzed with sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Primary antibodies are listed in TableS2.

Organoids from the same condition were pooled (Total 6) in an Eppendorf and rinsed with PBS. The PBS was removed, and the organoids were frozen at -80°C before extraction. Radio immunoprecipitation assay (RIPA) lysis buffer (ref. 89901, ThermoScientific) supplemented with Phosstop (ref. 04 906 837 001, Roche) and cOmplete tablets (ref. 4693116001) were used to lyse the organoids under sonication to perform protein extraction. The amount of protein was quantified using the DC protein assay kit (ref. 500-0116, Bio-Rad) according to the supplier’s recommendations.

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).