Supersignal west pico plus ecl kit

Cells were harvested and lysed using RIPA buffer (Thermo Fisher Scientific, Waltham, MA) supplemented with a protease inhibitor cocktail (Roche, Switzerland). After centrifugation, the protein concentration was determined using a BCA assay kit (Thermo Fisher Scientific, Waltham, MA) and mixed with sample buffer at 100 °C for 5 min. An equal amount of protein was separated via SDS PAGE and transferred onto NC membranes (Cytiva, Marlborough, MA). The membranes were blocked with 5% skim milk (BD Biosciences, San Jose, CA) in TBS-T buffer and subsequently incubated with primary and secondary antibodies. Protein bands were visualized using the SuperSignal™ West Pico PLUS ECL kit (Thermo Fisher Scientific, Waltham, MA) on an Amersham Imager AI680 (General Electric, Boston, MA). The following antibodies were used: anti-E-cadherin (Cell Signaling Technology, #3195), anti-N-cadherin (Cell Signaling Technology, #13116), anti-vimentin (Cell Signaling Technology, #5741), anti-α-SMA (Abcam, ab5694) and anti-β-actin (Sigma Aldrich, A5441).

Protein was extracted from enriched primary splenic NK cells using Pierce RIPA buffer (Thermo Fisher) with Halt protease inhibitor cocktail (Thermo Fisher) and protein concentration was quantified using the Pierce BCA Protein Assay kit (Thermo Fisher). Samples were electrophoresed on NuPage Novex 4-12% Bis-Tris Protein Gels, transferred to PVDF membranes, and blocked overnight at 4 °C with 5% wt/vol nonfat milk in 1× TBS and 0.1% Tween-20. Immunoblots were performed using rabbit anti-UTX (1:1,000 dilution; Cell Signaling Rabbit monoclonal antibody, 33510), rabbit anti-β-actin (1:10,000 dilution; Cell Signaling, CST4970) and goat anti-rabbit horseradish peroxidase secondary antibody (1:20,000 dilution; Thermo Fisher, 31466). Proteins were detected using the SuperSignal West Pico PLUS ECL kit (Thermo Fisher) and visualized using the Azure Biosystems c280 imager.