*2.3. Western Blotting*

In order to investigate the protein expression of Bcl10, western blotting was performed. The cell pellets were lysed in 10X RIPA buffer (abcam, Cambridge, UK, Cat. No. ab156034) diluted to 1X and supplemented with 1× Protease Inhibitor Cocktail (Sigma-Aldrich, Burlington, MA, USA, Cat. No. P2714) and 1 mM PMSF (Sigma-Aldrich, Burlington, MA, USA, Cat. No. P7626). The protein lysates were quantified using the Protein Assay Kit II (Bio-Rad, Hercules, CA, USA, Cat. No. 5000002) with bovine serum albumin (BSA) as standard. The lysates were boiled in 10× Laemmli Sample Buffer diluted to 1×, and total protein was separated using 4–20% Mini-PROTEAN TGX Precast Protein Gels (Biorad, Hercules, CA, USA, Cat. No. 4561095-6). Post electrophoresis, the proteins were transferred onto a 0.2 μm nitrocellulose membrane (Bio-Rad, Hercules, CA, USA, Cat. No. 1620112) and blocked in 5% non-fat dry milk for at least an hour at room temperature (RT) before incubating the membrane overnight at 4 ◦C with anti-Bcl10 antibody (331.3) (Santa Cruz, TX, USA, Cat. No. sc-5273). The membrane was subsequently incubated for 1 h at RT with the respective horseradish peroxidase (HRP)-linked secondary antibody. The blots were developed using the Clarity™ Western ECL Substrate (Bio-Rad, Hercules, CA, USA, Cat. No. 170-5060) in the ChemiDoc™ Touch Gel and Western Blot Imaging System (Bio-Rad, Hercules, CA, USA). Anti-β-actin (Sigma-Aldrich, Burlington, MA, USA, Cat. No. Cat# A5441) was used as the loading control. Image Lab software (Bio-Rad, Hercules, CA, USA) was used to detect and quantify the protein bands. Protein levels were normalized to β-actin and thereafter to healthy controls.
