Western Blotting:
Cells were lysed by adding RIPA buffer (Sigma-Aldrich) supplemented with protease and phosphatase inhibitors (Sigma, UK) followed by manual disruption. Protein concentration was determined by using a BCA protein assay kit (Thermo Fisher Scientific). Total cell protein (20 µg) was added to 4× Laemmli buffer (250 mM Tris–HCl, pH 6.8, 8 % SDS, 40 % glycerol, 0.004 % bromophenol blue, 20 % beta-mercaptoethanol) and heated at 72 °C for 10 min. Samples were then resolved on SDS-PAGE gels and transferred onto Hybond ECL nitrocellulose membranes (GE Healthcare, Buckinghamshire, UK). Membranes were blocked with 5% milk in Tris-buffered saline with Tween (TBS-T) (Tris-buffered saline, 0.1 % Tween-20) for 1h at RT and then incubated with the primary antibody (rabbit anti-phospho-BTK (Cell signalling 8714S), rabbit anti-total BTK (Cell signalling 8547S), rabbit anti-phospho-Akt (Cell signalling 4060S), rabbit anti-total Akt (Cell signalling 9272S) diluted 1:1,000 in 5% BSA/TBS-T overnight at 4°C. Next, membranes were incubated with an HRP-conjugated anti-rabbit secondary antibody for 1 h at RT. Protein bands were visualized by incubating the membranes for 5 min with Amersham ECL prime and subsequent exposure to X-ray film over a range of exposure times. Western blotting of stripped membranes with an anti β-actin antibody (Cell Signalling) was used as a loading control. Densitometry was preformed using the Licor Studio Lite software.