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.