Alexa fluor 488 conjugated α tubulin antibody

PhosFlow and immunoblotting studies were performed as previously described^{5 (link),6 (link)} using antibodies to p-SYK(Y525/Y526) (R&D Systems, MN); p-BTK(Y223), p-IRAK1(T209) (Abcam, MA); SYK, p-STAT3(Y705), STAT3, p-AKT(S473), AKT, BTK, IRAK1, p-IRAK4(T345/S346), IRAK4, and Alexa Fluor^® 647-conjugated p-SYK(Y525/Y526) (Cell Signaling Technologies, MA). Alexa Fluor^® 488-conjugated α-tubulin antibody (Cell Signaling Technologies) was used as an internal control for p-SYK levels among different cell lines. GAPDH antibody (Santa Cruz Biotechnology, TX) was used as a loading control for immunoblotting. Cell lines or WM patient BM mononuclear cells were treated with inhibitors for 1–2 h before signaling studies.

After treatments, cells were fixed in methanol at −20°C for 10 min and permeabilized with cold methanol. Thereafter, cells were blocked with 0.1% bovine serum albumin in PBS at room temperature for 1 h and then stained with Alexa Fluor 488 conjugated α-tubulin antibody (Cell Signaling Technology) at 4°C overnight. Cells were then washed with PBS three times and stained with DAPI (Merck). Mitotic morphology was observed under fluorescence microscopy.