Scrambled sc sirna

Purified CLL B-cells (4.0 × 10⁶/ml) treated with DMSO or TP-0903 (0.1μM) or ibrutinib/TP-4216 (0.75 μM) for 20–24 hours were lysed in NP40-lysis buffer, and whole cell extract was prepared as described previously(4 (link), 5 (link)). Normal B-cell lysates were also prepared in NP40-lysis buffer as control. For immunoprecipitation (IP) experiments, 0.2–0.3mg of total protein from CLL B-cell lysates was used to IP specific RTKs using appropriate antibodies as described elsewhere(4 (link)). The precipitated immune complex was electrophoresed on SDS-polyacrylamide gels, transferred onto nitrocellulose membranes, followed by detection of proteins of interest using specific antibodies. MDA-MB-231 cells were transfected with a specific siRNA to Axl (Santa Cruz) or scrambled (sc) siRNA (Santa Cruz) using Lipofectamine2000 (Invitrogen) according to manufacturer’s instructions. Cells were harvested after 48 hours, lysed and analyzed for the expression of Axl, XIAP, Bcl-2, Mcl-1 and phosphorylation status of AKT using specific antibodies by Western blots as described above.