α bcl2

Denatured proteins of cell lysates were subjected to Western blot analysis. The concentration of total proteins was determined by BCA protein assay (Pierce, Rockford, IL). 40 μg of total proteins was separated on a 7.5%–15% SDS polyacrylamide gel and transferred onto a PVDF membrane. The membrane was blocked in 5% non-fat dry milk for 1 h and probed overnight with primary antibody. Subsequently the membrane was incubated with infrared cw800-conjugated secondary antibody for 1 h at room temperature. The proteins were detected using a LICOR Odyssey Infrared Imager. Protein density quantification was performed in Image Studio Lite (LICOR). The antibodies used were α-DCLK1 (ABCAM, AB31704), α-phosphoDCLK1, α-C-MYC (Santa Cruz, SC-40), α-BCL2 (Santa Cruz, SC-492), and α-phosphohistone H3 (EMD-Millipore 07–492).

Cells were lysed in RIPA buffer supplemented with a protease inhibitor mix. Protein concentration was established using the Bicinchoninic Acid Protein Assay (Pierce™, Rockford, IL, USA). Equal quantities of protein were run on a 12% or a 15% polyacrylamide gel electrophoresis and transferred to PVDF membrane (Bio-Rad, Hercules, CA, USA). Membranes were blocked for 1 h with 5% skim milk in TTBS and incubated with primary antibodies for 16 h at 4 °C in blocking buffer (3% skim milk in TTBS). After incubation with the corresponding secondary antibody, protein bands were evidenced by chemiluminescence detection (SuperSignal West Femto System, Thermo Scientific, Rockford, IL, USA). Primary antibodies used: α-Actin (sc-1616), α-GAPDH (sc-47724); α-Bcl-2 (sc-7382); α-BclXS/L (sc- 634) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); α-Bcl-w (cat # MA5-15076) (Thermo Scientific, Rockford, IL, USA); α-Mcl-1(cat # 94296); α-Noxa (cat # 14766) (Cell Signaling Technologies, Danvers, MA, USA). Secondary antibodies: a horseradish peroxidase-conjugated α-rabbit IgG; α-mouse IgG and α-goat IgG (Thermo Scientific, Rockford, IL, USA).