Nitrocellulose membrane

Cell extracts were prepared in lysis buffer containing 20 mM Tris-HCl pH 7.4, 100 mM NaCl, 5 mM EDTA, 2 mM PMSF, 10 ng/mL leupeptin, and 10 μg/mL aprotinin. Equal amounts of extracted proteins (40 µg) were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH, USA). The membranes were blocked with nonfat milk solution for 1 h, and then washed and probed with the appropriate primary antibody. The membranes were rinsed with Tris-buffered saline containing 0.1% Tween 20, incubated with horseradish peroxidase-conjugated secondary antibody for 1 h, rinsed again, and developed using enhanced chemiluminescence (ECL) reagents (Amersham Biosciences, Piscataway, NJ, USA).

Isolated proteins and prestained protein standards (Bio-Rad, Munich, Germany) were subjected to 4.5-15% SDS-polyacrylamide gel electrophoresis under reducing conditions. Proteins were electrotransferred onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) for 30 min at 25 V and ≤1 A with the Trans-Blot Turbo Blotting System (Bio-Rad, Munich, Germany). Membranes were blocked for 30 min in blocking buffer (5% dry skim milk+1% BSA in TBS containing 0.1% Tween 20 (TBS-T)), then incubated overnight with primary antibodies (see Table 2), washed with TBS-T, and finally incubated at room temperature for 1 h with peroxidase-coupled anti-mouse IgG antibody from donkey (Dianova, Hamburg, Germany, dilution 1 : 1,000) or peroxidase-coupled anti-rabbit IgG antibody from donkey (Amersham Bioscience, Freiburg, Germany, dilution 1 : 10,000). Signals were visualized with Super Signal® West Femto Maximum Sensitive Substrate (Thermo Fisher Scientific, Schwerte, Germany) following the procedure recommended by the supplier and detected with the chemiluminescence detector Fusion-SL4.2 MP (Peqlab Biotechnology, Erlangen, Germany).

For preparation of cellular extracts, cells were washed with PBS and dissolved in 2xSDS-PAGE sample buffer (116 mM Tris-HCl pH 6.8, 3.3% [w/v] SDS, 12% [v/v] glycerol, 2% [v/v] beta-mercaptoethanol, bromophenol blue) and 1x complete cOmplete™ protease inhibitor cocktail (Roche). After separation by SDS-PAGE, proteins were transferred to nitrocellulose membranes (Schleicher & Schuell) and subjected to immune blot analysis.

For Western-Blot analysis, SKO-007(J3) cells were pelleted, washed once with cold phosphate-buffered saline, resuspended in lysis buffer [1% Nonidet P-40 (v/v), 10% glycerol, 0.1% SDS, 0.5% Sodium Deoxycholate, 1 mM phenyl-methyl-sulfonyl fluoride (PMSF), 10 mM NaF, 1 mM Na3VO4, complete protease inhibitor mixture (Roche) in PBS] and subsequently incubated 30 min on ice. The lysate was centrifuged at 14000g for 15 min at 4°C and the supernatant was collected as whole cell extract. Protein concentration was determined by the BCA method (Pierce). Thirty to 50 μg of cell extract was run on 12.5% denaturing SDS-polyacrylamide gels. Proteins were then electroblotted onto nitrocellulose membranes (Schleicher&Schuell), stained with Ponceau to verify that similar amounts of proteins had been loaded in each lane, and blocked in 3% milk in TBST buffer. Immunoreactive bands were visualized on the nitrocellulose membranes, using horseradish-peroxidase-coupled goat anti-rabbit or goat anti-mouse immunoglobulins and the ECL detection system (GE Healthcare Amersham), following the manufacturer's instructions. Antibodies against β-actin, IRF4 (H-140), Ikaros (H-100) and Aiolos (L-15) were purchased from Santa Cruz Biotechnology. Antibody against Blimp-1 was purchased from Cell Signaling.

Protein extracts were resolved on 12% and 10% SDS–PAGE gels, transferred onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) and blocked in 5% skim milk. The following primary antibodies were used according to the manufacturer's instructions: anti-CD133 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), ADAM17 (ab cam), NICD (Cell Signaling Technology, Beverly, MA, USA), MTSS1 (Santa Cruz Biotechnology), HES1 (Santa Cruz Biotechnology) and β-actin (Sigma-Aldrich Co.). After incubation with horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit secondary antibodies (Amersham Biosciences, Cardiff, UK), specific protein bands were visualized using enhanced chemiluminescence (Amersham Biosciences). The density of each band was measured using the TINA software.