Imagequant las 4000 mini

Neuroblastoma cell lines were seeded in a 10-cm dish at a final density of 1.5×10⁶ cells and allowed to attach overnight. The cells were then treated with DMSO or experimental compounds. After 48–72 h, cells were lysed in CHAPS lysis buffer with protease and phosphatase inhibitors (Roche, Basel, Switzerland), and 25 or 50 μg of total lysate were boiled for 5–10 min in CHAPS and sample buffer. Western blotting was performed using the following primary antibodies: anti-ALK (Cell Signaling Technology, Danvers, MA, USA), anti-Phospho-ALK (Cell Signaling Technology), anti-Shc (Cell Signaling Technology), anti-Phospho-Shc (Cell Signaling Technology), anti-PARP (Cell Signaling Technology), anti-cleaved Caspase-3 (Cell Signaling Technology), anti-GAPDH (Fuji Film-Wako Chemicals), and anti-Actin (Merck). Secondary antibodies used were as follows: HRP-linked anti-rabbit IgG (Cell Signaling Technology) and HRP-linked anti-mouse IgG (Cell Signaling Technology). The proteins were visualized using ImageQuant LAS 4000mini with enhanced chemiluminescence reagents (Thermo Fisher Scientific).

Cells treated with physciosporin for 48 h were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in lysis buffer [30 (link)]. Antibodies against ALDH1 (sc-166362; Santa Cruz Biotechnology, Dallas, TX, USA), CD133 (CA1217; Cell Applications, San Diego, CA, USA), CD44 (3570; Cell Signaling Technology, Danvers, MA, USA), Lgr-5 (ab75850; Abcam, Cambridge, MA, USA), and Msi-1 (ab52865, Abcam) were used to detect stemness factors. α-tubulin (2125, Cell Signaling Technology) antibody was used as an internal control. The luminescence photon from proteins were detected by an Image Quant LAS 4000 mini using horseradish peroxidase-conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) with an Immobilon Western Chemiluminescent HRP Substrate Kit (Merck Millipore, Billerica, MA, USA). Quantitation of bands were performed by using Multi-Gauge 3.0 software, and the relative density of each band was calculated based on the density of the internal control bands in each sample. Values are shown as arbitrary densitometric units corresponding to signal intensity. All results are representative of at least three independent experiments.

Overnight cultures grown in CYE medium were used to inoculate fresh CYE medium to an OD_600nm of 0.2. Strains were grown 6 h at 32°C under shaking, and 1 mL of OD_600nm 0.5 of cells was pelleted. Then, 50 μL of Laemmli buffer and 50 μL of water were added and heated for 9 min at 95°C. Ten microliters were electrophoresed onto a 15% acrylamide/bis-acrylamide gel and then transferred to a nitrocellulose blotting membrane, Amersham Protan 0.45 μm NC. To assay the production of 6His tagged versions of RisR, the membrane was washed with PBS-Tween 0.05%, followed by 1 h of blocking in PBS-BSA 3% at room temperature and washed again before adding the Penta-His Antibody (Qiagen) diluted at 1:1,000 in PBS-BSA 3%, which was used as the primary antibody at 4°C overnight. Then, an anti-mouse IgG (HRP) (Promega) was used as a second antibody diluted at 1:10,000 in PBS-Tween 0.05% and applied 1 h at room temperature. Signal was detected with the SuperSignal West HisProbe kit (Thermo Scientific) and an ImageQuant LAS 4000 mini.