Fusion solo

Cells were lysed in RIPA buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton-X-100, 0.5% sodium deoxycholate, 1 mM EDTA, 0.5 mM PMSF, 0.1% SDS, 1 mM sodium orthovanadate, 10 mM sodium fluoride, and 20 mM β-glycerophosphate plus Complete protease inhibitors without EDTA (Roche)] and lysates were clarified by centrifugation (16,000 × g, 10 min). Protein concentration was determined by Bio-Rad DC protein assay. Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Roth, Karlsruhe, Germany). Alternatively, lysates were loaded on 4–12% NuPAGE® Novex Bis–Tris gels (Invitrogen) and transferred to nitrocellulose membranes (iBlot®Gel Transfer Stacks; Invitrogen). Membranes were blocked with 0.5% blocking reagent (Roche) in PBS containing 0.05% Tween-20 and incubated with primary antibodies, followed by HRP-labeled secondary antibodies for ECL-based (Pierce, Rockford, IL) visualization with the Amersham600 system (GE Healthcare) or the Fusion Solo (VilberLourmat). Original western blots of all cropped blots are provided as Supplementary Information.

The protein lysates from 5,000,000 U87MG WT, U87MG EGFR, or U87MG EGFR have been treated with 5 μM afatinib for 6 h. The relative phosphorylation levels of 43 kinase phosphorylation sites were determined using the human phospho-kinase array kit (ARY003B, R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions. Briefly, the Proteome Profiler human phospho-kinase array kit is a membrane-based sandwich immunoassay. Cell lysates were incubated overnight with the Human Phospho-Kinase Array. Capture antibodies spotted in duplicate on nitrocellulose membranes bind to specific target proteins present in the sample. Then, the membrane was washed to remove unbound proteins, and the phosphorylation of captured proteins was detected with biotinylated phospho-specific detection antibodies. Streptavidin–HRP and chemiluminescent detection reagents were applied to produce a signal at each capture spot corresponding to the amount of phosphorylated protein bound.
The chemiluminescent signal was acquired using the blot documentation system Fusion Solo (Vilber, Collégien, France) and was analyzed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA) [47 (link)]. The pixel density of each spot was calculated following subtraction of the background (negative control) and normalization to the positive control spots.

Proteins were extracted from harvested cells using RIPA lysis buffer (Thermo Fisher Scientific, #89,900) containing a protease inhibitor cocktail (Thermo Fisher Scientific, #78,430). The total protein concentration of each cell lysate was measured using the Bradford method, with bovine serum albumin as a standard. Equal amounts of protein lysate (45 µg) were loaded onto denaturing polyacrylamide gels and transferred to a nitrocellulose membrane. The primary antibodies used for immunoblotting were anti-RAB27A (diluted 1:2000; #ab55667; Abcam), anti-RAB27B (diluted 1:2000; Cat # PA5-54,096; Thermo Fisher Scientific), and anti-MYCN (diluted 1:500; Cat # ab24193; Abcam) followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The immunoreactive bands were detected using enhanced chemiluminescence (Cat #32,109; Thermo Fisher Scientific). Digital chemiluminescent images were captured and analyzed using Fusion Solo (Vilber, Marne-la-Vallee Cedex, France).

To analyze protein expression, algal cells were harvested by centrifugation at 6000 rpm. To extract protein, the cells were resuspended in RIPA buffer supplemented with protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO, USA) and disrupted by bead-beating with 0.1 mm zirconia/silica beads for 5 min. Protein concentration of lysates were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The samples (50 μg/sample) were separated on 4-20% Mini-PROTEAN TGX™ Precast Protein Gel (Bio-Rad) according to standard SDS-PAGE procedure [42 ]. The gels were transferred onto a PVDF membrane using a Trans-Blot Turbo Transfer System (Bio-Rad). After transfer, the membrane was incubated with blocking solution (5% skim milk solution with 1× TBS-T) at room temperature for an hour. The membrane was then incubated with blocking solution supplemented with 1:5000-diluted mouse anti-FLAG M2 monoclonal antibody (Sigma Aldrich) at room temperature for 1 h. After antibody binding, the membrane was washed with 1× TBS-T four times (20 min/wash). To visualize the bands, the membrane was treated with ECL™ Prime (GE Healthcare, IL, USA) and the chemiluminescent signal was detected using a FUSION Solo (Vilber Lourmat, Collegien, France) imaging system.

The HUVECs grown in 6-well plates (3 × 10⁵ cells/well) were treated with the indicated concentrations of deoxyshikonin or shikonin for 24 h. Next, cell extracts were prepared using radio immunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.25% deoxycholate, 1% NP-40, and 1 mM EDTA) that contained 1 mM DTT, 1 mM phenyl methyl sulfonyl fluoride, and a protease inhibitor cocktail (Roche Diagnostics Corp., Indianapolis, IN, USA). Preparation of cell lysates, supernatant collection, and protein quantification were performed according to previous studies with some modifications [35 (link)]. Briefly, proteins were separated by 10% SDS-PAGE, transferred onto Immobilon-P polyvinylidene fluoride (PVDF) membranes, and allowed to be treated with 5% skim milk in TBS (tris-buffered saline, pH 7.4) containing 0.1% Tween 20 (TBS-T) for 2 h. After that, the membranes were probed with specific primary antibodies for 2 h and horseradish peroxidase-conjugated secondary antibodies for 1 h. The membranes were visualized with the Enhanced chemiluminescence (ECL) system (Thermo Scientific, Waltham, MA, USA) using Fusion Solo (Vilber Lourmat, Paris, France) following the instruction manual.

Total cellular proteins were collected through the lysis of harvested cells with RIPA buffer (GeneTex Inc., Hsinchu City, Taiwan). Afterward, 25 μg of proteins was separated via SDS-PAGE and transferred onto PVDF membranes (Immobilon-P, Merckmillipore, Danvers, MA, USA). The membranes were blocked with 5% skimmed milk, incubated with primary antibodies at 4 ºC overnight, and incubated with horseradish peroxidase (HRP)-conjugated specific secondary antibodies at room temperature for 1 h. The signals were developed by incubating with an enhanced chemiluminescence substrate (PerkinElmer, Waltham, MA, USA) and captured with a luminescent image analyzer (Fusion Solo, Vilber Lourmat Deutschland GmbH, Germany). The following antibodies were used in this study: mouse monoclonal IgG anti-Myc tag antibody (Proteintech Group Inc., Rosemont, IL, USA); mouse monoclonal IgG anti-NQO1 (Cat. No. sc-32793), anti-Nrf2 (Cat. No. sc-365949), and anti-JNK (Cat. No. sc-7345) antibodies (Santa Cruz Biotechnologies Inc., Dallas, TX, USA); anti-phosphorylated JNK antibody (Cat. No. 4668S; Cell Signaling Technology, Danvers, MA, USA); rabbit polyclonal IgG anti-GAPDH antibody (Cat. No. GTX100118; GeneTex Inc., Hsinchu City, Taiwan); and mouse monoclonal IgG anti-β-actin (Cat. No. A5441; Sigma-Aldrich, St. Louis, MO, USA).