Imagequant las 4000

Total protein extracts and microsome factions, separation on SDS-PAGE gels and western blotting were performed as in refs. ^{17 (link),19 (link)}. Briefly, total proteins (100 µl 2× Laemmli buffer for 20 mg fresh weight; 10 µg per lane) were separated on 8% SDS/PAGE gels and transferred onto nitrocellulose with 100 mM CAPS pH11 + 10% (v/v) ethanol. Original blot images for Figs. 1 and 3 can be found in Supplementary Figs. 5 and 6. For immunoprecipitation, we used the µMACS system (Miltenyi Biotec). The microsome fractions were incubated with 40 µl of µMACS GFP-tagged magnetic MicroBeads for 1 h on ice and then loaded on µcolumns, mounted on the µMACS separator, prepared with 200 µl extraction buffer. After four rounds of beads washing with extraction buffer, the eluates were extracted with 70 µl of 2× Laemmli buffer at 95 °C. Proteins were separated on 8% acrylamide SDS-PAGE gels. Anti-PKS4 antibodies were used at 1/500^{19 (link)}, anti-HA at 1/2000 (3F10 monoclonal, Roche) anti-phot1 at 1/5000^{17 (link)}, anti-NPH3 at 1/3000^{17 (link)} and anti-DET3 at 1/20,000^{17 (link)} dilutions in 1× PBS containing 0.1% Tween-20 and 5% non-fat milk. Chemiluminescence signals were generated using Immobilon Western HRP Substrate (Millipore). Signals were detected with a Fujifilm ImageQuant LAS 4000 mini CCD camera system and quantifications were performed with ImageQuant TL software (GE Healthcare).

Cells were harvested and lysed with ice-cold lysis buffer (Sigma, USA) and the protein concentration was determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Denatured proteins (100 μg) were separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels, transferred to Hybond membranes (Amersham, Munich, Germany), and blocked overnight in 5% skimmed milk in Tris-buffered saline with Tween 20 (TBST). For immunoblotting, the membrane was incubated with antibodies against TGFα (1:200, Bioss), EGFR, NF-kB, Cyclin D1, Survivin, Bax, MMP2 and SMARCD1, nanog, c-Fos (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membranes were then rinsed with TBST and incubated with anti-mouse, or anti-rabbit IgG antibodies conjugated to horseradish peroxidase (1:5000; Dako, Carpinteria, CA, USA) for 2 hours. Bands were visualized on X-ray film (Fuji film, Tokyo, Japan) using Image Quant LAS 4000 (Fuji film, Tokyo, Japan) and ECL Plus detection reagents (Santa Cruz Biotechnology). GAPDH (Sigma-Aldrich) was used as a loading control.

Cells were lysed and immunoblotted as previously described47 (link). Briefly, proteins were resolved by SDS-polyacrylamide gel electrophoresis, transferred to a PVDF membrane (Millipore, Billerica, MA, USA) and then detected by the proper primary and secondary antibodies before visualization with a chemiluminescence kits (Biological Industries, Kibbutz Beth HaEmek, Israel). Visualization was done with Image Quant LAS-4000 (Fujifilm, Tokyo, Japan) using image Multi-Gauge Software (Fujifilm).

For detecting 30-kDa myostatin-EGFP fusion and 36-kDa GAPDH proteins, 30 µg of protein extract was electrophoresed on 12% SDS polyacrylamide gels, using 1∶5000 mouse monoclonal anti-EGFP antibody (Sunshine Biotechnology, Nanjing, CHN) and 1∶10000 mouse monoclonal anti-GAPDH antibody (LifeSpan BioSciences, Seattle, WA). Goat anti-mouse antibody (Abmart, Shanghai, CHN), linked to horseradish peroxidase (HRP), was diluted 1∶5000 and used as the secondary antibody. Immunoreactive bands were visualized using ImageQuant LAS 4000 (Fujifilm, Tokyo, JPN). Band intensities were estimated by densitometry and normalized to GAPDH. Densitometric analysis of the bands was performed using the Image-Pro Plus (IPP) software version 6.0 (Media Cybernetics, Silver Spring, MD).

The cells were treated with (−)-asarinin (1) and incubated for 48 h. The cells were rinsed twice with ice-cold PBS and lysed with protein lysis buffer (Intron Biotechnology, Seoul, Korea) containing protease inhibitors (0.5 mM PMSF and 5 μg/mL aprotinin). The lysates were mixed with 5X sodium dodecyl sulfate (SDS) sample buffer and boiled for 5 min for denaturation. Total protein was run on 10–12% SDS-PAGE gels and electrotransferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was immunoblotted using specific primary antibodies overnight at 4 °C following blocking with 5% non-fat dry milk for 30 min–1 h. After washing, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (1:1000–2000) at room temperature for 1−2 h. After washing, immunepositive bands were visualized using an ECL chemiluminescent system and analyzed by Image Quant LAS-4000 (Fujifilm Life science, Tokyo, Japan). Anti-caspase-3 and b-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Caspase-9 antibody was purchased from Cell Signaling (Beverly, MA, USA). Caspase-8 antibody was obtained from BD Biosciences (San Jose, CA, USA).

Western blotting was performed as described previously [30 (link)]. The following primary antibodies were used: mouse anti-β-actin mAb (1:5000; Sigma-Aldrich, A5441); rabbit anti-IL-1β pAb (1:1000; Abcam, ab9722); rabbit anti-TNF-α pAb (1:1000; Abcam, ab9739); rabbit anti-GAPDH pAb (1:5000; Proteintech, 10494-1-AP); rabbit anti-alpha-synuclein pAb (1:2000; Cell Signaling, 2628); rabbit anti-TNFR2 pAb (1:1000; Beyotime, AF8199); rabbit anti-HA tag pAb (1: 1000; Abcam, ab9110); mouse anti-FLAG tag mAb (1: 5000; Sigma-Aldrich, F1804); The membrane was washed and incubated for 2 h at room temperature with the corresponding secondary antibodies: (a) HRP-conjugated goat anti-rabbit IgG (1:10,000; Jackson ImmunoResearch Laboratories, 115-035-003); (b) HRP-conjugated goat anti-mouse IgG (1:10,000; Jackson ImmunoResearch Laboratories, 115-035-003). Peroxidase activity was detected with SuperSignal WestPico chemiluminescent substrate (Pierce Biotechnology) and visualized and digitized with ImageQuant (LAS-4000, Fujifilm, Japan). Optical densities of bands were analyzed by using ImageReader software (Fujifilm, Japan). Protein levels, quantified by computer analysis as the ratio between each immunoreactive band and the levels of β-actin or GAPDH, were expressed as a percentage of vehicle-treated control.