Mini protean tgx precast gels 4 20

Mice were perfused with ice-cold Dulbecco’s PBS (-) and the brain was dissected and lysed in lysis buffer (10 mM Tris–HCl [pH 7.4], 150 mM NaCl, 1% [vol/vol] Nonidet P40 Substitute, 0.5 mM EDTA) plus EDTA-free protease inhibitor cocktail cOmplete (Roche) and phosphatase inhibitor phosSTOP (Roche). Samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (Mini-Protean TGX Precastgels 4–20%, Bio-Rad, Richmond, CA), and transferred to polyvinyldifluoride (PVDF) membranes using the Trans-Blot Turbo Transfer system (Bio-Rad, Richmond, CA, USA). The membranes were blocked in 5% skim milk in PBST (0.05% Tween 20 in PBS) for 1 h and probed with the primary antibodies overnight and secondary antibodies for 1 h at RT. The following antibodies were used for Western blotting: Peroxidase AffiniPure goat anti-mouse IgG (H + L) antibody (1:10,000, 115–035-146, Jackson) and anti-GAPDH antibody (1:1000, sc32233, Santa Cruz Biotechnology). Bands were detected using Amersham ECL Prime Western Blotting Detection Reagents (GE Healthcare) and images were captured using LAS-4000 mini.

Amounts of AMP-activated protein kinase (AMPK) and phosphorylated AMPK (AMPK-P) were analyzed by Western blot. Proteins from frozen liver samples were extracted using a nuclear extract kit (Active Motif, Carlsbad, CA). The amount of protein was determined by Bio-Rad DC™ protein assay reagents. Twenty micrograms of proteins, Laemmli 1× sample buffer (Bio-Rad), and 10 mM DTT were run on Mini-protean TGX Precast gels 4–20% (Bio-Rad), blotted on a PVDF membrane using iBlot gel transfer stack (Invitrogen, Carlsbad, CA), blocked in TBS (pH 8.0) with 3% nonfat milk (catalog number T8793; Sigma-Aldrich), and washed in TBS with Tween20 (Sigma-Aldrich). Blots were incubated with rabbit-anti-AMPK [1:500 or 1:1,000 (catalog number 2532, lot 19)] or rabbit-anti-AMPK-P [1:1,000 (catalog number 2535, lot 14)] and β-actin [1:1,000 (catalog number 4970, lot 7; Cell Signaling Technology, Danvers, MA)], overnight at 4°C, and anti-rabbit IgG HRP-linked secondary Ab [1:1,000 (catalog number 7074, lot 24; Cell Signaling) for 1 h at room temperature and developed with Supersignal™ ELISA Femto substrate for 1 min and analyzed using the FluorChem HD2 program (Alpha Innotech, Kasendorf, Germany). AMPK and AMPK-P were normalized to β-actin.

Cell lysates were harvested in NP-40 lysis buffer containing Complete Mini protease inhibitor cocktail (Roche Diagnostics). A small portion of each solid tumor (from NSG mice) or mouse spleen (from HIS mice) was collected in NP-40 lysis buffer containing Complete Mini protease inhibitor cocktail and homogenized using a BioMasher® II Micro Tissue Homogenizer (VWR). Protein quantification was carried out using the Pierce™ BCA Protein Assay Kit (Thermo Fisher) and a FilterMax F5 Multi-Mode Microplate Reader (San Jose, CA, United SA). Equal concentrations of protein were separated in Mini-PROTEAN® TGX™ Precast 4–20% Gels (Bio-Rad, Hercules, CA, USA) and transferred to nitrocellulose membranes. Membranes were blocked in PBS containing 5% milk and 0.1% Tween-20 and incubated with primary antibody overnight at 4°C. The following antibodies were used: anti-Symmetric Di-Methyl Arginine Motif [sdme-RG] (1:1000, Cell Signaling, Danvers, MA, USA), anti-PARP (1:1000, Cell Signaling), anti-β-actin (1:5000, Sigma, St. Louis, MO, USA), and anti-α-tubulin (1:250, Santa Cruz Biotechnology). Secondary antibodies used include IgG HRP anti-rabbit and anti-mouse (1:5000, Promega). Blots were developed using Pierce™ ECL Western chemiluminescence substrate (Thermo Scientific). Images were captured using the Amersham Imager 600 (GE Healthcare Life Sciences).

A standard western blot protocol was carried out to evaluate ALK protein expression in cell lysates of NB cell lines. After 48 and 72 hours from transfection, cells were washed twice with ice-cold PBS and lysed with 50-100 μl of lysis buffer (Invitrogen), supplemented with a cocktail of protease inhibitors and PMSF (phenylmethylsulfonyl fluoride) (Sigma-Aldrich). Lysates were vortexed every 10 minutes and incubated on ice, for a total of 30 minutes, and then cleared by spinning. Proteins were separated in Mini-PROTEAN® TGX™ Precast 4-20% Gels (Biorad), and transfer was performed by Trans-Blot Turbo transfer system (Biorad). Membranes were incubated with primary antibodies for ALK (Cell Signaling) and GAPDH (Cell Signaling), which was used as a control for equal protein loading. Goat anti-rabbit IgG HRP conjugated secondary antibody was from Santa Cruz Biotechnologies. Blots were developed with Amersham’s ECL Prime (GE Healthcare) and images acquired by a chemiluminescent detection system (Uvitec Cambridge). Quantification of western blot bands was performed by ImageJ [74 ].

Forty microliters of each SEC fraction were loaded and separated on Mini-Protean TGX precast 4–20% gels (Bio-Rad Laboratory), and proteins were blotted onto PVDF membranes using a Trans-Blot Turbo Transfer system (Bio-Rad Laboratory). Membranes were blocked with 5% non-fat dry milk in TBS containing 0.01% Tween-20 (TBST). Membranes were incubated with the following primary antibodies: CD81 (1:500 dilution; clone H-121, sc-9158, Santa Cruz Biotechnology, Santa Cruz, CA), TSG-101 (1:500 dilution; clone 4A10, ab83, Abcam, Cambridge, UK), flotillin-1 (1:1000 dilution; clone H-104, sc-25506, Santa Cruz Biotechnology), and Apo-A (1:1000 dilution; clone FL-267, sc-30089, Santa Cruz Biotechnology), diluted in TBST overnight at 4 °C. Membranes were washed three times before being incubated with the following secondary antibodies diluted in TBST; donkey anti-rabbit IgG HRP-linked F(ab′)₂ fragment (1:10,000 dilution; NA9340V), and sheep anti-mouse IgG HRP-linked F(ab′)₂ fragment (1:10,000 dilutions; NA9310V) (both from GE Healthcare, Buckinghamshire, UK). Blots were visualised with SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific) and a VersaDoc 4000 MP imaging system (Bio-Rad Laboratory) with Quantity One software.