Nupage novex 3 8 tris acetate protein gel

Myocytes were cultured in D-MEM with Sodium Pyruvate, without Amino Acids (WAKO, Osaka, Japan) for 1 h to lower mTORC1 activity. The cells were then cultured with DMEM containing amino acids and 100 nM insulin (Nacalai Tesque) for 10 min. After washing with cold PBS, the cells were lysed in RIPA buffer containing 1% (v/v) PIC and thoroughly sonicated on ice. Proteins were isolated from the lysate as described above. The isolated proteins (20 µg) were separated by electrophoresis on NuPAGE Novex 3–8% Tris-Acetate Protein Gel (Thermo Fisher Scientific) at 150 V for 60 min for S6K and pS6K, or on Extra PAGE One Precast Gel 15% (Nacalai Tesque) at 300 V for 30 min for 4E-BP1, p4E-BP1 and LC3, and transferred to a nitrocellulose membrane using an iBlot system (Thermo Fisher Scientific) with the program, P0, 9 min. The membrane was blocked with PBS-T containing 1% (w/w) skim milk and then incubated with primary antibody solution at 4 °C overnight. After washing with PBS-T three times, the membrane was incubated with secondary antibody solution for 1 h at room temperature. The blots were developed by Pierce Western Blotting Substrate Plus (Thermo Fisher Scientific) or ImmunoStar LD (WAKO). The bands were digitally detected by ChemiDoc XRS+ (Bio-Rad, Hercules, CA, USA) and quantified by Quantity One software (Bio-Rad).

The analysis of proteasome complexes and activity were performed by native gel electrophoresis, as reported [34 ,53 (link)]. Ten million fresh or rapidly thawed cells were used for each experimental point. Thirty micrograms of total protein were migrated per well in NuPAGE™ Novex™ 3–8% Tris-Acetate Protein Gel (Thermo Fisher Scientific, Walthan, MA, USA). Migrations were performed in native gel electrophoresis buffer (NG buffer) (90 mM Tris-borate, 0.1 mM EDTA, 5 mM MgCl₂, 0.5 mM ATP, 0.5 mM DTT) at 150 volts for 3 h. The intrinsic activity of native proteasomes was analysed in gel by 20 min incubation in NG buffer supplemented with 100 µM Suc-LLVY-AMC (Bachem, Bubendorf, Switzerland) at 37 °C. The amount of cleaved AMC fragment was imaged with Syngene NuGenius. Native gels were then washed twice for 10 min in 10× Tris-glycine–SDS Laemmli buffer (0.25 M Tris, 1.92 M glycine, 1% SDS, pH 8.6), followed by a final wash in 1× Tris-glycine–SDS Laemmli buffer. Gels were transferred in PVDF membranes (0.45 µm pore size, Immobilon-P, Merck, Kenilworth, NJ USA) overnight at 40 volts at 4 °C. Membranes were blotted and analysed for the proteins of interest.

To assess HIF1α protein stabilization, proteins were extracted from cultured cells as follows: cells were placed on ice, washed twice with ice-cold PBS, and lysed in protein extraction buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.5% Na-deoxycholate, 0.1% SDS, and 1× protease inhibitor cocktail (Roche)). Protein concentrations were determined using a bicinchoninic acid protein assay (BCA, Thermo Scientific) following the manufacturer’s protocol. An estimated 60 μg of protein was loaded per well on a NuPAGE Novex 3–8% Tris-Acetate Protein gel (Life Technologies), separated by electrophoresis and blotted on polyvinylidene fluoride membranes. Membranes were activated with methanol and washed with Tris-buffered saline (TBS; 50 mM Tris-HCl, 150 mM NaCl) with 0.1% Tween 20, and incubated with rabbit α-tubulin (2144S, Cell Signaling), rabbit β-actin (4967, Cell Signaling), rabbit HIF-1β/ARNT (D28F3) XP® (5537, Cell Signaling) at 1:1000 dilution, and rabbit HIF-1α (C-Term) Polyclonal Antibody (Cayman Chemical Item 10006421) 1:3000. Incubation with the secondary antibodies and detection were performed according to routine laboratory practices. Western blotting was done on 3 independent biological replicates.