Nupage bis tris gel

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 15 μg of protein from each sample on a 4–12% NuPAGE Bis-Tris Gel (Bio-Rad, USA). The proteins were blotted onto polyvinylidene difluoride (PVDF) membranes, blocked with 5% dry milk and incubated at 4°C overnight with anti-HE antibody (clone 8D2/E9; GrupoBios, Bios-Chile) diluted 1:2000 or anti-β-actin antibody (clone AC-74, Sigma-Aldrich) diluted 1:500, followed by a 1 h incubation with the secondary antibody [rabbit-anti-mouse horseradish peroxidase (HRP), Invitrogen, USA] at a dilution of 1:20000. The blots were developed using Novex® ECL HRP Chemiluminescent Substrate (Invitrogen, USA) and exposed to Carestream Kodak Biomax Light film (sigma Aldrich, USA). The films were scanned and used for expression analysis with ImageJ 1.43 μ software.

Mouse heart ventricles were homogenized in RIPA buffer containing protease and phosphatase inhibitors. Protein concentrations were determined using the BCA (Bicinchoninic Acid) Protein Assay Reagent Kit (Bio-Rad). Protein samples were separated on polyacrylamide gels (10% NuPAGE Bis-Tris Gel) and transferred to nitrocellulose membrane (Bio-Rad). The membranes were blocked with 5% BSA (Bovine Serum Albumin) and incubated with primary antibody diluted in 5% BSA at 4 °C overnight, followed by washing with TBST (TBS containing 0.1% Tween 20) solution. Membranes were incubated with near-infrared fluorophore-conjugated secondary antibodies diluted in TBST for 1 h at room temperature and followed by TBST washing three times. Target protein bands on the membranes were visualized using the Odyssey imaging system (LI-COR Biosciences). Primary antibodies include phospho-FOXO1 (Ser256)(E1F7T) Rabbit mAb (CST, #84192), phospho-FOXO3 (Ser318/321) (CST, #9465), FOXO1 (C29H4) Rabbit mAb (CST, #2880), and FOXO3 (75D8) Rabbit mAb (CST, #2497).

Adult worm lysates were resolved via SDS-PAGE on a 4 to 12% gel (Invitrogen NuPAGE Bis-Tris gel) and transferred to a 4.5-μm nitrocellulose membrane (Bio-Rad) at 100 mA for 1 h. The membrane was blocked using 3% (wt/vol) BSA in PBS with 0.01% Tween for 1 h, followed by overnight incubation at 4°C with an anti-ZFP-1 C-terminus-specific antibody [82 (link)], diluted 1:2,000 in PBST-3% BSA. The membrane was then washed 3 times with PBST, incubated for 1 h at room temperature with a horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (PerkinElmer) diluted 1:5,000 in PBST-3% BSA, and visualized by using SuperSignal West Pico chemiluminescence substrate (Thermo Scientific) and a series 2000A film processor (Tiba).