Tgx stain free fastcast acrylamide kit

The viable (non-infarcted) LV tissue samples were homogenized in RIPA buffer (Teknova, Hollister, CA, USA). The extracted protein was quantified using Pierce^TM BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Equal amounts of protein samples were separated on 7.5% sodium dodecyl sulphate-polyacrylamide gels (SDS-PAGE) using TGX Stain-Free^TM FastCast^TM Acrylamide Kit (Bio-Rad, Hercules, CA, USA). After electrophoresis, the separate proteins were transferred using the Trans-Blot Turbo Mini Nitrocellulose Transfer Packs and the Trans-Blot Transfer System (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% dry milk in Tris-buffered saline/0.1% Tween 20 (TBST) for 1 h at room temperature, and then incubated overnight at 4 °C with a primary anti-rat collagen type I (1:1000, CL50141AP, Cedarlane, Burlington, ON, Canada) or monoclonal anti-Na⁺-Ca²⁺ exchanger (1:1000, R3F1, Swant, Fribourg, Switzerland) in 1% dry milk. The membrane was washed with TBST and then incubated with secondary antibody IgG (1:10,000, Abcam, Cambridge, MA, USA) conjugated with horseradish peroxidase in 1% dry milk. The membrane blots were developed using Luminata^TM Forte Western HRP Substrate (MilliporeSigma, Burlington, MA, USA) and imaged on a Gel Doc^TM XR+ imager (Bio-Rad, Hercules, CA, USA).

SDS-PAGE, i.e., Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis—a common method for separating proteins based on their molecular weight—was used to separate the samples on a polyacrylamide gel. The TGX Stain-Free^TM Fast Cast^TM Acrylamide Kit (SDS-PAGE), supplied by Bio-Rad Laboratories Inc. Cat #161-0181, was used to create polyacrylamide gels. The manufacturer’s instructions were followed while preparing the SDS-PAGE TGX Stain-Free Fast Cast.

Again, ≈50 mg of muscle tissue were mechanically homogenized (6000 rpm for 2 × 30 s) in 1 × CST Cell Lysis Buffer (Cell Signaling, Frankfurt am Main, Germany) using Precellys ceramic beads. Samples were incubated on ice for 20 min and diluted 1:2 with 2× Laemmli (31.25 mM tris(hydroxymethyl)aminomethane, 1% sodium dodecyl sulfate, 5% glycerol). After denaturation (10 min, 94 °C) and centrifugation (2 min, 21,000× g, 4 °C), the protein concentration was also determined using the bicinchoninic acid method described earlier. The protein concentrations were then adjusted to 1 mg/mL with 1 × Laemmli, and beta-mercaptoethanol was added (to 0.4% finally). The samples (20 µg total protein) were separated by SDS-PAGE (Bio-Rad TGX Stain-Free FastCast Acrylamide kit; Bio-Rad Laboratories GmbH, Munich, Germany). After electrophoresis, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane by semi-dry blotting (60 min, current 80 mA/gel).

For electrophoresis, Bio-Rad TGX Stain-Free FastCast Acrylamide-Kit (Bio-Rad Laboratories GmbH, Munich, Germany) gels were used. All gels were blotted on polyvinylidene fluoride membranes (PVDF, pore size 0.45 µm, Carl Roth GmbH + Co. KG, Karlsruhe, Germany), blocked for 1 h with 3% powdered milk in PBS and incubated overnight with a primary antibody for AKT phosphorylated at serine 473 (CST #9271, dilution 1:1000, Cell Signaling Technologies). After washing, the membranes were incubated for 2 h with a secondary antibody (antirabbit IgG HRP, CST #7074, dilution 1:2000, Cell Signaling Technologies). Bands were visualized using Lumigen ECL Ultra (Lumigen Inc., Southfield, MI, USA) in a Bio-Rad Chemi-Doc MP system (Bio-Rad Laboratories GmbH). The images were analyzed using Image Lab Ver. 6.0.1 software (Bio-Rad Laboratories GmbH, Hercules, CA, USA) and normalized for total protein concentration. The molecular weight of phosphorylated human AKT is predicted to be 56–57 kDa.

A Western blot analysis was performed using standard techniques. In brief, conjunctivas were homogenized in RIPA lysis buffer with PMSF (RIPA: PMSF = 100: 1) (Beyotime, Shanghai, China), to extract total protein. After being quantified by a BCA protein assay kit (Beyotime), protein samples were separated using a 12% TGX Stain-Free FastCast Acrylamide Kit (Bio-Rad, Hercules, CA) and blotted to PVDF membranes (Merck-Millipore, Darmstadt, Germany). Membranes were blocked with 5% non-fatty milk and incubated with primary antibodies against GAPDH (1:5000, Cell Signaling Technology, Danvers, MA), IL-17A (1:1000, Abcam, Cambridge, UK). Membranes were then washed, incubated with appropriate peroxidase-conjugated secondary antibodies, and developed into an ECL kit (Najm Biotech ECL, Tehran, Iran).

Proteins from tissue and cell lines were extracted using a total protein extraction kit (BestBio). The protein concentration was determined using Micro BCA^™ Protein Assay Kit (Thermo Fisher Scientific) and after incubation for 1 hour, the protein concentration of samples and standards was determined at a wavelength of 562 nm using a microplate reader. All proteins were standardized to a concentration of 3 μg/μL and denatured at 99°C for 5 minutes. Total proteins were separated using TGX Stain‐Free^™ FastCast^™ Acrylamide Kit (BioRad) and transferred to the polyvinylidene difluoride membranes (PVDF, 16 20 177, BioRad). Subsequently, the membranes were blocked with 5% Bovine Serum Albumin (BSA) for 3 hours and incubated with primary antibody YY1 (1:12 000, Abcam) and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH, 1:5000, ZenBio) overnight at 4°C. The membranes were washed three times with TBST and then incubated with horseradish peroxidase (HRP)‐conjugated secondary antibody goat anti‐rabbit (1:10 000, HuaBio) and goat anti‐mouse (1:2000, HuaBio) for 1 hour at room temperature. Lastly, Immobilon Western Chemiluminescent HRP Substrate (Millipore) was used to detect the chemiluminescence intensity via the ChemiDoc^™ MP Imaging System (BioRad).

Protein extraction was performed with 100 μL RIPA lysis buffer (Beyotime, Shanghai, China) containing 1% phenylmethylsulfonyl fluoride (PMSF, Beyotime, Shanghai, China) by using the TGX Stain-Free FastCast Acrylamide Kit (Bio-Rad, 1610182, Hercules, CA, USA). The extracted proteins were separated by sodium lauryl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a semidry transfer membrane. The membrane was sealed with a sealing solution (Bio-Rad, P0023B, Shanghai, China) at 25 °C for 2 h and then incubated with the following antibodies at 4 °C for 12 h: purified anti-Vg antibody (1:1000), anti-30Kc19 antibody (1:1000), anti-V5 tag antibody (1:1000, AB_2533339, Thermo Fisher, Waltham, MA, USA), and anti-α-tubulin antibody (1:5000, T0033, Affinity). After three times washing with Tris-buffered saline (TBS) containing 0.05% Tween 20 (TBST; pH 7.5), the membrane was incubated with HRP-conjugated anti-mouse IgG (1:5000, Bioworld Technology, St Louis Park, MN, USA) at 37 °C for 2 h. After washing the membrane again with TBST three times, an appropriate amount of ECL (Bio-Rad) coloring solution (1:1) was added, and the membrane was kept incubated in dark. Image Lab software was used to process and analyze the generated images.