Trans blot turbotm transfer pack

Cells were lysed in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P40 Substitute). Protein concentration was determined using the Bradford reagent (Sigma Aldrich). Equal amounts of proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes by using Trans-Blot Turbo^TM Transfer Pack (Bio-Rad, Hercules, CA, USA). Western blot analysis was performed using primary antibodies against Pgp, PON2, TXNIP, CYP D (Thermo Fisher Scientific), SOD2, OPA1 (BD Biosciences, St. Diego, CA, USA), TRPM7 (Bethyl, Montgomery, TX, USA), MagT1 (Abcam, Cambridge, UK), SIRT2 (Merck Millipore, Burlington, MA, USA), DRP1 (Cell Signalling, Danvers, MA, USA), β-actin and GAPDH (Santa-Cruz Biotechnology, Dallas, TX, USA). Secondary antibodies conjugated with horseradish peroxidase (Amersham Pharmacia Biotech Italia, Cologno Monzese, Italy) were used. The immunoreactive proteins were detected with Clarity^TM Western ECL substrate (Bio-Rad) and images were captured with a ChemiDoc MP Imaging System (Bio-Rad). Densitometry of the bands was performed with the software ImageJ (National Institute of Health, Bethesda, MD, USA). The Western blots shown are representative and the densitometric analysis was performed on three independent experiments ± SD.

Fibroblast cells were lysed in RIPA buffer (R0278 Sigma) containing a protease inhibitor cocktail (Roche), and total protein was quantified using a Bradford Reagent protein assay (B6916 Sigma-Aldrich). Protein lysates were denatured by 1X SDS Sample Buffer. The resulting samples were incubated at 95°C for 5 min. Protein samples (100 μg) were then separated on TGX Stain-FreeTM Gels (Bio-Rad) and electroblotted onto a PVDF membrane (Trans-Blot^® TurboTM Transfer Pack/Bio-Rad). Membranes were incubated in blocking buffer (5% non-fat dried milk diluted in TBS + 0.1% Tween) for 1 h at room temperature, washed three times in TBS + 0.1% Tween for 5 min, and incubated with primary antibody (PRPF8 Abcam ab79237) at 1:500 dilution in blocking buffer overnight at 4°C. Thereafter, blots were washed three times in TBS + 0.1% Tween and incubated with secondary HRP-conjugated antibody in blocking buffer for 45 min at RT. Blots were washed another five times and protein bands were visualized using SuperSignal West Pico PLUS (Thermo Scientific) on X-ray films. β-ACTIN (monoclonal, 1:4,000,000, Sigma-Aldrich A3854) was used as a loading control. HiMark Prestained Protein Standard (LC5699, Thermo) was used as a molecular weight standard.

Total cell lysates and tissue were lysed on ice using a radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 5 mM EDTA, 1 mM DTT, 100 mM NaF, 2 mM sodium pyrophosphate, 20 mM β-glycerophosphate, 2 mM sodium orthovanadate) and 1× protease inhibitor cocktail (Sigma-Aldrich) for 30 min. The protein contents of cell and tissue lysates were quantified using the Pierce BCA Protein Assay kit (23209, Thermo Scientific, Chicago, IL, USA). Thereafter, proteins (10–50 μg) were separated on a 6–15% gel via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane using a Trans-Blot^® TurboTM Transfer pack (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% skim milk/TBST for 1 h and incubated with primary antibodies overnight at 4 °C. After three washes, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 40 min at RT. After washing for 2 h, the protein bands were visualized using Clarity Western ECL Substrate (1705061; Bio-Rad, Hercules, CA, USA), and chemiluminescent images were captured by Fusion Solo S (Vilber Lourmat, Paris, France), following the instruction manual.

The cells were lysed on ice by treating them with radio-immunoprecipitation assay lysis buffer and 1x protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO, USA) for 30 min. The lysates were quantified using the Pierce BCA Protein Assay kit (23209; Thermo Scientific, Rockford, IL, USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed to separate 10–40 μg of proteins using a 10–15% gel, which was subsequently transferred to a polyvinylidene difluoride membrane using a Trans-Blot® TurboTM Transfer pack (Bio-Rad, CA, USA). The membranes were blocked using 5% skim milk dissolved in phosphate-buffered saline with 1% Tween 20 (PBST) for 1 h and incubated with primary antibodies overnight at 4 °C. After washing the membrane three times with PBST, they were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Next, after washing for 2 h with PBST, the protein bands were visualized using Clarity Western ECL Substrate (1705061; Bio-Rad, CA, USA).

Pulled down proteins were separated on Mini-PROTEAN^® TGX^TM 4–20% precast gels (BIO-RAD Laboratories, Inc. USA) and then transferred onto a 0.2 µm nitrocellulose membrane by Trans-Blot^® Turbo^TM Transfer Pack (BIO-RAD Laboratories, Inc. USA) and Transfer Blot Turbo Transfer System (BIO-RAD). Each membrane was blocked with 5% BSA in PBS-T. A rabbit-anti-HLTF antibody (Abcam, ab17984) and rabbit-anti-heparanase antibody (InSight Pharmaceuticals, Rehovot, Israel) were applied for the assay. After washing, membrane was incubated with HRP-conjugated anti-rabbit secondary antibody. Detection was performed by applying enhanced chemiluminescence (EZ-ECL, Biological Industries, Israel).

Cellular protein extracts were isolated by directly adding Laemmli sample buffer containing 5% beta mercaptoethanol to the cells. Protein concentrations were measured using Bradford protein assay. Equal amounts of protein extracts (50μg) were separated by Mini Protean precast gels TGX (Biorad). Afterwards, protein extracts were transferred to nitrocellulose membrane using a Trans-Blot® TurboTM transfer Pack (biorad) and Biorad semi-dry Transfer System. Membranes were blocked for 1 h in 5 mL 5% BSA/0.1% Tween/TBS and probed overnight with primary antibody for pSTAT1 Y701 (Cell Signaling #9167), pSTAT1 S727 (Cell Signaling #9177), Total-STAT1 (Cell Signaling #9172), IRF-1 (Cell Signaling #14028), Phospho-AKT (Cell Signaling #9271) or beta-actin (Cell Signaling #3700). membranes were washed 3 times with 5 mL 0.1% Tween/TBS and incubated with anti-rabbit IgG or anti-mouse IgG with HPR-linked antibody (Cell signaling #7074, #7075) for 1 h. Membranes were developed using 1X SignalFireTM ECL Reagent (Cell signaling) and analyzed with a compatible imager. Western-blots were quantified by measuring protein band intensity using imageJ software. Samples were normalized with beta-actin band intensity.

The total protein concentration of the tissue homogenate from cultured nasal fibroblasts was measured using a Bicinchoninic acid Protein Assay Kit per the manufacturer’s instructions (Thermo Fisher Scientific), and 20 ng of protein was loaded for electrophoresis on a Mini-PROTEAN^® TGXTM Gel (Bio-Rad Laboratories, Hercules, CA, United States). The protein was then transblotted using a Trans-Blot^® TurboTM Transfer Pack and Trans-Blot^® TurboTM system (Bio-Rad Laboratories). Following transfer, the blots were blocked with 5% non-fat dry milk in 0.1% Tween 20/Tris-buffer saline (TBS) and TBS alone, and then incubated with anti-leptin receptor antibody (1:100; Novus Biologicals, Centennial, CO, United States). They were then incubated with a horse radish peroxidase-conjugated (HRP-conjugated) anti-rabbit IgG antibody (Dako; Agilent Technologies, Santa Clara, CA, United States). Subsequently, the blots were developed using chemiluminescent Western blot detection reagents (Amersham ECL Prime; GE Healthcare, Chicago, IL, United States) according to the manufacturer’s instructions. The blot density was scanned using Fusion Solo S (Vilber, Marne-la-Vallée cedex, France).