Clarity maxtm western ecl substrate

Samples were lysed in a lysis buffer (1% TritonX-100, 10 mM Tris-HCl, 50 mM NaCl, 5 mM EDTA, 30 mM Na₄P₂O₇, 50 mM NaF, 0.1 mM Na₃VO₄) with the addition of protease and phosphatase inhibitors (1%). Protein content was determined and equal amounts of protein were loaded and electrophoresed on a 4–20% precast polyacrylamide gel (Bio-Rad Laboratories AB, Solna, Sweden). The fractionated proteins were blotted on a Trans-Blot Turbo Transfer System (Bio-Rad Laboratories AB, Solna, Sweden) and this was followed by blocking of the membrane in 5% (w/v) milk in Tris-buffered saline Tween-20 and overnight incubation with primary antibodies at 4 °C: anti GAPDH mAb, anti TLR3 mAb, anti RIG-I Rabbit mAb and anti MDA5 Rabbit mAb (Cell Signaling Technology, Leiden, The Netherlands). Membranes were washed and incubated for 1 h with secondary antibodies: anti Rabbit IgG HRP-linked Ab (Cell Signaling Technology, Leiden, The Netherlands). Chemiluminescent detection was performed using Clarity Max^TM Western ECL Substrate (Bio-Rad Laboratories AB, Solna, Sweden) and immunoblots were visualized by LI-COR Odyssey Fc Imager (LI-COR Biosciences, Lincoln, NE, USA) and Image Studio (v3.1.4; LI-COR Biosciences, Lincoln, NE, USA).

Protein samples were loaded on 10% SDS-PAGE for separation on the basis of molecular weight and then transferred on 0.45 μm nitrocellulose membrane (Merck-Millipore) by wet transfer for 1 h at 90V with TE series transphor electrophoresis unit (Hoefer) at 4 °C. The 5% bovine serum albumin solution was used to block the membrane for 3 h at 37 °C, after that polyclonal anti-rabbit LmChaC_2a or LmChaC_2b antibody was applied for overnight incubation at 4 °C. Then membrane was washed thrice with 1× Tris-buffered saline with Tween-20 at 5 min interval and membrane was incubated for 2 h with AP-conjugated anti-rabbit secondary antibody (1:16,000) (Sigma) or horseradish peroxidase (HRP) conjugate secondary antibody (1:16,000) (Merck). Anti-α-tubulin antibody (Merck) and AP or HRP-conjugated antimouse secondary antibody (1:15,000) (Sigma) was used to detect α-tubulin, which was measured as a loading control. Clarity Max TM Western ECL substrate (Bio-rad) and NBT-BCIP (Roche) was applied to develop the band for HRP conjugate antibody and AP conjugated antibody, respectively.

The effects of BEOs on the expression of important proteins involved in the inflammatory response in RAW 264.7 macrophages were assessed by Western blotting. The cells (4 × 10⁵ cells/well in 6-wells plate) were cultured for 24 h before incubating with BEOs for 2 h. The cells were then stimulated by LPS (1 µg/mL) for 24 h before being harvested and resuspended in radioimmunoprecipitation assay lysis buffer (Thermo Scientific, Rockford, IL, USA) to obtain the total protein. Total protein samples (20 µg each) were analyzed using SDS-PAGE before being transferred to a PVDF membrane. The membrane was blocked with T-TBS (Tris-buffered saline, 0.1% Tween 20) containing 2% bovine serum albumin (Biobasic, Markham, Ontario, Canada) for 1 h. The membrane was then incubated with primary antibodies (1:1000 dilutions) for detecting β-actin, iNOS, COX-2, IκBα, p-IκBα, NF-κB p65, and p-NF-κBp65 for 16 h at 4 °C. The membranes were then washed thrice with T-TBS before being incubated with HRP-conjugated goat anti-rabbit IgG for 1 h at 25 °C. The membranes were washed thrice and then incubated with Clarity Max^TM Western ECL substrate (Bio-Rad, Milan, Italy) and the signals were detected using a ChemmiDoc^TM imaging system (Bio-Rad, Hercules, CA, USA). Protein quantities from Western blot results were calculated by Image Lab software Version 6.1 (Bio-Rad, Hercules, CA, USA).

Patient-derived tumor cells were treated with cell lysis buffer (Cell Signaling Technologies, Danvers, Massachusetts, 9803S), complete mini protease inhibitor mixture tablets (Roche, 11836153001), and PhosSTOP Phosphatase Inhibitor Cocktail Tablets (Roche, 4906845001). Lysates were spun at 8,000 xg for 10 minutes at 4°C, mixed 3:1 with 4x Laemmli Sample Buffer (Bio-Rad, 1610747) with b-ME, and heated at 95°C for 5 minutes. Lysates were run on Criterion TGX Precast Midi Protein Gel (Bio-Rad, 5671083), transferred to a polyvinylidene difluoride membrane (Bio-Rad, 1704157), and blocked for 1 hour in TBS-T with 5% milk. Blots were probed overnight at 4°C with PERK (C33E10) Rabbit, Phospho-p44/42 MAPK (Erk1/2) Rabbit, Phospho-Akt (Ser473) (D9E) XP Rabbit, Akt (pan) (C67E7) Rabbit, or a-Tubulin (DM1A) Mouse (Cell Signaling Technology). HRP conjugated secondary antibodies, anti-rabbit, or anti-mouse IgG were used for rabbit and mouse primary antibodies, respectively. Blots were developed using Clarity^TM or Clarity Max^TM Western ECL Substrate (Bio-Rad, 1705060 and 1705062) and imaged using a Bio-Rad ChemiDoc touch MP Imaging System. For all western blots the same blot was probed repeatedly.

SDS-PAGE and Western blot analysis were performed as previously described [26 (link)]. The following primary antibodies were used: cleaved caspase-3 antibody (1:3000), α-tubulin antibody (1:3000), LaminB1 antibody (1:3000), importin alpha 5 antibody (1:3000), p63(ΔN) antibody (1:3000), and β-actin antibody (1:4000). HRP-labeled anti-rabbit IgG antibody and HRP-labeled anti-mouse IgG antibody were used as secondary antibodies (1:10,000). The antigens were visualized using Clarity^TM Western ECL Substrate or Clarity MAX^TM Western ECL Substrate (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Blot stripping was performed using stripping solution (Wako Pure Chemical Industries, Ltd.). Images were captured using the Cool Saver AE-6955 (ATTO, Tokyo, Japan) or iBright 1500 Imaging System (Thermo Fisher Scientific, Inc.). Quantification of the bands was performed using iBright Analysis Software (Thermo Fisher Scientific, Inc.).

SDS-PAGE and western blot analysis were performed as previously reported [35 (link)]. The samples were probed with primary antibodies against cleaved caspase-3 (1:3000) and β-actin (1:4000), followed by HRP-conjugated anti-rabbit secondary antibodies against IgG (1:10,000). The antigens were visualized using Clarity MAX^TM Western ECL Substrate (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

The total protein in the uterus was extracted using RIPA lysis buffer containing phosphatase and protease inhibitor cocktails. Equal amounts of protein (15 μg) were separated on 10% sodium dodecyl sulfate polyacrylamide gels and then transferred on to polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk for 1 h and then probed with specific primary antibodies at a final dilution of 1 : 1000. The immunoreactive protein bands were detected using an enhanced chemiluminescence western blot detection kit (Clarity Max^TM Western ECL Substrate; Bio-Rad Laboratories, Hercules, CA, USA) and visualized using the Chemidoc Touch image system (ChemiDoc^TM XRS + system; Bio-Rad Laboratories).