Amersham ecl plus western blotting detection reagent

Sciatic nerves from adult control littermates (n = 6) and SC-p75^NTR-KO (n = 6) mice were dissociated in lysis buffer (2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, 140 mM NaCl and 1% Triton X-100, pH 7.8, with protease inhibitors from Roche) and centrifuged at 10.000 × g for 10 min at 4°C. Total protein concentration was determined using the Bicinchoninic Acid kit from Sigma. Protein lysates were run on 12% SDS-PAGE (20 μg/lane) and electro-blotted for 1.5 h onto polyvinylidenedifluoride (PVDF) filters (Amersham) in 192 mM glycine, 25 mM Tris–HCl, pH 8.0. Membranes were then blocked and incubated overnight at 4°C with the primary antibodies: rabbit anti-p75^NTR (1:500, Promega, Cat. #G323A) and mouse anti-β-actin (1:5000, Sigma, Cat. #A5441). Following a washing step, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:1000, swine anti-rabbit, Dako, Cat. # P0217; rabbit anti-mouse, Dako # P0260) and blots visualized with the Amersham ECL plus western blotting detection reagents (GE Healthcare) and Fuji film LAS1000. Densitometry was performed with QuantityOne software (Bio-Rad).

Matrix metalloproteinase 9 (MMP-9) or neutrophil elastase (NE) protein expression was analyzed by immunoblotting as described previously (5 (link)). Briefly, ear skin lysates were separated in polyacrylamide gel under denaturing conditions (SDS-PAGE) and transferred onto nitrocellulose membrane (Bio-Rad). The membrane was incubated in blocking buffer (TBS) containing 3% non-fat milk, followed by incubation with antibodies to MMP-9 (1:1000; Abcam), NE (1:1000; Cell Signaling Technology) or β-actin (1:1000; Cell Signaling Technology) at room temperature. HRP-coupled goat anti-mouse or anti-rabbit secondary antibodies (1:2000; BioLegend or 1:2000; Sigma-Aldrich, respectively) were used. Amersham ECL Plus Western Blotting Detection Reagents (GE Healthcare) were used to visualize the reaction. Protein levels relative to β-actin were measured by densitometry (Image Lab 6.1.0).

Immunoreactive proteins were detected as previously described [9 (link)]. Briefly, 20 µg of cytosolic and nuclear protein extracts were fractionated in SDS-PAGE gels at 6% for the detection of DOCK9, DOCK10, and DOCK11 and at 10% for the detection of HDAC1 and actin, and electroblotted onto nitrocellulose membranes. Blots were blocked, incubated with primary Abs (Table 2) in TBST with 0.5% skim milk for 2 h, washed, and incubated with horseradish peroxidase (HRP)-conjugated secondary Abs—rabbit, mouse or goat immunoglobulins (Igs) (Table 2)—in TBST with 2.5% skim milk for 1 h. After final washes, immunoreactive proteins were detected using the Amersham ECL Plus Western Blotting Detection Reagents (GE Healthcare) in the Molecular Imager ChemiDoc™ XRS+ with Image Lab software (Bio-Rad Laboratories, Hercules, CA), which provides the tools to measure the intensity of the bands. Abs and their dilutions are listed in Table 2. The primary (1ary) Abs that were obtained from the mouse or rat (Actin, HA) were monoclonal (Mo) and those obtained from the rabbit or goat (DOCK9, DOCK10, DOCK10.1, DOCK11, HDAC1) were polyclonal (Po).

The sample was concentrated to 2–10 times its original concentration using Amicon Ultra Centrifugal Filter, 10 kDa (Merck Millipore, Burlington, MA, USA). The sample was mixed with an equal volume of double the concentration of sample buffer (0.1 M Tris-HCl (pH 6.8), 4% SDS, 12% β-mercaptoethanol, 20% glycerol, and bromophenol blue) and subjected to heat treatment (95 °C, 5 min). SDS-PAGE was conducted using a 15% running gel and a 4.75% stacking gel subjected to electrophoresis at 40 mA/gel for 3 h, followed by silver staining. Western blotting was carried out using a semi-dry method, employing PVDF membrane (Clear Blot membrane-P, AE-6666, ATTO, Tokyo, Japan). The Horizon blot (AE-6677, ATTO) was utilized to transfer at 2 mA/cm² for 60 min. Blocking was conducted using Blocking One (Nacalai tesque, Kyoto, Japan). G1_P6 antibody (a peptide antibody recognizing the C-terminus of gamone 1 in B. japonicum, Thermo Scientific, Waltham, MA, USA) [25 ] was used as the primary antibody. The detection of the signal was achieved using Amersham ECL Plus Western blotting detection reagents (GE healthcare, Chicago, IL, USA), and exposure was performed on X-ray film.

PureLink DNAse set and PureLink RNA mini kit were purchased from Invitrogen (Grand Island, NY, USA). High-Capacity cDNA Reverse Transcription kit and Power SYBR green Mastermix were purchased from Applied Biosystems (Foster City, CA, USA). Polyvinylidene difluoride (PVDF) membranes were purchased from Bio-Rad (Berkeley, CA, USA). Amersham ECL Plus Western Blotting Detection reagents were purchased from GE Healthcare (Rockford, IL, USA). Xuesaitong injection (containing 400 mg PNS per ampule) was obtained from the Kunming Pharmaceutical Group Co., Ltd. (Yun Nan province, China). Rabbit anti-Notch3, mouse anti-SM22α, and rabbit anti-OPN were purchased from Abcam (Cambridge, MA, USA). TRITR-phalloidin, FITC-phalloidin, and mouse anti-α-SM-actin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse anti-β-actin and mouse anti-β-tubulin were purchased from Sigma (St. Louis, MO, USA).

Proteins were subjected to 8% to 20% gradient gels of SDS‐PAGE (GE Healthcare) or 4% to 15% gradient gels (Invitrogen) and transferred to polyvinylidene difluoride membranes. Membranes were blocked (PBS, 0.1% Tween 20, 5% bovine serum albumin) for 1 hour. Membranes were probed in blocking buffer containing primary antibodies of 1:1000 dilution: phosphorylated eNOS at serine 1177 (BD Biosciences), followed by the appropriate horseradish peroxidase–conjugated secondary antibody. Immunoreactions were visualized with Amersham ECL Plus Western Blotting Detection Reagents (GE Healthcare). Membranes were stripped (62.5 mmol/L Tris‐HCl [pH 6.8], 100 mmol/L mercaptoethanol, 2% SDS) for 30 minutes at 50°C and reprobed with O‐GlcNAc (RL2; Abcam), eNOS/NOS type III, antiactin, or glyceraldehyde‐3‐phosphate dehydrogenase antibodies of 1:1000 dilution to verify equal protein loading. The bands were quantified by densitometry (LAS‐3000 mini; FUJIFILM).