Horseradish peroxidase hrp conjugated secondary antibody

Western blotting was performed as described previously^{3 (link)}. Briefly, retinal proteins were separated on an sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, USA). The membrane was incubated with primary antibodies against STAT3 (1:400, Cat# sc-8019, Santa Cruz Biotech, Dallas, TX, USA), p-STAT3 (1:1000, Cat# 9145, Cell Signalling Technology, Danvers, MA, USA) and β-actin (1:200, Cat# sc-47778, Santa Cruz Biotech) overnight and then with horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signalling Technology). The bands were visualised with Immobilon Western Chemilum HRP Substrate (Millipore, Darmstadt, Germany) and an image capture system (Bio-Rad). Relative protein expression levels were measured by calculating the band density with ImageJ software (National Institutes of Health, USA).

Protein concentration was quantified using the Quantum Protein BCA Assay kit (Euroclone, Pero, Italy) in 80% confluent male and female AFCs after cell lysis. Western blotting was performed on 25 μg of solubilized protein as previously described [28 (link)]; protein expression was evaluated using the following primary antibodies: actin (1:1000; Sigma-Aldrich, Milano, Italy), ERα, ERβ (1:1000; Thermo Fisher Scientific, Rodano, Italy), LC3 (1:1000; MBL, Eppendorf Italia, Milano, Italy), p62 (1:1000), and LAMP1 (1:1000); these were obtained from Cell Signaling Technology (Milano, Italy) as primary antibodies.
After 1 h incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000; Cell Signaling Technology, Milano, Italy), the binding was detected by chemiluminescence with the Bio-Rad ChemiDoc instrument (Bio-Rad, Milano, Italy). Band volume analysis was performed using the Image Lab 4.0 software (Bio-Rad, Milano, Italy). A pilot study (5 samples of each sex) suggested that in female and male AFCs, the expression of α-actin was very similar (827,366.79 ± 578,040.86 optical density (OD) and 839,428.71 ± 713,143.57 OD for females and males, respectively; p = 0.49). Therefore, it was used for the normalization of Western blot analysis.

Protein was extracted from MSCs and quantified as described above. Equal amounts of protein were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). The polyvinylidene difluoride (PVDF) membranes were incubated with primary antibodies against GAPDH, OCN, Runx2, AKT, p‐AKT, Smad1, p‐Smad1/5/9, total catenin, p‐catenin, ERK1/2, and p‐ERK1/2 (all diluted 1:1000, Cell Signaling Technology) for 24 h and were then washed and incubated with a horseradish peroxidase (HRP)‐conjugated secondary antibody (1:3000, Cell Signaling Technology) for 1 h. Specific antibody‐antigen complexes were detected using Immobilon Western Chemiluminescent horseradish peroxidase (HRP) Substrate (Millipore).

RAW264.7 cells (1 x 10⁶ cells) were seeded into a 5.5-cm culture dish and cultured for 24 h. Cells were pre-treated with various concentrations of HCFP aqueous (25–750 μg/mL) and methanolic (4–12 μg/mL) extracts for 2 h and then incubated with LPS (1 μg/mL) for 24 h. The treatment with diclofenac (DCF; 25 μg/mL) was used as a positive control. The treated cells were harvested and lysed with lysis buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 5mM EDTA, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) for 1 h on ice. The protein concentration was determined by Bradford protein assay (Bio-Rad, USA). Equal amounts of protein (30 μg) were loaded and separated on 12% SDS-polyacrylamide gel and afterward the proteins were transferred to the PVDF membrane. The membrane was blocked with a blocking solution, 5% skim milk in phosphate-buffered saline containing Tween-20 (PBST), for 1 h at room temperature, and then incubated with monoclonal anti-iNOS, anti-COX-2, anti-β-Actin (1:1000 dilutions, Cell signaling, Germany) for overnight at 4°C. The blots were washed twice with PBST and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:1000 dilutions, Cell signaling, Germany) for 2 h at room temperature. Blots were washed again twice with PBST and PBS, respectively. The protein bands were visualized using ECL detection reagent (GE healthcare, UK).

To isolate the proteins, cellular total proteins were lysed with RIPA lysis buffer (CWBIO, Beijing, China) and using a Protein BCA Assay Kit (Bio-Rad, Hercules, California, USA) to quantify content of protein. Protein samples were separated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore Corporation, Billerica, MA, USA). After blocking in 5% powdered milk for at least 1 h at room temperature, the membranes were incubated by using rabbit anti-ROCK1 and anti-VEGFA antibodies (1 : 1000, Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. Afterward, washing and incubating the membranes with a horseradish peroxidase- (HRP-) conjugated secondary antibody (1 : 10000, Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. Subsequent visualization was detected using a chemiluminescent HRP substrate (Millipore Corporation, Billerica, USA) and imaged with an E-Gel Imager. Protein levels were normalized to GADPH.

HMEC-1 cells were seeded at 3 × 10⁵/well in 6-well plates overnight and infected with GAS as described above. Harvested cells were lysed in lysis buffer containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma-Aldrich). After centrifugation, the lysates were boiled in sample buffer for 10 min. Samples were then subjected to SDS-PAGE and proteins transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). After blocking with 5% skim milk in PBS, the membranes were incubated overnight at 4°C with primary antibodies, including anti-p70s6k antibody (cs9202), anti-phospho-p70s6k antibody (Thr389) (cs9205), anti-AKT antibody (cs9272), anti-phospho-AKT antibody (Ser473) (cs9271), anti-ERK (p44/42 MAPK) antibody (cs9102), anti-phospho-ERK antibody (Thr202/Tyr204) (cs9101), anti-β1 integrin antibody (cs4706), and anti-β-actin antibody (AC-74; Sigma-Aldrich). The above-named antibodies were purchased from Cell Signaling Technology, except for anti-β-actin antibody. After incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (Cell signaling Technology) at RT for 1 h, membranes were soaked in ECL solution (PerkinElmer Life and Analytical Sciences, Inc.) and the images were captured by a luminescence imaging system (LAS-4000; Fujifilm).