Horseradish peroxidase hrp

Incubated cells were treated with serum-free medium, Pep1, Pep2, DC, DOX/Pep2, DOX/Pep1, DC/Pep2, or DC/Pep1 for 24 h. RIPA lysis buffer was used to extract total protein from cells. SDS‒PAGE was used to isolate and transfer 20 μg protein samples onto 0.45 μm PVDF membranes. E-cadherin (dilution 1:1000, Beyotime), Vimentin (dilution 1:3000, Beyotime), Caspase-3 (dilution 1:3000, Beyotime), and β-actin (dilution 1:3000, Beyotime) rabbit monoclonal antibodies were added for incubation overnight at 4 °C after blocking. Samples were incubated with secondary antibodies coupled with horseradish peroxidase (HRP) at a dilution of 1:1000 (Beyotime) for a period of 1–2 h, followed by development using enhanced chemiluminescence (ECL) reagents.

Tumor samples were formalin-fixed and embedded in paraffin. Paraffin sections were stained with the first antibody for CD24 (1:200; sc-7034) and Notch1 (1:200; sc-376403) (Santa Cruz Biotechnology) by incubating overnight at 4°C. Secondary staining with biotinylated secondary antibodies with horseradish peroxidase (HRP; Beyotime Biotechnology, Nanjing, People’s Republic of China) was performed for 30 min at room temperature. Then, the sections were counterstained with hematoxylin (Beyotime).

Fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Horseradish peroxidase (HRP) and 3,3′,5,5′-tetramethylbenzidine (TMB) were purchased from Beyotime Biotechnology (Jiangsu, People’s Republic of China). Endo Free Plasmid Kit was purchased from QIAGEN. GC cell lines MKN-45 and HGC-27, gastric mucosal cell lines GES-1 and RGM-1, CRC cell lines HCT-116 and SW480, and intestinal epithelial cell lines HIEC-6 and NCM-460 were purchased from BeNa Culture Collection (Shanghai, People’s Republic of China). Cells were cultured in RPMI1640 medium (Gibco-BRL, Grand Island, NY, USA) supplied with 10% FBS, penicillin (100 U·mL⁻¹), and streptomycin (100 μg·mL⁻¹) at 37°C in a cell incubator with 5% CO₂. Radioimmunoprecipitation assay buffer, extraction buffer, and protein A/G beads were purchased from Beyotime Biotechnology. All other chemicals and reagents (which were of analytic grade) were purchased from Sino Pharm Chemical Reagent Co. Ltd. and used as received.
The chemiluminescence signal of TMB was detected with an iMARKT Microplate Reader (Bio-Rad, Hercules, CA, USA). The ultraviolet−visible light measurements were performed on a NanoDrop 2000 spectrometer (Thermo Fisher Scientific). The Bio-Rad 1575 Plate Washer was purchased from Bio-Rad.

The expressions of related proteins were examined using WB. Briefly, total protein was extracted using RIPA lysis buffer (Beyotime), and protein lysis products were quantified using a bicinchoninic acid (BCA) kit (Solarbio). Subsequently, 20 μg of protein was loaded onto SDS PAGE gels, followed by electrophoresis, membrane transfer, and closure. Then, the membranes were incubated with correspondent primary antibodies (ABclonal). The membranes were incubated with the secondary antibody solution labeled by horseradish peroxidase (HRP) (Beyotime), and the protein bands were detected using enhanced chemiluminescence (ECL) kit (Fdbio science) and scanned by gel imaging system. Using the Image J software, the blots (n = 3 per group) were quantified by a densitometric method. The results were expressed as mean ± SD.

First,
we extracted cardiac tissue samples and then placed them in a cold
RIPA lysis buffer (beyotime biotechnology) containing proteinase and
phosphatase inhibitors (bimake). Next, we used a homogenizer to grind
the tissue and performed ultrasonic treatment using an ultrasonic
disruptor. We centrifuged at 4 °C (12 000 rpm, 10 min) and then
determined the total protein content in the supernatant using the
BCA protein assay kit (beyotime biotechnology). Subsequently, we balanced
the protein content in each group using 4x SDS loading buffer. The
tissue protein samples were subjected to electrophoresis in 8%–10%
SDS-PAGE (voltage 80–120v), stopping when bromophenol blue
reached the bottom to separate total proteins. Then, the constant
current wet transfer method was used at 300 mA for 60–90 min.
Next, we incubated the membrane with the primary antibody overnight
at 4 °C, followed by incubation with the secondary antibody conjugated
with horseradish peroxidase (HRP) from the appropriate species (beyotime
biotechnology) for 1 h, and finally, we visualized using a chemiluminescent
imager (Azure 300).^{15 (link)} The primary antibodies
used included the following: anti-P-ERK1/2 (1:2 000), anti-ERK1/2
(1:2 000), anai-P-AKT (1:2 000), anti-AKT (1:2 000), anti-P-eNOS (1:1
000), anti-eNOS (1:1 000), and GAPDH (1:50 000). Signal results were
quantified by using ImageJ software.