Hrp conjugated goat anti mouse igg

The western blot analysis was conducted according to a previously reported
method19 (link). The concentration of
extracted proteins was measured by BCA Protein Quantification Kit (wellbio).
After denaturation in boiling water, protein samples (30 μg) were loaded onto
polyacrylamide gel and separated. Following electrophoresis, the proteins were
transferred onto polyvinylidene difluoride (PVDF) membrane, and then the
membranes were blocked with Tris-buffered saline Tween-20(TBST) containing 5%
skimmed milk powder at room temperature for 1.5 h. Subsequently, the membranes
were incubated with each primary antibody against IL-1β (1:1000), IL-6 (1:1000),
TNF-α (1:1000), TLR4 (1:500), NF-κB (1:50000), Bax (1:2000), casapase-3
(1:1000), casapase-9 (1:500), ZO-1 (1:500), occludin (1:500) and Claudin-1
(1:2000) overnight at 4 °C. After three washings with TBST for 15 min each, the
membranes were incubated with the appropriate horseradish peroxidase (HRP)
conjugated goat anti-rabbit IgG (1:6000, Proteintech Group) or HRP conjugated
goat anti-mouse IgG (1:5000, Proteintech Group) for 1 h at room temperature.
Immunoreactivity was reacted with a PierceTM ECL substrate (thermo) for 3 min
and then exposed to X-ray films. The results were standardized to the intensity
levels of β-actin and analyzed using the Quantity-One software (Bio-Rad,
Hercules, CA, United States).

The Poly(A) + RNAs were firstly denatured by heating at 65 °C for 5 min and transferred onto a nitrocellulose membrane (Amersham, GE Healthcare, USA) with a Bio-Dot apparatus (Bio-Rad, USA). The membranes were then UV cross-linked, blocked, incubated with m6A antibody (1:1000, Abcam, USA) overnight at 4 °C and subsequently incubated with HRP-conjugated goat anti-mouse IgG (1:3000, Proteintech, USA). Finally, the membrane can be visualized by the chemiluminescence system (Bio-Rad, USA). The membrane stained with 0.02% methylene blue (MB) in 0.3 M sodium acetate (pH 5.2), was used to ensure consistency among different groups.

Recombinant human matrix metalloproteinases‐7 protein was obtained from AmyJet Scientific. The chemical inhibitors for the MAPK pathway (ie U0126, SB203580 and JNK‐IN‐8) were purchased from Selleck Chemicals. Antibodies (Abs) against phospho‐ERK, phospho‐JNK, phospho‐p38, phospho‐β‐catenin, phospho‐AKT, phospho‐mTOR, phospho‐p65, ERK, JNK, p38, β‐catenin, AKT, PI3K p110, PI3K p85 and mTOR, p65 were obtained from Cell Signaling Technology. Anti‐AQP‐1(AF5231) and Anti‐MMP‐7(AF0218) were brought from Affinity Biosciences LTD. HRP‐conjugated Goat Anti‐Rabbit IgG and HRP‐conjugated Goat Anti‐Mouse IgG were bought from Proteintech.

A. sinica cyst samples were collected at different developmental stages (0–5 d). Samples from 0, 5 h and 10 h were dechorionated using 20% NaOCl. All the samples were fixed using an equivalent volume of heptane and PEM-FA (contains PIPES, EGTA, MgSO₄ and oxymethylene) for 20 min. The pellets were washed with methanol three times and stored at −20 °C.
After thawing, the samples were washed in PT (1 × PBS with Triton X-100) before ultrasonication. For the 0 h to 10 h samples, the cysts were sonicated for three times (3 s each time); 15 h and 20 h samples were sonicated five times; and 40 h–5 d samples were sonicated seven times. Samples from different stages without ultrasonication were used as controls. After washing in PT, the samples were blocked by incubating in 3% H₂O₂ for 30 min, and 1 mg/ml BSA for 25 min. The CDH1 primary antibody (Santa, Texas, USA) was added at a 1:50 dilution and the samples were overnight incubated at 4 °C. Controls were incubated in 1 × PBS buffer. All the samples were incubated in HRP-conjugated goat-anti-mouse IgG (Proteintech) antibodies at 37 °C for 1 h, then in SABC (streptavidin biotin complex) for 30 min; washed in PT before being stained with DAB. Finally, the samples were washed and stored in 70% glycerol at −20 °C until microscopic observation.

Total proteins in each group of tissues were extracted and denatured. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteins were transferred to nitrocellulose membranes. The membranes were incubated overnight at 4°C with primary antibodies to Occludin (1:2,000, cat# 27260-1-AP, Proteintech), Claudin5 (1:2,000, cat# ab131259, Abcam), NLRP3 (1:1,000, cat# ab263899, Abcam), ASC (1:1,000, cat# AG-25B-0006-C100, Adipogen), Casp-1(1:1,000, cat#24232, Cell Signaling Technology), and β-actin (1:5,000, cat# 60008-1-Ig, Proteintech). The membranes were then exposed to secondary antibody horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5,000, cat# SA00001-1, Proteintech) or HRP goat anti-rabbit IgG (1:6,000, cat# SA00001-2, Proteintech) was incubated for 90 min at room temperature. β-actin was used as an internal control. After ECL color exposure, the protein bands were analyzed using an Odyssey infrared imaging system (Li cor Biosciences).

After the cells were lysed by sodium dodecyl sulfate (SDS) lysis buffer, the lysate was performed by ultrasonic and centrifuge. Protein in the lysate was then separated by SDS-polyacrylamide gel electrophoresis. The primary antibodies were rabbit anti-CD44 (1:5000 dilution, CST, USA) and mouse anti-β-actin (1:5000 dilution, CST, USA) which was used as an internal reference. The secondary antibodies were HRP-conjugated goat anti-mouse IgG (1:5,000 dilution, Proteintech, USA) and HRP-conjugated goat anti-rabbit IgG (1:5,000 dilution, Proteintech, USA). Protein bands were colored using Immobilon™ HRP Substrate (Millipore, MASS, USA), and photographed using Tanon-4500 Gel Imaging System (Tanon Science & Technology, Shanghai, China).