Hrp conjugated anti mouse igg

UCMSCs transferred in the ovarian tissue were detected by IHC as described previously [32 ]. Briefly, the paraffin-embedded sections of the ovarian tissue were deparaffinized and blocked with 5% goat serum for 1 h at room temperature, and then incubated with anti-human mitochondria antibody (Boster Biological Technology Co., Ltd., Wuhan, China) at 4°C overnight. After washing thrice with PBS, the sections were incubated with HRP-conjugated anti-mouse IgG (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer's instructions. The sample was finally stained with DAB reaction, HE counterstained, and photomicrographed under a microscope. Positive expressing cells were counted every fifth section of every ovarian tissue. As controls for immunostaining procedures, some sections were incubated with normal mouse IgG or PBS in place of the primary antibodies. No false-positive reaction was detected in the sections.

Ileum samples were lysed on ice in RIPA lysis buffer (Beyotime) supplemented with 1 mM phenylmethylsulfonyl fluoride (Beyotime). The protein concentration was measured using BCA Protine Assay Kit (Beyotime). Protein were separated by 8% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Millipore). Then, membrane was blocked with 5% non-fat milk and incubated with primary antibodies overnight at 4°C and secondary antibodies for 1.5 h at room temperature. Signals were detected with WESTAR NOVA 2.0 (Cyanagen). The antibodies used were: rabbit anti-APN (1:2000, a generous gift from YW Huang¹¹ ), mouse anti-β-actin (1:2000, Beyotime), HRP-conjugated anti-mouse IgG (1:10000, A0216; Beyotime) or HRP-conjugated anti-rabbit IgG (1:10000, A0208; Beyotime).

The replication-competent HBV genome construct pREP-HBV1.2 (pHBV-1.2) was provided by Lin Xu. Plasmids were transfected into HepG2 or HL-7702 cells using Lipofectamine 3000 (Invitrogen, Thermo Fisher, USA) according to the manufacturer’s instructions. Anti-IFIT3 antibody (catalog no. ab236243) was purchased from Abcam (USA); anti-STAT2/phospho-STAT2, anti-Mx1, anti-PKR, anti-OAS1, and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were obtained from Cell Signaling Technologies (USA); and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and HRP-conjugated anti-mouse IgG antibodies were purchased from Beyotime Biotechnology (China).

Antibody LC3 was obtained from Sigma–Aldrich (Sigma, St. Louis, MO, USA). phospho-ERK1/2 and anti-ERK1 were from BD Biosciences (San Jose, CA, USA). Antibodies including SQSTM/p62, phospho-AKT, AKT, phospho-mTOR, mTOR, Beclin1, phospho-JNK, and phospho-p38 were obtained from Cell Signal Technologies (Danvers, Massachusetts, USA). SAPK/JNK, p38, β-actin, HRP-conjugated anti-mouse IgG, and HRP-conjugate anti-rabbit IgG were from Beyotime (Shanghai, China). Alexa Fluor 488-conjugated secondary antibody to rabbit IgG was purchased from Life Technologies (Gaithersburg, MD, USA). Autophagy inhibitors chloroquine, 3-MA, and the activator rapamycin were purchased from Sigma–Aldrich (Sigma, St. Louis, MO, USA).

A172 cells were treated with 5 μM PGE2 for 24 hours. Cells were lysed with HEPES‐NP40 buffer (20 mM HEPES, 150 mM NaCl, 0.5% NP‐40, 10% Glycerol, 2 mM EDTA, and 1% Protease Inhibitor Cocktail (Sigma, Munich, Germany), pH 7.5) on ice for 45 minutes. After centrifugation at 12 000 g for 15 minutes, the supernatants were mixed with 2× SDS loading buffer and incubated at 95°C for 5 minutes. The protein samples were separated using 8% SDS‐PAGE and transferred to polyvinyldifluoride (PVDF) membranes (Millipore, Burlington, MA, USA). After blocked with 10% nonfat milk in TBST for 1.5 hours at room temperature, the membranes were incubated with primary antibody [Anti‐TrpM7, 1:500 (University of California, Davis); anti‐GAPDH, 1:1000 (Beyotime), Anti‐ATP1A1, 1:800 (Proteintech, Chicago, IL, USA)] in Immunoreaction Enhancer Solution for primary antibody (TOYOBO, Osaka, Japan) overnight at 4°C. The membranes then washed with 0.3‰ TBST for three times and incubated with HRP‐conjugated anti‐mouse IgG (1:1000; Beyotime) for 2 hours at room temperature. Chemiluminescent signals were developed using enhanced chmiluminescence (ECL) reagents (Bio‐Rad) and detected using the ChemiDoc XRS+ System (Bio‐Rad). Image Lab software (Bio‐Rad) was used for quantification of immunoblotting data.

Whole-cell lysates were obtained using RIPA lysis buffer containing complete protease inhibitors (Beyotime Biotechnology). The protein concentration was assayed using a BCA Protein Assay Kit (Beyotime Biotechnology). The antibody against mouse IRF5 (1:1,000; CST), antibody against β-actin (1:1,000; Beyotime Biotechnology), antibody against IL-1β (1:1,000; CST), antibody against IL-6 (1:1,000; Santa Cruz), antibody against IL-12 (1:1,000; Santa Cruz), antibody against TNFα (1:1,000; CST), antibody against mouse TRAF6 (1:1,000; Abcam), horseradish peroxidase (HRP)-conjugated antirabbit immunoglobulin G (IgG) (1:5,000; Beyotime Biotechnology), and HRP-conjugated antimouse IgG (1:5,000; Beyotime Biotechnology) were used to detect the expression of IRF5, IL-1β, IL-6, IL-12, TNFα, TRAF6, and β-actin. The signals were detected using the Immobilon Western Chemiluminescent HRP Substrate (Wanleibio) and analyzed using the GE Amersham Imager 600 machine.

Total protein was recovered from rat footpad tissues at different developmental stages and HEK293T/Hela cells using RIPA lysis buffer (Beyotime). Samples were separated by 10% SDS-PAGE electrophoresis and transferred to PVDF filters. Immunoblottings were probed with rabbit monoclonal anti-Foxa1 (Abcam, 1:2000 dilution), mouse monoclonal anti-NKA α2 (Santa Cruz, 1:3000 dilution), and rabbit monoclonal anti-GAPDH (Abcam, 1:5000 dilution) antibodies for 2 h at room temperature. Following three 10-min washes in TBST buffer (10 mM Tris-HCl (pH 7.4), 300 mM NaCl, and 0.1% (v/v) TWEEN20), membranes were incubated with either the HRP-conjugated anti-mouse IgG (Beyotime, 1:2000 dilution) or the HRP-conjugated anti-rabbit IgG (Beyotime, 1:2000 dilution) at room temperature for 1 h. The membranes were then subjected to another three 10-min washes in TBST buffer and developed. Finally, the signal was detected using an enhanced chemiluminescence reagent (Millipore, USA) and recorded with the ChemiDoc Touch Imaging System (Bio-Rad, USA).