Rabbit anti mouse igg h l hrp

RIPA buffer was used to extract total protein from HUFs, human and rat urethral tissues. BCA protein assay (Thermo Fisher Scientific) was performed to detect the protein concentration. Equal amounts of protein (20 µg) were separated by 10% SDS-PAGE gels and transferred to PVDF membrane (Millipore, USA). Next, the membranes were blocked in 5% fat-free milk for 2 h, and incubated with the following antibodies at 4 °C overnight: anti-Wnt3a (1:1000, Abcam), anti-β-catenin (1:1000, Abcam), anti-DKK1(1:1000, Abcam), anti-collagen I (1:1000, Abcam), anti-collagen III (1:1000, Abcam), anti-α-SMA (1:1000, Abcam), anti-p-GSK-3β (1:1000, Abcam), anti-GSK-3β(1:1000, Abcam), anti-p-Smad2(1:1000, Abcam), anti-p-Smad3 (1:1000, Abcam), anti-Smad2/3 (1:1000, Abcam) and anti-GAPDH (1:5000, Abcam, Cambridge, UK). Membranes were cut horizontally based on the differences of molecular weight. Then membranes washed with TBST (Beyotime Biotechnology, Shanghai, China) and incubated with corresponding secondary antibodies (goat anti-rabbit IgG H&L (HRP), 1:10000; rabbit anti mouse IgG H&L (HRP), 1:10000; Abcam, USA) at room temperature for 1 h. Additionally, membranes were stripped with Western Blot Stripping Buffer (Cell Signaling Technology), and re-probed with target proteins. Target blots signals were observed by an enhanced chemiluminescence-detecting kit (Thermo Fisher, MA, USA).

Orbital blood sampling was used to collect serum from mice at 7, 14, and 21 days after immunization. Indirect ELISA was then used to detect IgG antibody levels in mouse sera. ELISA coating solution (Solarbio, China) was used to dilute the antigen to a working concentration; and FC (4.5 ng/ml), GS (2.6 ng/ml), and FCGS (3.0 ng/ml) were added to 96-well plates and kept overnight at 4°C. Each well was washed three times with PBST, 5% skim milk was added, and the plates incubated at 37°C for 2 h. The liquid in each well was then discarded, wells were washed with PBST, and mouse serum (1:2,000) was added and again incubated at 37°C for 1 h. After being washed with PBST, rabbit anti-mouse IgG H&L (HRP) (1:5,000) (Abcam, Cambridge, UK) was added to each well and incubated at 37°C for 1 h. After being washed with PBST, a one-component TMB substrate color developing solution (Solarbio, China) was added, and plates were maintained at room temperature in the dark for 15 min. An ELISA stop solution (Solarbio, China) was then added to each well, and the absorbance at 450 nm (OD value) was measured with a microplate reader within 5 min.

RIPA Cell Lysate (Beyotime, Shanghai, China) was used to lyse SMSCs. Then, we centrifuged the lysate cells at 12,000 rpm for 10 min and collected the supernatant. BCA protein quantification kit (Beyotime, Shanghai, China) was used to determine protein concentration. Next, we subjected the protein sample to polyacrylamide gel electrophoresis, and then transferred it to PVDF (Solarbio, Beijing, China). We used 5% skimmed milk powder to seal the PVDF membrane, which was closed at 25 °C for 1.5–2 h. We then added the primary antibody and incubated it overnight at 4 °C. The next day, we used 1X TBST (Solarbio, Beijing, China) to wash the PVDF membrane. After wash-over, we incubated cells with the secondary antibody at 25 °C for 1 h. We used Electrochemiluminescence (ECL) for imprinting display. The ChemDoc^TMTouch Imaging System (Bio-Rad, Hercules, CA, USA) was used for relative expression of the protein obtained. The antibodies used in this study and their dilution ratios are as follows: Anti-CDK2 antibody (Abcam, Cambridge, UK, 1:1500), Anti-MYHC antibody (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), Anti-GAPDH antibody (Abcam, Cambridge, UK, 1:1000), Goat Anti-Rabbit IgG H&L(HRP) (Abcam, Cambridge, UK, 1:5000), Rabbit Anti-Mouse IgG H&L(HRP) (Abcam, Cambridge, UK, 1:5000).

Cell culture was the same as previously described. Whole-cell lysates were prepared for western blotting as we previously performed [22 ]. The samples (2 μg for ABCB1 measurement, 30 μg for others) were equally subjected to 4–15% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad, Hercules, California, USA) and transferred to Immun-Blot PVDF Membrane (Bio-Rad). After blocking with Blocking One buffer (Nacalai Tesque, Inc., Kyoto, Japan) for 2 h, the membranes were incubated with the indicated antibodies for overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. The bands were further dealt with Chemi-Lumi One L solution (NACALAI) and analyzed. The primary antibodies for caspase-8 were obtained from Medical & Biological Laboratories CO., LTD. (Nagoya, Japan) and those for caspase-3, caspase-9, survivin, NF-kappa B p65, p-NF-kappa B p65, Bcl-xL, and Bcl-2-associated X protein (Bax) were from Cell Signaling Technology, Inc. (Danvers, Massachusetts, USA). Primary antibodies for ABCB1 and GAPDH and secondary antibodies goat anti-rabbit IgG H&L (HRP) and rabbit anti-mouse IgG H&L (HRP) were purchased from Abcam (Cambridge, Massachusetts, USA). The density of bands was measured using a LAS4000 fluorescence image analysis system (FUJIFILM, Tokyo, Japan). All experiments were performed three times.

Mouse testicular tissue was lysed using nuclear and cytoplasmic protein extraction kit (Beyotime, P0027) according to the manufacturer’s instructions. The lysates were ultracentrifuged at 12000 g for 10 min at 4 °C. The supernatants and remaining sediment were collected separately. The concentration of protein in the sediment and supernatants were measured using a bicin-choninic acid assay (Beyotime Biotechnology, P0012S). For western blot assay, 50 μg of protein samples was loaded on 10% SDS-PAGE gel and runed 1.5 h at 100 V before transferring to PVDF membranes. The antibodies used were as follows. Rabbit anti-CDKN2AIP (1:1000, Proteintech, Cat No.16615-1-AP), Mouse anti-SYCP3 (1:1000, Abcam, Cat No. ab97672), Rabbit anti-histone H3(1:1000, Abcam, Cat No.ab1791), Rabbit anti-PRM1(1:1000, Affinity, Cat No.DF5045), Rabbit anti PRM2(1:1000, Proteintech, Cat No.14500-1-AP), Rabbit anti-SUN1(1:1000, Abcam, Cat No.ab103021), Mouse anti-GAPDH (1:10,000, ABclonal, Cat No.AC002), Goat Anti-Rabbit IgG H&L (HRP) (1:8000, Abcam, ab6721), Rabbit Anti-Mouse IgG H&L (HRP) (1:8000, Abcam, ab6728).

Antibodies for NF-κB p65 (#6956), p-NF-κB p65 (#3033) and β-Actin (#4970) (Cell Signaling Technology), p-TBK1 (#AF8190) and p-STAT3 (#AF3293) (Affinity Biosciences), STAT3 (#GB12176, Servicebio), HK2 (#22029-1-AP) and IRF3 (#11312-1-AP) (Proteintech Group), p-IRF3 (#MA5-14947, Invitrogen), TBK1 (#ab227182), Goat Anti-Rabbit IgG H&L (HRP) (#ab6721) and Rabbit Anti-Mouse IgG H&L(HRP) (#ab6728) (Abcam) were used for blotting. The anti-PDCoV-N antibodies were prepared in our laboratory. The Alexa Fluor 488-conjugated goat anti-mouse (#4408S) was purchased from Cell Signaling Technology. The STAT3 inhibitor static (#HY-13818) was purchased from Med Chem Express. The selenomethionine (SeMet) (#S3132) was purchased from Sigma. The miR-9-5p mimic, miR-125b-5p-1 mimic, and miR-125b-5p-1 inhibitor were purchased from Gene Pharma.