Enhanced chemiluminescent substrate

Total protein was separated from the treated mammary epithelial cells using RIPA lysis buffer (Beyotime); and then centrifuged at 4°C; and 12,000 g for 15 min. The supernatant containing the total protein was collected. Similarly, nuclear proteins were obtained using the Nucleus and Cytoplasm Protein Extraction Kit (Beyotime). The protein concentration was subsequently determined using a BCA protein concentration assay kit (Beyotime). Protein samples were added to 4%-15% SDS-PAGE; and separated in a 110 V electric field for 90 min, and then the separated proteins were transferred to PVDF membranes (Millipore) at 75 V; for 60 min. The PVDF membranes containing proteins were closed at room temperature for 2 h in TBS-T containing 5% skimmed milk. The primary antibodies were then incubated overnight at 4°C, and each primary antibody was incubated using a TBS-T solution containing 5% bovine serum protein(Gentihold). After the primary antibody incubation was completed, the membrane was washed 5 times with TBS-T, and then the PVDF membrane was incubated for 1 h at room temperature (1:5000 dilution) with the corresponding species of enzyme-labeled secondary antibody (Bosterbio). Finally, the immunoreactive blots were visualized with an enhanced chemiluminescent substrate (Beyotime).

Total proteins were separately isolated from the immune-relevanttissues (livers, gills, kidneys, supraneural myeloid bodies and lymphocyte-like cells) of control and LPS-stimulated animalsusing tissue lysis buffer (Beyotime, Shanghai, China). The protein concentrations were determined using a BCA Protein Assay kit (Beyotime, Shanghai, China). The total protein samples from five lamprey tissues were separated by 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% BSA (Beyotime, Shanghai, China) and separatelyincubated with rabbit anti-rLj-BLNK (200-fold) or mouse anti-VLRB antibodies (200-fold) overnight at 4 °C; this was followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or HRP-conjugated goat anti-mouse IgG (Sigma-Aldrich, St. Louis, MO, USA, 500-fold)41 (link). The membranes were developed using an enhanced chemiluminescent substrate (Beyotime, Shanghai, China). Densitometric analysis was performed using Gel-Pro Analyzer software (Exon-Intron, Inc. Loganville, PA, USA). The optical density data in triplicate from three independent experiments were normalized to lamprey β-actin detected with a rabbit polyclonal antibody against human β-actin (Sigma-Aldrich, St. Louis, MO, USA, 5000-fold) and calibrated to the levels in control samples.

Protein levels of IL-27Rα in hPMSCs were determined using Western blot with CD3⁺T cells serving as a positive control. Expressions of IL-27Rα in hPMSCs as determined on different culture days for one generation were then analyzed by Western blot, as were levels of phosphorylated STAT1 (P-STAT1) and STAT1 in these hPMSCs. HPMSCs were pretreated with the Janus kinase 1/2 (JAK1/2) inhibitor INCB018424 (20 ng/ml, Selleck, Shanghai, China) for 1 h before stimulation with IL-27 and incubated in the presence or absence of INCB018424 for an additional 1 h; P-STAT1 and STAT1 expressions were then measured by means of Western blot. After adding RIPA lysis buffer to hPMSCs, the cells were lysed on ice for 40 min, centrifuged, subjected to SDS-PAGE electrophoresis, and then transferred to PVDF membranes. Rabbit anti-human IL-27Rα (Bioss, Beijing, China), β-actin (Bioworld, Nanjing, China), phosphorylated STAT1 (Abcam, Cambridge, UK), or STAT1 (Proteintech, Wuhan, China) antibodies were incubated with membranes at 4 °C overnight. Secondary goat anti-rabbit antibody (1: 1000) (Santa Cruz, CA, USA) was added on the following day. Blots were further washed and developed with enhanced chemiluminescent substrate (Beyotime, Shanghai, China), and protein bands were then visualized using a Western blot imager.

Total protein was extracted by cell samples and colonic tissue of different treatment groups in the lysis buffer. The cells were collected and incubated in an RIPA buffer (with the addition of phosphorylation inhibitor and protease inhibitor) for 20 min in an ice bath. After centrifugation at 15,000 rpm for 15 min at 4°C, the upper-clarified lysate was collected. For the colon tissue, 700 μL RIPA lysate was added to a 100-mg sample to extract the protein. Protein concentration was determined using a BCA kit (Beyotime Biotechnology).
For western blotting, the extracted protein of each group was separated by 8%–12% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. The membrane was blocked with a 5% BSA solution. Then, the membrane was cut into different blots and probed with specific primary antibodies (Table 2) overnight at 4°C (12 h). Next, the membrane was washed with 1 × PBST and incubated with secondary antibodies (Beyotime Biotechnology) at room temperature for 1 h. Then, it was incubated with an enhanced chemiluminescent substrate (Beyotime Biotechnology). In this experiment, β-actin, α-tubulin, HSP90, and GAPDH were used as an internal control to confirm that the amounts of loaded protein were equal. All experiments were repeated at least three times. Western blotting plots were quantified using ImageJ software (United States).