Ecl chemiluminescent substrate reagent kit

Total proteins were quantified using BCA method (Beyotime Institute of Biotechnology, Shanghai, China). After that, proteins (30 μg proteins per lane) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene difluoride (PVDF) membrane (Thermo Fisher Scientific). After blocking with 5% skimmed milk in TBST for 1 h, the membrane was incubated with primary antibodies against α-SMA (1:1000, Abcam), Collagen III (1:1000, Abcam), Fibronectin (1:1000, Abcam), Vimentin (1:1000, Abcam), p-STAT1 (1:1000, Abcam), STAT1 (1:1000, Abcam), Fas (1:1000, Abcam), Active caspase 8 (1:1000, Abcam), Active caspase 3 (1:1000, Abcam), and GAPDH (1:1000, Abcam) at 4 °C overnight. Subsequently, the membrane was incubated with secondary antibodies (1:5000, Abcam) for 1 h at room temperature, and bands were detected with the ECL Chemiluminescent Substrate Reagent Kit (Thermo Fisher Scientific). GAPDH was acted as the internal control.

After 24 h of treatment, whole protein of each treated group was harvested with 1 mM phenylmethylsulfonyl fluoride (PMSF) in RIPA buffer (Cell Signaling Technology, USA). The extracted proteins were centrifuged at 14,000 ×g in a cold microfuge for 10 min. The supernatants were collected for further experiments. The total protein concentration was determined by using a BCA Protein Assay Kit (Thermo Fisher Scientific). For each treatment, 30 μg of crude protein was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. Shortly afterward, the membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween 20 detergent (TBS-T) (Gibco) for 3 h. The blots were incubated overnight at 4°C with primary antibodies against MMP-2, MMP-9, Bcl-2, BAX, Caspase3, JNK, p-JNK, p38, and phospho-p38 (Cell Signaling Technology). After washing with TBS-T, the blot membranes were then incubated with mouse anti-rabbit HRP antibody for 2 h. Chemiluminescent signal was visualized by using an ECL Chemiluminescent Substrate Reagent Kit (Thermo Fisher Scientific). Band intensity was determined by ImageJ software. β-Actin was used as an internal control.

Western immunoblotting was performed following standard protocol. Sf9 cells expressing Ss-CYP450 enzymes were pelleted and lysed in Laemmli sample buffer by sonication followed by 5 min in a boiling water bath and 2 min on ice. The samples were then analyzed by 10% SDS–PAGE transferred to nitrocellulose membrane, and immunoblotted with the mouse anti-HA antibody [HA.C5] (Abcam, Cambridge, MA) followed by HRP-conjugated anti-mouse antibody (Abcam, Cambridge, MA). The membranes were then imaged by ImageQuant LAS4000 luminescent Image Analyzer (GE Healthcare, Chicago, IL) following an incubation with ECL chemiluminescent substrate reagent kit (Thermo Fisher, Waltham, MA).

The purified proteins were separated by SDS-PAGE, and the proteins in the gel were then transferred to a membrane under a constant voltage of 100 V for 1.5 hours. Following the completion of the transfer, the membrane was washed with PBS 4 times for 5 minutes per wash. The membrane was blocked with 5% skimmed milk at 37°C for 1 hour, followed by incubation with the primary mouse anti-His-tag monoclonal antibody (1 : 1000 dilution) and type A FMDV VP1 monoclonal antibody (prepared and stored in our laboratory, 1 : 1000 dilution) for 1 hour at 37°C, respectively. The membrane was washed with TBS-Tween (50 mM Tris, 150 mM NaCl, and 0.05% Tween 20, pH 7.6) 4 times for 5 minutes per wash, followed by incubation with the secondary horseradish peroxidase- (HRP-) labeled goat anti-rabbit IgG antibody (Sigma, USA) at a 1 : 5000 dilution for 1 hour at 37°C. The membrane was washed and visualized using an ECL chemiluminescent substrate reagent kit (Thermo Scientific, USA).

Western blot was used to detect YWHAZ, E-Cadherin, N-Cadherin and cleaved-caspase3 expression. Briefly, we harvested transfected NCl-H1299 and PC9 cells with 0.25% trypsinization, and centrifuged for 15 min at 3000 rpm. Total proteins in cells were extracted by RIPA lysis buffer (Beyotime, China) and then measured using a Nano-Drop 2000c spectrophotometer (Thermo Fisher Scientific, USA). After separated by 10% SDS-PAGE gel electrophores in equal amounts, proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. Then, we blocked the PVDF membranes using 5% skim milk at room temperature for 60 min and probed the PVDF membranes by primary antibody for a night at 4 °C. After that, the PVDF membranes was washed with PBS and co-incubated with the corresponding secondary antibodies at room temperature for 1 h. Specific protein signals were detected after washing with PBS for three times using ECL Chemiluminescent Substrate Reagent Kit (Thermo Fisher Scientific, USA). Imaging system (Bio-Rad, USA) was performed to analyze protein bands. To normalize relative protein expression levels, GAPDH expression was employed as an internal reference.

P3 rAd-PEDV-S was used to infect HEK293A cells, and the cells were harvested after 120 hours. The cells were lysed using the lysis buffer (Thermo Scientific, Waltham, MA) containing protease inhibitors (Invitrogen, USA), frozen/thawed 3 times, and then centrifuged and collected at 4°C and 12000 rpm for 10 minutes. Then, the proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes under a constant voltage of 80 V for 2 hours. The blots were blocked with 5% skim milk at 37°C for 1 hour and then incubated with a primary mouse anti-His-tag monoclonal antibody (mAb) (1 : 1000 dilution; Abcam, USA) for 1 hour at 37°C. After washing 3 times for 5 minutes per wash with TBS-Tween 20 (50 mM Tris, 150 mM NaCl, and 0.05% Tween 20, pH 7.6), the membrane was incubated with a secondary horseradish peroxidase- (HRP-) labeled goat anti-mouse IgG antibody (1 : 2000 dilution; Abcam, USA) for 1 hour at 37°C. Finally, the membrane was washed and visualized using an ECL chemiluminescent substrate reagent kit (Thermo Scientific, USA).

miR-28-5p and miR-NC groups cells were collected with the addition of RIPA buffer (Thermo Fisher, USA). The cells were lysed on ice for 1 h and centrifuged at 4°C for 15 minutes. The concentration of the supernatant of cell lysate was measured using the BCA kit (Thermo Fisher, USA). Each lysate sample containing 30 μg protein was mixed with SDS loading buffer and boiled for 10 minutes, followed by loading onto a 5% concentrating +12% separating gel. The initial voltage was 80 V at the concentrating gel and then adjusted to 120 V when the samples entered the separating gel. The electrophoresis was continued for about 1 h, and the proteins were transferred onto a PVDF at 100 V voltage for 1 h. The PVDF membrane was incubated in 5% bovine serum albumin for 1 h, then washed with TBST three times, added with GAGE12I antibody (1 : 100), or GAPDH antibody (1 : 100) as a loading control, left at 4°C overnight, washed with TBST three times, and then incubated with the secondary antibody for 1 h. After being washed with TBST, the ECL chemiluminescent substrate reagent kit (Thermo Fisher, USA) was used for luminescence and the image was taken by gel imager. The gray value of each band was analyzed, and the protein expression level was semiquantitatively analyzed using ImageJ.