Protease inhibitor

Endoglycosidase assays were performed as previously published [8 (link)]. In brief, COS-7 cells (Cell Resource Center, Peking Union Medical College, Beijing, China) with 60–70% confluence were transiently transfected with 300 ng of plasmid containing myc-tagged WT or mutated FGFR1 cDNA in 6-well plates using Lipofectamine 3000 reagent (Thermo Fisher Scientific, Waltham, MA, USA). Forty-eight hours post transfection, cells were washed with phosphate-buffered saline (PBS), and then, lysed with 100 μL of RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing 1× protease inhibitor (Solarbio, Beijing, China). For deglycosylation analysis, all protein lysates were diluted to 10 μg/μL, and 9 μL of diluted lysate (90 μg of total protein) was subjected to PNGasef and EndoH digestion according to the manufacturer’s recommendations (New England Biolab, Ipswich, MA, USA).

The transcription levels of EtROP35 in four stages (unsporulated oocysts, sporulated oocysts, sporozoites, and second-generation merozoites) of E. tenella were detected by employing qRT-PCR using SYBR Green I (Vazyme Biotech Co., Ltd., Nanjing, China) (primers: F: 5′-ATGAGCCTGCGCGCC-3′; R: 5′-CAGGAACTTGTAGGGAGTCTGG-3′) with the 18S rRNA as the standard reference gene (Primers: Et18S-F: 5′-TGTAGTGGAGTCTTGGTGATTC-3′; Et18S-R: 5′−CCTGCTGCCTTCCTTAGATG-3′). The translation levels of EtROP35 were detected by western blot using the anti-EtROP35 polyclonal antibody (1:1000). Parasites of four stages were ground in liquid nitrogen and the protein lysates were prepared in RIPA buffer including protease inhibitor (Solarbio Science & Technology Co., Ltd., Beijing, China). The ImageJ 1.8.0 software (National Institutes of Health, Bethesda, MD, USA) was used to measure Grey values.

Total protein was extracted from the liver, kidney, jejunum and ileum (100 mg) of fowl using 1 mL of RIPA cell lysate (Biosharp, Shanghai, China), 10 μL of protease inhibitor (Solarbio, China), and 20 μL of phosphatase inhibitor (Solarbio, China), and the resulting mixture was homogenized for 2 min. The cells were lysed at 4°C for 20 min. The lysates were then centrifuged at 12,000×g for 10 min at 4°C. The supernatant was aspirated and stored at −80°C. The protein concentration was determined using the BCA method [26 (link)] (Biosharp, China).
For western blotting, 50 μg of the treated sample was elec trophoresed at 80 V for 40 min on a 5% sodium dodecyl sulfate (SDS) -polyacrylamide stacking gel, followed by electrophoresis at 120 V for 80 min on a 10% SDS-polyacrylamide running gel. The proteins were electrophoretically transferred on to a polyvinylidene fluoride membrane at 120 V for 26 min. The rabbit anti-GLUT9 antibody (diluted 1:1,000; Novus, Littleton, CO, USA) and a goat anti-rabbit immunoglobulin G (IgG) secondary antibody (diluted 1:5,000; ImmunoWay, Plano, TX, USA) were used. The proteins were visualized using a Western Blot Detection Kit (Advansta, Menlo Park, CA, USA). The density of the bands was analyzed using the Image Pro Plus software version 6.0 [27 (link)] (Media Cybernetics, Washington, MD, USA), and the protein expression was normalized to β-actin band.

RIPA lysis buffer (P0013B, Beyotime, Shanghai, China) with protease inhibitor (P0100, Solarbio life sciences, Beijing, China) was added to rat myocardial tissue or H9C2 cells to extract protein according to the manufacture’s protocol. The protein concentration was detected using a BCA Protein Assay Kit (P0012, Beyotime, Shanghai, China). Protein samples, which were mixed with loading buffer and heated at 100°C for 8 min, were separated by 12% SDS-PAGE gel electrophoresis and transferred to a polyvinylidene fluoride membrane (PVDF) pre-activated with methanol. The membrane was then blocked with 5% bovine serum albumin (BSA) for 1 h and immersed in the primary antibody solution overnight at 4°C. The primary antibodies were anti-NRF2, anti-HO-1 (E3F4S, CST, United States), anti-PKC (ab23511, Abcam, United Kingdom), anti-PKC (phosphor T497, ab59411, Abcam, United Kingdom), anti-α-tubulin (ab7291, Abcam, United Kingdom), and anti-H3 (17168-1-AP, Proteintech, Wuhan, China). The membranes were incubated with secondary antibodies (ZB-2301 or ZB-2305, ZSGB-BIO, Beijing, China) and the protein signals were detected using enhanced chemiluminescence (ECL) detection system. Images were analyzed using Image J software (National Institutes of Health, United States).