Enhanced chemi luminescence (ecl)

Soluble worm somatic crude and ES proteins of diverse T. spiralis phases, and purified rTsPKM were separated on 10% SDS-PAGE [24 (link),42 (link)]. The proteins were transferred onto nitrocellulose (NC) membrane (Millipore, USA) in the semi-dry transfer cell (Bio-Rad, USA) [43 (link)]. The membrane was blocked with 5% skimmed milk in Tris-buffered saline containing 0.05% Tween (TBST) at 37°C for 2 h, and cut into strips. The strips were probed by various sera (1:100; anti-rTsPKM serum, infection serum and pre-immune serum) at 37°C for 2 h. After washes with TBST, the strips were incubated at 37°C for 1 h with HRP-anti-mouse IgG conjugate (1:10000; Southern Biotech, USA). After being washed again, the strips were colored using 3, 3’-diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich) or an enhanced chemiluminescent kit (ECL, Solarbio, China) [44 (link),45 (link)].

Retinal tissue was extracted and lysed with RIPA (Solarbio, R0010, Beijing, China) lysis buffer for 30 min on ice. After centrifuging the lysate at 12,000 rpm for 15 min at 4 °C, the supernatant was collected. The protein concentration was determined using the BCA protein assay kit (Solarbio, PC0020, Beijing, China). The same amount of protein was loaded on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel. Then, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were immersed in a blocking solution of 50 g L⁻¹ nonfat dry milk for 2 h. The corresponding primary antibodies (1/1000, LC3B, 18725-1-AP, Proteintech, USA; 1/1000, P62, AF5384, AffinitY, USA) were added to the membrane overnight at 4 °C. Then, the membranes were incubated with the corresponding secondary antibody (1/1000, MDL, MD912565, Hebei, China) for 1 h at 37 °C on a shaker. Finally, immunoreactive bands were visualized using enhanced chemiluminescence (ECL, Solarbio, PE0010, Beijing, China) and detected using an automated image analysis system (170-8280, ChemiDoc MP Chemiluminescence Imaging System; Bio-Rad, CA, USA). Statistical analysis was performed with β-actin as an internal reference.

Kidney tissue was added to RIPA lysate (Thermo Fisher Scientific, United States) containing phosphatase inhibitor and protease, followed by being disrupted by an ultrasound. Then, the sample was centrifuged at 14,000 g at 4°C for 15 min, and the supernatant was collected. The BCA protein detection kit (Pierce, United States) was utilized for measuring the protein concentration. 20 μg protein was loaded. After 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, the product was transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Germany). The membrane was blocked with 5% bovine serum albumin at 37°C for 2 h, and was incubated with primary antibodies overnight at 4°C. Antibodies included EIF4B (1/10000; ab134138; Abcam, United States), RICTOR (1/1000; ab219950; Abcam, United States), PRKCB (1/2000; ab195039; Abcam, United States), and GAPDH (1/10000; ab8245; Abcam, United States). After being washed 3 times with TBST, the membrane was incubated with HRP-labeled secondary antibody (1/2000; ab7097; Abcam, United States) for 2 h at 37°C. After being washed three times, the protein expression was detected by a gel quantitative analysis system following treatment with ECL (Solarbio, Beijing, China) with GAPDH as the reference control.

The cells were cultured in 1 mM FFA for 24 h as NAFLD model cells. After 24 h, the normal medium continued to incubate for 24 h. The normal group consisted of the cells cultured in a normal medium for 48 h. After the collected cells were lysed on ice with RIPA lysis buffer for 30 min, the supernatant was centrifuged and the protein concentration of each sample was determined using the BCA method. Then, we added an appropriate amount of 5× protein loading buffer and denatured the protein in a metal bath at 95 °C for 10 min, and stored it at −20 °C after sealing. It was then added to each well, after the electrophoresis proteins of different molecular weights were separated by SDS-PAGE and were transferred to polyvinylidene difluoride membranes. The detection was performed on the Fluor Chem FC3 system (Protein Simple, Waltham, MA, USA) using the enhanced chemiluminescence reagent (ECL, Solarbio, Beijing, China) (Figure S1). Image Lab software was used to analyze the protein levels, and β-actin was used as the internal control.

Cell lysates were prepared in radioimmunoprecipitation (RIPA) buffer with protease inhibitor PMSF and Halt^TM phosphatase inhibitor cocktail (Roche, Switzerland). Protein concentrations were confirmed by BCA Protein Assay Reagent (CWBIO, China). Equivalent amounts of protein samples were separated by 12% SDS-PAGE, and the target proteins were transferred to PVDF membranes (Millipore, United States). Membranes were blocked in TBST, a blocking buffer containing 5% skim milk for 2 h at room temperature, followed by incubation with primary antibodies, including rabbit anti-GPNMB polyclonal antibody, rabbit anti-Cyclin A polyclonal antibody (1:1000, Santa Cruz Biotechnology, United States), and rabbit anti-β-actin polyclonal antibody (1:2000, CWBIO, China) at 4°C for overnight. After washes with TBST for five times, membranes were incubated with horseradish peroxidase HRP-conjugated goat anti-rabbit (or mouse) IgG (1:1000, CWBIO, China) for 2 h at room temperature. After washes with TBST for five times, immunoreactive bands were detected using chemiluminescent reagent ECL (Solarbio, China) by GeneGnome XRQ Chemidoc System (Syngene, Cambridge, United Kingdom). The cellular protein β-actin was measured as an internal control.