Total protein extraction kit

According to a previous description, the levels of Bax, B‐cell lymphoma‐2 (Bcl‐2), TLR4, MyD88, p‐p65, and p65 were detected.
⁴⁴ (link) Total protein was extracted from H9c2 cells and myocardial tissues with the help of Total protein extraction kit (BB‐3101; BestBio), the concentration of which was quantified using the Protein quantitative kit (BCA; DQ111‐01; TransGen Biotech). Afterwards, sodium dodecyl sulfate–polyacrylamide gel (SDS‐PAGE) was prepared by SDS‐PAGE gel preparation kit (BB‐3702; BestBio), and 20 μL of protein samples were electrophoresed. The separated proteins were transferred onto the polyvinylidene fluoride membrane (LC2002; Thermo Fisher Scientific) with Western Transfer Buffer (BB‐35112; BestBio). Subsequently, the membrane was blocked with Western Blocking Buffer (BB‐3512; BestBio) at room temperature for 1 h, and incubated with the primary antibody working dilution at 4°C overnight. Post washing with Western Wash Buffer (P0023C3; Beyotime), the membrane was incubated with secondary antibodies at room temperature for 1 h, rinsed with Western Wash Buffer, and visualized by ECL working solution (32209; Thermo Fisher Scientific). Finally, the Western blot imaging system (FluorChem M; Alpha Innotech) and ImageJ software (Version. 5.0; Bio‐Rad) were used to analyze the results of Western blot. In this test, all information of antibodies was listed in Table 2.

Twenty New Zealand rabbits were purchased from Chengdu Tatsuo Biological Technology Co. Ltd. (Chengdu, China) and infested with Sarcoptes scabiei for a month. Sarcoptes scabiei was harvested by the Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University. In brief, live mites including larvae, nymphs and adults were collected and isolated from severely affected rabbits by exposing the infested crust to 37 °C for 2 h. Partial mites were used for RNA and protein extraction and the remaining mites were used for a challenge test in a vaccination trial. RNA isolation from mites was performed using RNAprep pure Tissue Kit (TIANGEN, Beijing, China) and RNA was transcribed into cDNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Vilnius, Lithuania). Total crude protein was extracted from mites using Total Protein Extraction Kit (BestBio, Shanghai, China). cDNA and total crude protein were stored at -70 °C until assay.

After 72 h of transfection, SMSCs were collected using a Total Protein Extraction Kit (BestBio, Shanghai, China). Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (BestBio). The samples were separated by SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Corporation, Billerica, MA, United States). Subsequently, the membranes were blocked using a Quickblock solution (Beyotime, Shanghai, China) for 1 h at room temperature. The membranes were incubated with specific primary antibodies, including anti-MyoG (Biorbyt, Cambridge, United Kingdom; diluted 1: 500), and anti-β-tubulin (Zenbio, Chengdu, China, 1: 1000) at 4°C overnight. The next day, PVDF membranes with proteins were incubated with a specific secondary antibody [anti-mouse/rabbit immunoglobulin G (IgG)] (ZenBio, 1:2000). Finally, the enhanced chemiluminescence (ECL) luminous fluid (Beyotime) was used for detection. The relative gray scales of MyoG were detected using the ImageJ software, and three independent replicates were set for each treatment (National Health Institute, Bethesda, MD, United States).