Stripping buffer

Total tissue protein was obtained from the border zone of the left ventricular myocardial tissue; cellular protein was obtained from the treated cardiac fibroblasts. The protein concentration was determined with a BCA. Equal amounts of total protein were separated with 8–10% SDS-PAGE and blotted onto PVDF membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 5% BSA, incubated overnight at 4 °C with the antibodies of interest, washed (10 min, 3×) with TBST, incubated with the corresponding secondary antibody (2 h, room temperature), and washed with TBST buffer (10 min, 3×); this was followed by detection with a Western Blotting Luminol Reagent (ECL) kit (Amersham, NJ, USA). Detection of phosphorylated proteins was performed first, and then PVDF membranes were stripped with stripping buffer (Beyotime Biotech, Haimen, Jiangsu, China) according to manufacturer’s instructions; the second hybridization was carried out to measure the levels of total protein. The protein bands were visualized (Protein Simple system, Santa Clara, California), and the intensity of the immunoblot bands was quantified with densitometry image analysis software (Quantity One, Bio-Rad, Hercules, California). Densitometry values were normalized to the GAPDH level in each sample.

At 48 h post-infection, cells were lysed with NP-40 lysis buffer (Beyotime) to extract total proteins. The proteins were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore, Bedford, MA, USA) which was then incubated with a specific primary antibody against human PEG10, MMP-2, MMP-9 or TIMP-1 (1:1000; Santa Cruz) at 4°C overnight. Following incubation with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5000; Beyotime), the target protein bands were visualized with the ECL solution (7SeaPharmTech, Shanghai, China). To verify equal loading and transfer, the membrane was stripped with the stripping buffer (Beyotime) and re-probed with anti-β-actin antibody (Santa Cruz).

Protein was extracted from NCM, NCF, and homogenized myocardium tissue in lysis bu?er. Protein lysate concentrations were determined via Pierce BCA Protein Assay Kit (Termo Scientific, USA). Equal amount of protein sample (20–30 μg/lane) from each group was subjected to 10% SDS-PAGE and transferred onto nitrocellulose membranes. After blocking with 5% bovine serum albumin (BSA), membranes were incubated overnight with primary antibodies (1:1,000, Cell Signaling Technology, USA) against p-JAK2, p-STAT3, STAT3, p-p38, GAPDH, and pan-actin. On the second day, the membranes were incubated with fluorescent secondary antibody DyLight 800-Goat Anti-Rabbit IgG (H+L) (KPL, USA). The membranes were scanned by ODYSSEY infrared imaging system (LI-COR Biosciences, USA). After incubation of phosphorylated proteins, we used stripping buffer (beyotime, China) to extract antibodies. Then we did the blocking and the following incubation procedures to obtain total protein quantification.

qPCR was performed to analyze TsASP2 mRNA transcription in siRNA-treated ML as described above. The TsASP2 protein expression in treated worms was also evaluated by western blot analysis [34 (link)]. In brief, the crude proteins extracted from siRNA-treated ML were separated by SDS-PAGE and then transferred onto a PVDF membrane. Anti-rTsASP2 serum (1:100) was first used to recognize the membrane and then visualized using an enhanced chemiluminescent kit (Beyotime Biotech, China). The membrane was washed with stripping buffer (Beyotime Biotech, China) and then incubated with mouse anti-GAPDH IgG for quantitative protein control.

Proteins were obtained from BMDMs, CFs, and myocardial tissues. Cells or tissues were homogenized in RIPA buffer containing a protease and phosphatase inhibitor. After measuring the protein concentration, the proteins were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was then blocked with non-fat dry milk (5%) and incubated with primary antibodies, followed by secondary antibodies. Finally, the membrane was visualized using an ECL substrate (Thermo Fisher Scientific). After phospho-Akt or phospho-Smad2/3 detection, the membranes were stripped with stripping buffer (Beyotime, Shanghai, China, cat#P0025) and reblotted with antibodies against the anti-Akt, anti-Smad2 and Smad3, or β actin antibodies.
To conduct cell immunofluorescence, the cell monolayers were fixed with 4% paraformaldehyde followed by treatment with 0.3% Triton X-100. After three washes with phosphate-buffered saline, the cells were blocked with 5% bovine serum albumin for 40 min. The cells were then incubated with primary antibodies, followed by fluorescently labelled secondary antibodies and Hoechst. The final results were observed using a fluorescence microscope (Leica).

After washed twice with PBS, ICP2 cells were lysed for 30 min on ice, using RIPA lysis buffer (Beyotime, Nanjing, China) with PMSF (Beyotime). Total cell extracts were obtained by centrifugation. Protein concentrations were determined using Compat-Able^TM Protein Assay Preparation Reagent Set (Thermo Fisher Scientific, Shanghai, China). Proteins were separated by 10% SDS-PAGE and transferred to PVDF membrane (Merck-Millipore, Billerica, MA, United States). After blocking with 5% non-fat dry milk, the membrane was incubated with anti-c-Myc tag (1:1000; Clontech, Palo Alto, CA, United States). Anti-β actin (1:1000; Beyotime) was used to ensure protein loading control equally. After incubated with anti-c-Myc tag, the membranes were stripped using the stripping buffer (Beyotime), and re-hybridized with Anti-β actin. The BeyoECL Plus kit (Beyotime) was used for detection.