Ap0060

The RIPA lysis buffer (Thermo Scientific, Waltham, MA, USA, 89901) was used to extract the total protein. In brief, 10% SDS-PAGE was adopted to separate the protein and the protein was electrophoretically transferred onto 0.2 μm PVDF membranes (Immobilon-PSQ Transfer membrane, Merck Millipore, ISEQ00010). Then 5% skim milk was applied to block the membranes for 2 h at room temperature; then, we incubated them at 4 °C for at least 12 h with the following antibodies: anti-Stat5 antibody (CST, 9363S, 1:1000), anti-phospho-Stat5 antibody (CST, 4322S, 1:1000) (endogenous levels of Stat5a only when phosphorylated with Tyr694 and endogenous levels of Stat5b when phosphorylated with Tyr699), anti-P-NF-kB antibody (CST, 3033S, 1:1000), anti-NF-κB antibody (CST, 8242S, 1:1000), anti-β-tubulin antibody (Bioworld Technology, AP0064, 1:2000), and anti-β-actin antibody (Bioworld Technology, AP0060, 1:2000). Corresponding secondary antibodies were chosen for combination with the membranes for 1 h at room temperature and were visualized with the Chemiluminescent HRP Substrate (Millipore, Burlington, MA, USA, WBKLS0500). ImageJ (the National Institutes of Health, version 1.8.0_172) was used to quantify the bands with a gray value. The relative protein expression was expressed with the band intensity for the β-tubulin or β-actin.

Cells were homogenized in radioimmunoprecipitation assay buffer supplemented with protease and phosphatase inhibitors and centrifuged at 15,000 g for 15 min at 4°C. The protein concentration was determined according to the manufacturer’s instructions of the Pierce BCA Protein Assay kit (Rockford, IL, United States). Western blots were performed with 20–50 μg of protein. Proteins were separated in 8–10% SDS-PAGE and transferred to a nitrocellulose membrane. Western blot analysis for TLR2 (SC10739, Santa Cruz, United Kingdom, diluted 1:500), TLR4 (SC30002, Santa Cruz, United Kingdom, diluted 1:1,000), NFκB (ab16502, Abcam, United Kingdom, diluted 1:1,000), p-p65 (ab86299, Abcam, United Kingdom, diluted 1:1,000), PCK2 (ab70359, Abcam, United Kingdom, diluted 1:1,000), β-actin (AP0060, Bioworld Technology, United States, diluted 1:10,000), AKT (BS1007, Bioworld Technology, United States, diluted 1:1,000), mitochondrial transcription factor A (TFAM) (BS7319, Bioworld Technology, United States, diluted 1:1,000), COX Ⅳ (AP0707, Bioworld Technology, United States, diluted 1:1,000) were carried out according to the recommended protocols provided by the manufacturers. Images were captured by Versa Doc 4000MP system (Bio-Rad, United States) and the band density was analyzed with Quantity One software (Bio-Rad, United States).

Total protein extraction was conducted as previously reported [15 (link)]. A mitochondrial isolation kit was purchased for (GENMED, China) mitochondrial protein isolation. Twenty micrograms of total protein or mitochondrial protein from cells, mice tissues and human samples were resolved and then transferred to PVDF membranes. Afterwards, 5% blotting-grade blocker (Bio-rad, USA) or BSA albumin fraction V (Biofroxx, USA) was provided for 90 min to block the PVDF membrane. Primary antibodies for immunoblotting against SIRT1 (1:10000, ab189494, Abcam), Akt (1:1000, 9272S, Cell Signaling), p-Akt (1:1000, 9271S, Cell Signaling), PDK1 (1:1000, 5662S, Cell Signaling), p-PDK1 (1:1000, AF3018, Affinity), cytochrome C (1:1000, ab133504, abcam), Bcl2 (1:1000, 12,789–1-AP, Proteintech), BAX (1:1000, ab32503, abcam), VDAC (1:10000, CY5416, Abways) and β-actin (1:10000, ap0060, Bioworld Technology), the goat anti-rabbit HRP-conjugated secondary antibody was then incubated (1:10000, a0045, sigma). The antibodies used in our study have been previously validated in former works [15 (link), 16 (link)]. Diaminobenzidine was used to detect protein signals and ImageJ software (NIH, Bethesda, MD, USA) was used to perform measurement of protein expression in a blind fashion.

The protein samples were extracted from milk sEVs, milk somatic cells, intestinal tissues, and IPEC-J2 cells using radio immunoprecipitation assay lysis buffer containing PMSF protease inhibitor. The protein samples were homogenized, and centrifuged (12,000× g, 5 min, 4 °C). The supernatant was obtained to further analysis. Protein concentration of the samples was measured by BCA Protein Assay Kit. Protein samples were mixed with sodium dodecyl sulfate loading buffer and denatured in a boiling water bath. The protein samples were added to 10% SDS-PAGE gel for separation and then transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk for 2 h and incubated with specific antibodies for 12 h at 4 °C. The following antibodies were used: anti-CD63, anti-TSG101, anti-Alix, and anti-Calnexin (Sangon Biotech, Shanghai, China), anti-pIgR-mice (AF2800-SP, R&D Systems, Minneapolis, MN, USA), anti-pIgR-pig (bs-6061R, Bioss, Beijing, China), and anti-β-actin (AP0060, Bioworld, Technology Inc., Bloomington, MN, USA). Then, the membranes were incubated with HRP-conjugated corresponding secondary antibodies for 1 h. Images of protein bands were visualized using FluorChem M (ProteinSimple, San Jose, CA, USA). Gray value of protein bands were analyzed using Image J software.