Sheep anti mouse igg hrp

The antibodies used included anti-GAPDH (Bioword, AP0063), anti-NUCB2 (Proteintech, 26712-1-AP), anti-E-cadherin (Cell Signaling Technology, 24E10), anti-N-cadherin (Cell Signaling Technology, D4R1H), anti-Vimentin (Cell Signaling Technology, D21H3), anti-HisG (Cell Signaling Technology, D3I1O), anti-HMGCR (Abcam, ab242315), anti-SREBP2 (Abcam, ab30682), horseradish peroxidase (HRP) labeled sheep anti-Rabbit IgG (Cell Signaling Technology, 7074P2), and HRP labeled sheep anti-Mouse IgG-HRP (Sigma, A9917). The primary antibody titers for Western Blot or Immunohistochemistry are listed in Additional file 2: Table S1. Recombinant human purified Nesfatin-1 was purchased from Peprotech Company, and Rapamycin was obtained from Selleck Chemicals. Cholesterol was purchased from Beyotime, China.

Immunity was assessed on day 10 after immunisation with 20μg MPO in FCA and at day 21, at the end of the autoimmune anti-MPO glomerulonephritis model. The spleen and draining lymph nodes were harvested and a single cell suspension was obtained. IFN-γ and IL-17A ELISPOT was performed according to the manufacturer’s instructions (Ebioscience, San Diego, CA) with 5x10⁵ cells per well. Cells were incubated for 18 hours at 37°C with 10μg/ml heat inactivated recombinant murine MPO (rMPO). Total anti-MPO IgG was measured by ELISA on MPO coated plates, with IgG detected with sheep-anti mouse IgG-HRP (Sigma-Aldrich). Antibody subclasses were measured using subclass specific goat anti-mouse IgG-HRP (Southern Biotech, Birmingham, AL). Serum BAFF was measured by ELISA (RnD Systems, Minneapolis, MN). For assessment of B cell development, bone marrow was flushed from tibiae and femora of naïve mice and analysed by flow cytometry.

EV pellets were lysed in RIPA assay buffer (FUJIFILM Wako Pure Chemical Corporation) for 30 min on ice. Protein samples were electrophoresed on a 10% SDS–polyacrylamide gel and blotted onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA). The membranes were washed with TBS-Tween and were shaken in Can Get Signal PVDF Blocking Reagent (TOYOBO) at room temperature for 1 h. After washed, the membranes were incubated with primary antibodies diluted in Can Get Signal Solution 1 (TOYOBO) at room temperature for 1 h. After washed, the membrane was incubated with secondary antibodies conjugated to horseradish peroxidase (HRP) diluted in Can Get Signal Solution 2 (TOYOBO) at room temperature for 1 h. After washed, the membranes were incubated with Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, KGaA, Darmstadt, Germany) for a few seconds, and chemiluminescence was detected using the ChemiDoc Touch system (Bio-Rad). The following antibodies were used: mouse anti-CD63 (Santa Cruz Biotechnology, Santa Cruz, CA) and sheep anti-mouse IgG HRP (Sigma).