Horseradish peroxidase conjugated goat anti rabbit igg

Piperine (≥98% purity) was procured from the Shanghai De Bai Chemical Technology Co., Ltd. Nimodipine tablets were purchased from Bayer, Germany. TTC was bought from Solarbio Life Sciences (Beijing Solarbio Science & Technology Co., Ltd). BCA protein quantitative kits and total protein extraction kits were purchased from Ken Gen Biotech. Co. Ltd. Primary antibodies of Caspase‐3, Caspase‐9, Bax, Bcl‐2, Cyt‐c, and β‐actin, as well as the secondary antibody of horseradish peroxidase‐conjugated goat antirabbit IgG were obtained from Proteintech Group, Inc. SuperSignal West Pico was bought from Thermo Scientific, US.

The protein lysates were prepared from fresh kidney tissue. Samples were separated on electrophoresis gels and then transferred onto polyvinylidene difluoride membrane (Millipore, Billerica, MA). The primary antibodies were rabbit antibodies to α-SMA, fibronectin, or collagen I (Abcam), and rabbit antibodies to glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology). The secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit IgG (Proteintech Group). The signal was measured by an enhanced chemiluminescence kit (Millipore). The intensities of blot bands were quantitated by ImageJ software and normalized to glyceraldehyde-3-phosphate dehydrogenase values.

Jejunum and ileum were extracted with total protein extraction reagents (Thermo Fisher Scientific Inc., New York, NK, USA) in accordance with the manufacturer’s instructions. The relative expression of protein was determined by Western blot technique as described previously [22 (link)]. The following antibodies were used for protein quantification: PXR (1:500; Proteintech Group, Inc.), RXRα (1:500; Proteintech Group, Inc.), CYP2B6 (1:200; Proteintech Group, Inc.), CYP3A4 (1:500; Proteintech Group, Inc.), CYP3A5 (1:2000; Proteintech Group, Inc.), NF-κBp65 (1:1000; Cell Signaling Technology, Danvers, MA, USA), phosphorylated NF-κBp65 (Ser536) (1:1000; Cell Signaling Technology, Danvers, MA, USA), IKKα(1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), IκB (1:1500; Proteintech Group, Inc.), IL-10(1:1000; Abcam, Cambridge, LON, UK) and β-actin (1:4000;Proteintech Group, Inc.)and secondary antibody horseradish peroxidase-conjugated goat anti-rabbit IgG (1:6000; Proteintech Group, Inc.) or anti-mouse IgG (1:4000; Proteintech Group, Inc.). All protein measurements were normalized to β-actin (1:4000; Proteintech Group, Inc.) and data are expressed relative to the values in control piglets. In addition, porcine protein CYP2B22, CYP3A29, CYP3A46 have a high homology similarity (over 90 percent) with human protein CYP2B6, CYP3A4, CYP3A5, respectively.

Cells were lysed using the protein-extraction reagent RIPA (GIBCO BRL, USA) and were supplemented with protease inhibitors. Approximately 50 μg protein extractions were subsequently subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, for transfer onto nitrocellulose membranes (Millipore, USA), and incubated with specific primary antibodies against AKIP1, Bax, Bcl-2, cleaved caspase-3, cleaved caspase-9, p-ASK1, p-JNK, p-p38 and GAPDH (Proteintech Group, Inc., PRC). Horseradish peroxidase-conjugated goat anti-rabbit IgG (Proteintech Group, Inc., PRC) and ECL detection systems (PerkinElmer, NEL100001EA, USA) were used to visualize the specific binding.

We obtained 293T cells from American Type Culture Collection (Rockville, MD, USA). Lentiviral vector expressing APN or TXNIP was synthesized by Genechem (Shanghai, China). Dulbecco's Modified Eagle Medium (DMEM)/F12 (Gibco, Grand Island, NY, US), Hoechst 33342/propidium iodide (PI) double stain kit (Solarbio, Beijing, China), Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA), miR-135a-5p mimic/inhibitor (Ribobio, Guangzhou, China), dual-luciferase reporter assay system (Promega, Madison, WI, USA), lactate dehydrogenase (LDH) assay kit (Beyotime, Shanghai, China), enzyme-linked immunosorbent assay (ELISA) kits (R&D systems, Minneapolis, MN, USA), polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA), and horseradish peroxidase-conjugated goat anti-rabbit IgG (Proteintech, Chicago, IL, USA) were obtained as indicated. Fetal bovine serum (FBS), trypsin, type II collagenase and LPS were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit antibodies against APN, TXNIP, NLRP3, caspase-1, Gasdermin N-terminal fragment (GSDMD-N), and β-actin were supplied by Abcam (Cambridge, UK).