Anti mouse antibodies

The various tissue samples of the pigs and Cells were split by radio immunoprecipitation assay (RIPA) buffer (Beyotime, China) add with protease inhibitor (Pierce, WA, USA). The total protein sample was pointed into and separated in the SDS-polyacrylamide gel. Then transferred it into a PVDF membrane (Millipore, Bedford, MA, USA). Nest, the membrane was blocked in 5% defatted milk for 2 h. After that, the membrane was incubated with primary antibodies at 4 °C overnight followed by a secondary antibody at room temperature for 1.5 h. Protein bands were exposure by chemiluminescence reagents (Millipore, Bedford, MA, USA) and quantified using the Image Lab Image Document. Following primary antibodies were used: PDGFRα (1:300; Boster, Wuhan, China), FABP4 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), β-actin (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), β-tubulin (1:500; Santa Cruz Biotechnology, Dallas, TX, USA). The secondary antibodies were anti-rabbit, anti-goat and anti-mouse antibodies (Santa Cruz Biotechnology, Dallas, TX, USA). The targeted proteins were detected using the Gel Doc XR System (Bio-Rad, Hercules, CA, USA) as the instructions of the manufacturer.

The concentrations of proteins extracted with TRizol reagent (Thermo Fisher Scientific, Inc.) were determined using Bradford’s method with bovine serum albumin as a standard. Samples containing equal protein amounts and prestained molecular weight markers were separated by Tris-SDS-PAGE and transferred onto polyvinylidene fluoride membranes (0.45 µm, Merck Millipore). The membranes were blocked with 3% BSA in Tris-buffered saline with 0.05% Tween-20 for 1 h at room temperature, and incubated overnight at 4 °C with the following antibodies: rabbit polyclonal anti-brain-derived neurotrophic factor (BDNF; 1:3000; Abcam, Cambridge, MA, USA), rabbit monoclonal anti-cAMP response element binding protein (CREB; 1:2000; Abcam), rabbit monoclonal anti-Ser133-phosphorylated CREB (pCREB; 1:2000; Abcam), and mouse monoclonal anti-β-actin (1:5000; Santa Cruz Biotechnology, Dallas, TX, USA). Subsequently, the membranes were washed and incubated for 1 h at room temperature with secondary HRP-conjugated anti-rabbit (1:5000; Santa Cruz Biotechnology) or anti-mouse antibodies (1:10,000; Santa Cruz Biotechnology). Chemiluminescence detection was performed using the EzWestLumi plus kit (ATTO, Tokyo, Japan) and AE-9300 Ez-Capture (ATTO). Densitometric analyses were performed using the public domain NIH Image Program, ImageJ.

Whole-cell extracts (see
Cell culture), scraped out and extracted using RIPA Lysis and Extraction Buffer, were run on 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane using a transfer apparatus following the standard protocols (Bio-Rad). After incubation with 5% nonfat milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 60 min, the membrane was washed once with TBST and incubated with rabbit antibodies against human ABCB11 (Affinity, Catalog #DF 9278) 1: 2000 dilution; human β-actin (Santa Cruz Cat.# SC4778), dilution 1:1000; 4°C overnight. The membrane was washed with TBST buffer and incubated with a 1:5000 dilution of horseradish peroxidase-conjugated anti-rabbit (Santa Cruz Cat# SC-2004)/anti-mouse antibodies (Cat.#SC-2005) for 2 h at room temperature. Blots were washed with TBST four times and developed with the ECL system (Bio-Rad, US Cat.#170-5060) according to the manufacturer’s protocols. Raw, uncropped images from western blotting are available as Underlying data
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