Horseradish peroxidase hrp conjugated goat anti rabbit

MIA PaCa-2 and PANC-1 cells were lysed in RIPA buffer supplemented with cOmplete™ mini protease inhibitor cocktail (Roche). Following incubation for 30 min on ice, lysates were centrifuged at 20,000×g for 10 min at 4 °C and the supernatant was collected for quantification of protein content using the BCA protein assay kit (Thermo Scientific). Samples were denatured for 10 min at 95 °C, separated by SDS-PAGE and transferred onto a PVDF membrane (Millipore). After immersion in tris-buffered saline containing 0.1% (v/v) Tween 20 and 5% (w/v) bovine serum albumin (blocking solution) for 1 h at room temperature, the membrane was probed with rabbit anti-EGFR (Cell Signaling Technology; 1:1000 in blocking solution) or rat anti-tubulin (Abcam; 1:1000 in blocking solution) primary antibodies overnight at 4 °C. Following incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Cell Signaling Technology; 1:10,000 in blocking solution) or rabbit anti-rat (Abcam; 1:10,000 in blocking solution) secondary antibodies for 1 h at room temperature, the membrane was overlaid with Immobilon® Forte Western HRP substrate (Millipore) and protein expression was imaged using the ChemiDoc XRS+ system (Bio-Rad).

Approximately 30 μg of total protein was loaded on an SDS–polyacrylamide gel as described previously [22 (link)]. The primary antibodies were used as follows: rabbit anti-myelin basic protein (MBP) and rabbit anti-IRAK1 (1:1000; Abcam, Cambridge, MA, USA), mouse anti-IL-6 (1:500; Abcam, Cambridge, MA, USA), mouse anti-Olig1 and mouse anti-TNF-a (1:300; Santa Cruz, USA), mouse monoclonal anti-β-actin (1:3000; Cell Signaling Technology, USA). Membranes were incubated with the secondary antibodies for 1 h at room temperature: horseradish peroxidase (HRP)-conjugated goat anti-rabbit or HRP-conjugated goat anti-mouse IgG antibody (1:3000; Cell Signaling Technology, USA). The bands were quantified by densitometry with ImageJ software (ImageJ 1.4, NIH, USA).

Protein levels were measured using a BCA reagent after IPEC-J2 cells, and colonic tissues were homogenized (Cwbio, Beijing, China). According to the manufacturer's recommendations, the cytoplasmic and nuclear extractions were carried out (keygen, Jiangsu, China). SDS-PAGE was used to load and electrophorese the samples, which were subsequently transferred through polyvinylidene fluoride (PVDF) membranes. After 30 min of blocking in QuickBlock™ Blocking Buffer (Beyotime, Shanghai, China), the membranes were incubated with primary antibodies. The following primary antibodies were used: TLR4, P65, phospho-P65, IκBα, phospho-IκBα, phospho-ERK1/2, ERK 1/2, phospho-P38, P38, phospho-JNK, JNK, occludin, ZO-1, claudin1, and β-actin at 1:1,000 dilution. Secondary antibodies include horseradish peroxidase (HRP)-conjugated goat anti-rabbit, and anti-mouse IgG at 1:1,000 dilution (Cell Signaling Technology, Danvers, MA, USA).