Goat anti rabbit igg hrp

Four cytokines (IL-1β, TNF-α, IL-17 and IL-6), four DEPs (BPGM, GPD1, APOE and APOC₂) were identified by the integrated Omic analysis, and five key proteins (LRP5, DVL1, β-catenin, Cacybp, Skp1) involved in the Wnt/β-catenin signaling pathway were further validated by Western blot analysis. Briefly, total proteins were extracted using Cell lysis buffer (PMSF 1:100) for Western and IP (Beyotime, China). The total protein concentrations were quantified with a BCA assay (Bio-Rad, Hercules, CA). Next, the target proteins were separated by 8–20% Precast-Glgel Tris-Glycine PAGE (Sangon, Biotech), transferred to PVDF membrane. Then the PVDF membrane was blocked for 1 h. Next, the PVDF membrane was incubated with β-actin, IL-1β, TNF-α, IL-17, IL-6, BPGM, GPD1, APOE, APOC, LRP5, DVL1, β-catenin, GSK-3β, Cacybp and Skp1 overnight at 4 °C. Subsequently, the PVDF membrane was washed three times in TBST and incubated with either secondary antibody goat anti-mouse IgG-HRP (diluted 1: 2000, absin, abs20001, China) or goat anti-rabbit IgG-HRP (diluted 1: 2000, absin, abs20002, China) for 1 h. Protein bands were detected with the help of a FluorChem Q scanner (Proteinsimple, CA, USA).

Panc-1 and MiaPaCa-2 cells were collected and lysed for 15 min on ice in RIPA buffer (Beyotime Institute of Biotechnology). Quantification of protein concentration was measured by bicinchoninic acid kit (Beyotime Institute of Biotechnology). A total of 30-50 µg protein was loaded per lane, separated by SDS-PAGE on 8-12% gels and transferred to 0.45-µm polyvinylidene difluoride (PVDF) membranes (Immobilon-P; MilliporeSigma). Subsequently, 5% skim milk (cat. no. 70166; Sigma-Aldrich; Merck KGaA) was used to block PVDF membranes for 1 h at room temperature. The membranes were incubated with primary antibodies overnight at 4°C. Horseradish peroxidase-conjugated secondary antibodies were then used to incubate membranes at room temperature for 1 h. Protein expression was detected using an enhanced chemiluminescence detection system (Thermo Fisher Scientific, Inc.). The following primary antibodies were used: Rabbit anti-human HNF1A (1:1,000; cat. no. ab96777; Abcam), 53BP1 (1:1,000; cat. no. ab87097; Abcam), mouse anti-human GAPDH (1:2,000; cat. no. abs830030; Absin Bioscience, Inc.) The secondary antibodies were goat anti-rabbit IgG-HRP (1:10,000; abs20002) and goat anti-mouse IgG-HRP (1:10,000; cat. no. abs20001) (both from Absin Bioscience, Inc.).

At P10 and P17, the HI injured brains were carefully removed as completed and rapidly as possible. The tissue was homogenized by a magnetic grinder in protein extract solution (T-PER Tissue Protein Extraction Reagent, Halt Protease Inhibitor Cocktail; Thermo Fisher Scientific, Waltham, MA, USA). The supernatants were reserved after centrifugation. Supernatant with 50 µg of total protein was then denatured at 95 ℃ for 5 min and separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (12%). Next, the protein was transferred to a nitrocellulose membrane and saturated for 2 h with blocking buffer (7% defatted milk) followed by the primary antibody (mouse anti-MBP, 1:1,000, BioLegend; mouse anti-actin, 1:1,000, Origene Technologies, Rockville, MD, USA) and incubation overnight at 4 ℃. After rewarming for 30 min, the membranes were incubated in the secondary antibody [goat anti-mouse IgG-horseradish peroxidase (HRP), 1:500; goat anti-rabbit IgG-HRP, 1:500, Absin, Shanghai, China] for 1 h. A ChemiDocXRS+ imaging system and ImageLab software (Bio-Rad, Hercules, CA, USA) were used for visualization and densitometric analysis after 5 min of immersion in enhanced chemiluminescence substrate (Thermo Fisher Scientific). The strips were analyzed using ImageLab version 5.0 (Bio-Rad).