Goat anti rabbit igg hrp sc 2004

Cells were harvested and lysed in NP40 Cell Lysis Buffer (FNN0021, Invitrogen Life Technologies (Grand Island, NY, USA)). Lysates were centrifuged at 15,300× g for 10 min at 4 °C, and the supernatants were obtained. The protein concentrations of the supernatants were determined by using the Bradford assay (500-0006, Bio-Rad Laboratories (Hercules, CA, USA)). Proteins were electrophoresed on NuPAGE 4%−12% Bis-Tris gels (Invitrogen (Waltham, MA, USA)) and transferred to Protran nitrocellulose membranes (10600001, GE Healthcare Life science (Pittsburgh, PA, USA)). Membranes were blocked in 5% (w/v) bovine serum albumin (BSA) (9048-46-8, Santa Cruz Biotechnology (Dallas, TX, USA)) in TBS-T (TBS (170-6435, Bio-Rad Laboratories (Hercules, CA, USA)) and 1% (v/v) Tween-20 (170-6531, Bio-Rad Laboratories (Hercules, CA, USA)) for 30 min at 25 °C. Membranes were incubated with primary antibody diluted in 1% (w/v) BSA in TBS-T for 16 h at 4 °C and then with the horseradish peroxidase (HRP)-conjugated secondary antibody (goat anti–rabbit IgG–HRP (SC-2004) (1:2500), goat anti–mouse IgG–HRP (SC-2005) (1:2500), goat anti–rabbit IgG–HRP (SC-2004) (1:2500) from Santa Cruz Biotechnology (Dallas, TX, USA)) diluted in TBS-T for 30 min at room temperature. The immunoreactive bands were detected by the SuperSignal West Pico Chemiluminescent Substrate (34078, Thermo Scientific (Waltham, MA, USA)).

Western blot analysis of mucosal jejunum samples was performed by SDS/PAGE and transferred to membrane. For the detection, primary antibodies against zonula occludens 1 (ZO-1, H-300 Sc-10804, Santa Cruz Biotechnology) and claudin-1 (sc-166338, Santa Cruz Biotechnology) were utilized and the corresponding secondary antibodies (goat anti-rabbit IgG HRP, sc-2004; Santa Cruz Biotechnology or rabbit anti-mouse IgG HRP, AS09 627, Agrisera AB). Chemiluminescent reagents (Pierce ECL Western Blotting Substrate, Thermo Fisher Scientific) were applied, and blots were exposed to hyperfilms (GE Healthcare). hyperfilms and Ponceau S-stained membranes were scanned and individual bands or lanes, respectively, were quantified using ImageJ (v1.49).

For Western blotting analysis, cells were washed twice with cold PBS and then whole cell lysates were prepared by direct lysis of cell pellets in heated 2X SDS sample buffer. Samples were resolved on 4–12% Tris-Glycine gels (Life Technologies, Carlsbad, CA) followed by transferring onto nitrocellulose membranes (Bio-Rad, Hercules, CA). Primary antibodies used include anti-Setbp1 (16841-1AP, Proteintech, Chicago, IL), anti-Myb (05–175, Millipore, Temecula, CA), anti-Hoxa9 (07–178, Millipore, Temecula, CA), and Anti-β-Actin (ab8224, Abcam, Cambridge, MA). Secondary antibodies used include goat anti-rabbit IgG-HRP (SC-2004, Santa Cruz Biotechnology, Dallas, TX) and anti-mouse IgG-HRP (A-9044, Sigma Aldrich). Protein bands were visualized by incubation with SuperSignal West chemiluminescent substrate (Pierce, Thermo Fisher Scientific, Rockford, IL) and quantified using Image Lab software (Bio-Rad, Hercules, CA).