Rabbit anti β tubulin antibody

Human fibrinogen (plasminogen, von Willebrand factor, and fibronectin-depleted) and human zymogens Glu-plasminogen, α-thrombin, prothrombin, factor X zymogen, and activated factor Xa (all >95% purity) were purchased from Enzyme Research Laboratories, South Bend, IN, USA. Human plasmin and tranexamic acid (TXA) were purchased from MilliporeSigma, Burlington, MA, USA. Recombinant human pro-uPA zymogen [70 (link)] was kindly provided M. Ploug (Finsen Laboratory, Copenhagen, Denmark). Recombinant human uPA and recombinant mouse testisin proteins were from R&D Systems, Minneapolis, MN, USA. Fluorogenic substrates for thrombin (thrombin substrate III, Bz-FVR-AMC), FXa (Boc-IEGR-AMC [36 (link)]), uPA (Glt-GR-AMC [36 (link)]), plasmin (Boc-EEK-AMC [71 (link)]), and trypsin-like (Boc-QAR-AMC) proteases were from BACHEM, Bubendorf, Switzerland. Rivaroxaban (BAY 59-7939) was from Thermo Fisher Scientific, Waltham, MA, USA. Antibodies used were mouse anti-testisin antibody (MAbD9.1) [12 (link)], mouse anti-uPA antibody (IM15L, MilliporeSigma, Burlington, MA, USA), and rabbit anti-β-tubulin antibody (Santa-Cruz Biotechnology, Dallas, TX, USA). Secondary antibodies were goat anti-mouse-HRP, mouse anti-rabbit-HRP (Jackson ImmunoResearch, West Grove, PA, USA), and donkey anti-sheep-HRP (R&D Systems, Minneapolis, MN, USA).

The duodenum proteins were extracted using RIPA buffer. After centrifugation, protein concentration in supernatant was assayed using the bicinchoninic acid (BCA) method (Thermo Scientific Pierce, IL, USA). Aliquots of the extract containing 200 μg of protein were separated by reducing SDS-PAGE (10%) and electroblotted onto nitrocellulose membranes. The blots were blocked by using 7% non-fat milk in a solution of Tris-buffered salt with Tween-20. The blots were incubated with rabbit anti-SLC40A1 antibody (1:100, abcam, Cambridge, UK) overnight at 4°C, then they were incubated in goat-anti-rabbit secondary antibody-conjugated horseradish peroxide (1:1000, SantaCruz Biotechnoloy, TX, USA). Immunoreactive proteins were detected by using the enhanced chemiluminescence method (ECL; WesternBright, Advansta, CA, USA). The blots analysis was performed by densotometry (Bio1D++ version99, Vilber Lourmat). To ensure even loading of the samples, the same membrane was probed with rabbit anti-β-tubulin antibody (SantaCruz Biotechnology, TX, USA) at 1:200 dilution. The protein concentration in each sample was normalized for Sham group.

E13.5 CM+ectoderm was collected by manual dissection. Protein was isolated using RIPA buffer (Cell Signaling 9806). Protein species were separated by SDS-PAGE using Mini-PROTEAN TGC gels (Bio-Rad #456-1084). Western blots were performed with polyclonal rabbit primary antibodies against H3K27me3 (1:1000, Cell Signaling 9733), EZH2 (1:500, Cell Signaling #5246), and SUZ12 (1:1000, Cell Signaling 3737). Species-specific HRP-conjugated secondary antibodies were used at a 1:10,000 dilution. Immunoblots were probed with a rabbit anti-β-tubulin antibody (1:400, Santa Cruz 9104) as a loading control. Signals were detected using an Amersham ECL Western Blotting Analysis System (GE Healthcare RPN2109), and imaged using an Odyssey FC Imaging System (Li-Cor). Relative protein levels were quantitated using ImageJ/Fiji software (Schindelin et al. 2012 (link); Schneider et al. 2012 (link)).