Anti rabbit igg hrp

Recombinantly produced IgG1 WT and REW Fc fragments were coated (1 μg/mL/100 uL per well) in 96-well EIA/RIA plates (CorningCostar) for 1 h at RT. Then Rh+ human serum (Lee BioSolutions) was diluted 1:1–1:10⁶-fold in PBS, added to the plates and incubated for 1 h at RT. NHS was added at 1:1 dilution as a negative control. The plates were washed 4 times with PBS/T/S. Serum antibodies bound to the coated Fc fragments were detected using a pan anti-human IgG light chain antibody from rabbit (ReMab Biosciences, 32-1031-00) (diluted 1:3000 in PBS/T/S) and visualized by addition of anti-rabbit IgG-HRP (Cytiva Life Sciences, NA935) (diluted 1:3000 in PBS/T/S). The coating efficacy of WT and REW Fc fragments were controlled using an AP conjugated polyclonal anti-human IgG (Fc specific) antibody from goat (Sigma-Aldrich, A5944) (diluted 1:5000 in PBS/T/S). The 450 nm or 405 nm absorbance values were recorded using a Sunrise plate reader (Tecan).

Immunoblotting was conducted as described [72 (link)]. In brief, cells were resuspended in RIPA with fresh prepared EDTA-free protease inhibitor (Roche) and incubated on ice for 15 min, followed by sonication. Forty micrograms protein from each sample was subjected to 7.5% SDS–PAGE gels. Primary antibodies were rabbit monoclonal anti-TOP3B (1 : 500) (Abcam) or mouse monoclonal anti-TOP3B (1 : 250) (Santa Cruz Biotechnology) or mouse monoclonal anti-α-tubulin (1 : 1000) (Sigma). Secondary antibodies were anti-rabbit IgG-HRP (1 : 10 000) (Amersham) or anti-mouse IgG-HRP (1 : 10 000) (Amersham). Western Blotting Luminol Reagent (Santa Cruz Biotechnology) was used according to the manufacturer's instructions. Intensity was measured by Fiji distribution of ImageJ.

Standard methods were used to perform Western blot. Briefly, cytoplasmic or nuclear extracts were diluted (1:2) in 2× sample buffer and boiled for 5 min. Twenty micrograms of each extract was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Amersham Biosciences, USA). The membrane was washed with phosphate-buffered saline-Tween 20 (TPBS), blocked in a solution of TPBS containing 5 % nonfat dry milk (blocking buffer), and then washed three times. The membrane was then incubated with primary antibody diluted 1:200 in blocking buffer overnight at 4 °C, washed three times with TPBS, and incubated with the secondary antibody conjugated with horseradish peroxidase (HRP) diluted 1:10,000 in blocking buffer for 1 h at RT. Samples were washed three times with TPBS, and then the signal was detected with the chemiluminescent protein detection system according to the manufacturer’s directions (Amersham Biosciences, USA). Antibodies used for Western blot include anti-NF-κB p65 (Santa Cruz Biotechnology, USA), anti-rabbit IgG-HRP (Amersham Biosciences, USA), and anti-actin (Sigma, USA).

Total cell lysates were prepared with RIPA buffer (Thermo Scientific, Waltham, MA, USA), protease inhibitor mix (Complete Mini, Roche, Basel, Switzerland) and phosphatase inhibitor (PhosphoStop, Roche), followed by the addition of 40 ul of 1X sample buffer and boiled. The samples were subjected to SDS-PAGE and assayed by western blot using the corresponding antibodies. The following primary antibodies were used for immunoblot: anti-β-tubulin (T8328, Sigma), anti-p-ERK (#4370, CST, Danvers, MA, USA), anti-LSD1 (ab17721, Abcam), and anti-p-LSD1 (S131) (#37177, CST). The secondary antibodies used were anti-mouse IgG HRP and anti-rabbit IgG HRP (1:2000 dilution, Amersham, Little Chalfont, UK). Immunoreactive proteins were visualized using ECL reagent (Amersham).

Small intestinal tissues were homogenized and lysed on ice in buffer containing 0.5% NP-40, 40 mM Tris-HCl (pH 8.0), 120 mM NaCl, 1 mM PMSF, and 10 μg/ml leupeptin. Proteins in lysates were measured with a modified bicinchoninic acid method. Proteins were denatured with SDS sample buffer, subjected to 10% SDS-polyacrylamide gel electrophoresis, and transferred to PVDF membrane. Membranes were blocked with TBS buffer (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 0.1% Tween-20) containing 5% BSA and were incubated overnight with rabbit polyclonal antibodies against ZO-1 (catalog no. 61–7300, Invitrogen, Carlsbad, CA, USA; diluted 1:1000), or rabbit monoclonal antibodies against occludin (catalog no. ab168986, Abcam, Cambridge, UK; diluted 1:1000). Antigen-antibody complexes were detected with anti-rabbit IgG-HRP (diluted 1:1000) using enhanced chemiluminescence in accordance with the manufacturer’s instructions (Amersham, Arlington Heights, IL, USA). Bands were quantified using laser-scanning densitometry and the expression level of each protein was normalized against that of β-actin. Full-length Western blots of ZO-1 and occludin are shown in Supplementary Fig. S3.

HUVECs and fibroblasts were treated with BMP9 (10 ng/ml), BMP4 (100 ng/ml) or TGF-β (10 ng/ml) for 60 min, and lysed with the cell lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Triton X-100) containing protease inhibitor cocktail (Roche). Equal amounts of proteins were loaded onto NuPAGE Novex 4-12% Bis-Tris Gels (Life Technologies), and blotted onto a nitrocellulose membrane (Immobilon; Millipore). After blocking with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 30 min at room temperature, the membranes were incubated with anti-phospho-Smad1/5/8 antibody (1:1000; CST, 9511L) or anti-β-actin antibody (1:2000; Sigma, AC-74) overnight at 4°C. The membranes were then washed with TBST, incubated with anti-rabbit-IgG-HRP (Amersham) or anti-mouse-IgG-HRP (Amersham) for 1 h, washed again with TBST, and the chemiluminescent signals were detected using ECL Prime (GE Healthcare).

For all lysate samples, protein concentrations were measured via Lowry, DC and loaded by equal protein amounts onto 12.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose. Transferred protein and amount loaded was confirmed with Ponceau S stain. Membranes were blocked with 5% nonfat milk in TBS-T. Rac1 antibody (Millipore, Billerica, MA) or β-actin antibody (Cell Signaling) was used at a dilution of 1:3000 or 1:5000, respectively, in 0.5% milk TBS-T. Anti-mouse IgG-HRP (Amersham, Amersham, UK) secondary was used at 1:10,000 in TBS-T for Rac1 and Anti-rabbit IgG-HRP (Amersham) secondary antibody was used at 1:10,000 in TBS-T for β-actin. In order to detect biotin, blots were incubated with 10 ml of Streptavidin-HRP (1:10,000 in TBS-T) for 1 h. HRP-conjugated signal was visualized via chemiluminescence using SuperSignal West Dura (Thermo Scientific, San Jose, CA), detected using a FluorChem M camera imaging system (ProteinSimple). Protein bands were quantified by analysis of pixel density using Alpha ViewSA version 3.4.0 software (ProteinSimple, San Jose, CA).