Control rabbit igg

Maxisorp™ EIA plates (Thermo Scientific) were coated with 0.5 μg/ml goat anti-human IgG-Fc (Jackson ImmunoResearch Laboratories) in PBS at 4 °C overnight. Thereafter the plate was blocked with 2 % bovine serum albumin (BSA) for 1 h. Recombinant human FGFRs-Fc (R&D systems) (500 ng/ml) or the control human IgG-Fc (Abcam) was then added for 2 h, followed by incubation with IMB-R1 or the control rabbit IgG (Invitrogen) at the indicated doses for 1 h. The bound antibodies were detected with 0.5 μg/ml HRP-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories) and visualized with TMB substrate (Thermo Scientific). The color was read at 450 nm using the Victor³ multilabel plate reader (PerkinElmer). All reactions were performed at room temperature, unless indicated otherwise, and protected from light, with each step followed by extensive washing in blocking buffer. The readings were normalized against the controls.

Chromatin immunoprecipitation (ChIP) assays were performed as previously described (Forsberg et al, 2000; Wilson et al, 2010) using antibodies against total STAT5 (Santa Cruz, sc835), ERG (Santa Cruz, sc354x), CTCF (Millipore, 07‐592), and control rabbit IgG (Invitrogen, 9172). ChIP DNA samples were amplified for sequencing with the Illumina Chip‐Seq Sample Prep Kit (Illumina) and sequenced with the Illumina GA2 Genome Analyser platform. The ChIP‐Seq data from this study have been submitted to the NCBI Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE70697.

To assess the phosphorylation of c-Met and EGFR in vivo, immunocompetent mice were infected with the various C. albicans strains as described above. After 1-d of infection, the mice were sacrificed, and the tongues were excised, snap frozen, and embedded into OTC. Thin sections were prepared and transferred to glass slides. The samples were air dried, fixed in 100% methanol, rinsed, and then blocked with 5% goat serum in PBS. The slides were incubated with a rabbit anti-phospho-c-MET antibody (Tyr1003, #MBS9600900, My Biosource Inc.) or control rabbit IgG (#026102, Invitrogen) followed an Alexa Fluor 568-conjugated goat anti-rabbit antibody. The C. albicans cells were labeled with an anti-Candida antibody conjugated with Alexa Fluor 488 and the nuclei were labeled with DAPI. Phosphorylated EGFR was detected similarly, except that the slides were incubated with an anti-rabbit phosho-EGFR conjugated with phycoerythrin (Tyr1068, #14565, Cell Signaling Technologies) and control slides were stained with rabbit IgG conjugated with phycoerthrin (#5742, Cell Signaling Technologies). The slides were imaged by confocal microscopy, and z-stacks were combined using LAS X software.

RIP experiments were performed by using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA). Approximately 10⁷ cells were pelleted and re-suspended with an equal pellet volume of RIP Lysis Buffer (about 100 μl) plus protease and RNase inhibitors. The cell lysates (100 μl) were incubated with 5 μg of AGO2 (Cell signaling technologies, #2897), MSH2 (ab70270, Abcam, Cambridge, UK), or control Rabbit IgG (Thermo Fisher Scientific, #31235) coated beads with rotation at 4 °C overnight, respectively. After treating with proteinase K, the immunoprecipitated RNAs were extracted by RNeasy MinElute Cleanup Kit (Qiagen) and reversely transcripted using PrimeScript RT Master Mix (TaKaRa).

A549 cells were harvested using a cell scraper and washed three times in 1x PBS. The cell pellet was resuspended in 1x PBS containing 0.5 mM DTME and 0.5 mM DSP and incubated at room temperature for 30 min, followed by incubation in quenching buffer (20 mM Tris-HCl (pH 7.5), 5 mM cysteine) at 25 °C for 5 min. After washing in ice-cold PBS, the pellet was sonicated in RIPA buffer briefly and centrifuged at 14,000 × g at 4 °C for 5 min. The supernatant was subjected to anti-NRF2 affinity purification. An anti-NRF2 antibody (#12721, Cell Signaling Technology) and control rabbit IgG (#55944, Thermo Fisher Scientific) were crosslinked to a 1:1 mixture of Dynabeads protein A and protein G (Thermo Fisher Scientific) with DMP and incubated with the supernatant at 4 °C for 2 h. After washing in RIPA buffer, the NRF2 complex was eluted from the beads by incubation in elution buffer (50 mM Tris-HCl (pH 8.0), 0.2 M NaCl, 2 w/v% SDS, 50 mM DTT) at 37 °C for 30 min. The eluate was analyzed by immunoblot analysis with anti-NRF2 antibody (sc-13032X, Santa Cruz; 1:1,000), anti-FOSL2 antibody (#19967S, Cell Signaling Technology; 1:1,000), and anti-CEBPB antibody (sc-150 X, Santa Cruz; 1:1,000).

As described in Supplementary Fig. 4a, 9-week-old female C57BL/6J mice were injected twice with purified hSeP protein (1 mg/kg intraperitoneally ip)^{16 (link)}. Control mice were injected ip with an identical volume of PBS (vehicle control). Treatment hSeP was made 12 and 2 h before tolerance tests or tissue sampling. Tissue samples were taken for western blotting after perfusion with saline. AE2 mAb (rat IgG1) and control IgG (20 mg/kg body weight) were administered 2 h before the first injection of SeP. Control rat IgG1 mAb (R&D systems) was used as a control.
Mouse models of diabetes were treated as described in Supplementary Fig. 8a. Nine-week-old male KKAy mice or 9-week-old male C57BL/6J mice fed HFHSD for 11 weeks were injected ip with mFHR pAb and Control rabbit IgG (25 mg/kg body weight), and then underwent a glucose tolerance test (24 h after Ab injection), insulin tolerance test (48 h after Ab injection) and had tissue sampling (72 h after Ab injection). Control rabbit IgG (Thermo Fisher Scientific) was used as a control.