Control rabbit igg

Antibodies specific for GATA-2 (H-116, sc-9008) and CITED2 (JA22, sc-21795) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A specific alpha-tubulin antibody was purchased from Calbiochem (Darmstadt, Germany). CITED2-specific antibody (EPR3416, ab108345) and rabbit control IgG were purchased from abcam (Cambridge, MA, USA).

To identify HIF-1α- and HIF-2α-expressing cells by cytometry, cells were detached with a scraper and collected with cold PBS. Cells were then fixed with 4% paraformaldehyde (PFA) at pH 7.4 for 15 min at 4 °C and 5 min at room temperature (RT). Cells were permeabilized with 0.3% Triton X-100 for 15 min on ice and blocked with 5% FBS at 4 °C for 30 min. Cells were incubated at 4 °C for 1h with anti-HIF-1α 1:100 (Abcam), anti-HIF-2α 1:100 (Abcam), anti-HIF-1α (OH P402) 1:100 (Abcam), and anti-NANOG 1:500 (Cell Signaling). Cells were then washed 3 times in PBS and incubated with anti-mouse Alexa fluor 488 1:500 (Invitrogen,), anti-rabbit Alexa fluor 633 1:500 (Invitrogen,), and anti-rabbit IgG-PE 1:500 (Abcam) for 30 min at RT. Cells were then washed 3 times at 4 °C with PBS and resuspended in 500 mL of PBS. Fluorescence analysis was carried out by flow cytometry (FACSCalibur; BD Biosciences). In this assay, we use as an isotype control the following antibodies: anti-rabbit IgG phycoerythrin (PE) goat isotype (Abcam); rabbit control IgG (Abcam), and mouse IgG1 isotype control mAb (Cell Signaling). The percentage of positive HIF-1α and Hif-2α cells was calculated by FACSCalibur software after we selected a correct cell gate. The program analyzes the percentage of positive HIF-1α and Hif-2α cells.

For co-immunoprecipitation experiments, 100 μl of protein-G Dynabeads (Life Technology, #10004D) was crosslinked to 20 μg of anti-Tbx1 antibodies (Abcam, # ab18530). Crosslinking was performed as per the manufacturer's instructions. As a negative control, 20 μg of Rabbit Control IgG (Abcam, #ab46540) was added to reaction mixture to control for proteins interacting nonspecifically with the beads. Following crosslinking, nuclear extracts of P19Cl6 cells (D1) were added to the beads and incubated overnight at 4 °C, washed six times with PBS-0.1% Tween-20. Two consecutive elutions were performed with 0.1 M Glycine pH2.5 and immediately neutralized with Tris-HCl, pH 8.0. Samples were subjected to SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and proteins were transferred into polyvinyldene difluoride (PVDF) membrane for western blotting analyses with anti-KMT2C/MLL3 antibodies (Abcam #ab71200 or #ab71200, 1:1,000) and Amersham ECL Rabbit IgG, HRP-linked whole Ab (GE Heathcare, #NA934V; 1:10,000) as secondary antibody.

ChIP assay were performed as describied previously^{43 (link),46 (link),47 (link)}. Cells were crosslinked with final concentration 1% formaldehyde for 10 min at room temperature. Then, 125 mM glycine was added to quench unreacted formaldehyde. Cells were gathered and sonicated to make DNA fragments with a size range of 200–1000 bp. Cell extracts were immune-precipitated using 2 μg anti-Nrf2, anti-HIF-1α, or rabbit IgG control (Abcam, Cambridge, UK) for each sample suspended in 450 μl ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl, pH 8.1, 167 mM NaCl) purchased from Cell signaling Technology (Cat# 20-153, Danvers, MA). For all ChIP experiments, PCR analysis were performed by using multiple sets of primers spanning the transcription factor-binding site on hCXCR4 gene promoter.

ARPE-19 cells were cultured on glass plates in a cell culture plate (AGC Techno Glass, Shizuoka, Japan) for 24 h, then co-cultured with MT2, TL-Om1 cells using cell culture inserts (Thermo Fisher Scientific, San Jose, CA) for 48 h. ARPE-19 cells were permeabilized and fixed by cold fixing buffer (methanol/acetone, 1:1) at −20°C for 20 min. After blocking with 5% normal goat serum in phosphate-buffered saline for 15 min, cells were incubated in the diluted primary antibodies for 1 h at room temperature, followed by incubation with Alexa Fluor 488-labeled anti-rabbit secondary antibody (Abcam, Cambridge, MA) for 1 h at room temperature. After nuclear staining with 4',6-diamidino-2-phenylindole, cells were scanned using a TCS-SP8 microscope (Leica Micro Systems, Wetzlar, Germany). TNF-R1 polyclonal antibody (Bioss Antibodies, Woburn, MA) and TNF-R2 polyclonal antibody (Proteintech, Chicago, IL), or rabbit IgG control (Abcam) were used as primary antibodies.