Mouse anti human hif 1α

Cells were harvested and lysed as described before [20 (link)]. The protein concentration was determined using the Bradford assay (#39222, Serva, Heidelberg, Germany), and equal amounts were loaded on a gel. Protein levels were analyzed by Western blot as described before [21 (link)]. The following primary antibodies were used: mouse anti-human HIF1α (BD Biosciences, #610959), mouse anti-human PD-L1 (Cell Signaling Technology, #29122), mouse monoclonal β-Actin Antibody (C4) (#sc-47778). The following secondary antibodies were used: Donkey-anti-mouse IRDye 800CW (Li-cor Biosciences, Lincoln, NE.) and Donkey-anti-Rabbit IRDye 680CW (Li-cor Biosciences). All antibodies were diluted in 3% non-fat milk in TBST. Immunoblots were analyzed by Odyssey infrared imaging system (Li-cor Biosciences).

HL-60 and KG-1 cells (10 × 10⁶ cells) were incubated with the indicated compounds for 24 h. Cells were harvested and lysed in RIPA buffer. The protein extract was subjected to SDS-polyacrylamide gel (BioRad) electrophoresis and transferred to a nitrocellulose membrane (BioRad). Mouse anti-human HIF-1α and goat anti-β-actin antibodies were purchased from BD and Abcam respectively.

AT-MSCs were plated in black 96-well clear-bottom plates (# 6005182, Perkin-Elmer) at a cell-density of 1 × 10⁴ cells/well and allowed to adhere overnight. Cells were cultured for 5 and 72 h in MM with 312.5, 625, and 1563 µM of DMOG, or 16, 32, 162, and 624 µM of baicalein, or with equal volumes of DMSO. The HIF-1α stabilization was quantified using the In-Cell ELISA Near Infrared Detection Kit (# 62201, Thermo Fisher Scientific) following the manufacturer’s instructions. Cells were fixed in 4% methanol-free formaldehyde (# 28906, Thermo Scientific), followed by washing, permeabilization, and blocking steps. HIF-1α was probed using mouse anti-human HIF-1α (1:50, # 610959, BD Biosciences), and an antibody for housekeeping protein rabbit polyclonal beta actin (2 µg/mL, # PA5-16914, Thermo Fisher Scientific). After overnight incubation at + 4 °C, AT-MSCs were washed with 1 × wash buffer before incubation with species-specific near-infrared DyLight-conjugated secondary antibody mix for 1 h at RT. After washing steps, Odyssey FC Imager (LI-COR) served to scan the plates with excitation/emission maxima of 692/712 nm for DyLight 680 Dye and 777/794 nm for DyLight 800 Dye. Measured signals were analyzed with Image Studio Software (LI-COR).