Pierce ecl western blotting substrate

HINT1_WT (25 μM) or HINT1_H114A were mixed with either 700 μM Ap₃A, Ap₄A, Ap₅A, AMP, ATP, or blank buffer and incubated at 4 °C rotating gently for 2 h. The working buffer includes 25 mM HEPES pH 8.0, 400 mM NaCl, and 2 mM EDTA. Samples were separated by 4–20% gradient SDS-polyacrylamide gel electrophoresis (PAGE) without pre-heating. RealBand 3-color High Range Protein Marker (BBI Life Science, Rockville, MD) was used to identify molecule weight of target bands. Gel under 25 kDa marker was cut for Coomassie brilliant blue staining. Gel above 25 kDa was transferred onto nitrocellulose membranes. Blottings were blocked in 5% non-fat milk for 30 min at room temperature, then incubated in Anti-His antibody (TransGen Biotech, catalog number HT501-02, dilution 1:3000) overnight at 4 °C and follow by 1 h incubation in horseradish peroxidase (HRP)-conjugated Goat Anti-Mouse IgG (BBI Life Science, catalog number D110087, 1:5000 dilution) at room temperature. Blottings were then treated with Pierce ECL Western Blotting Substrate and visualized with Amersham TM Imager 600 (GE Healthcare, Piscataway, NJ). The molecular weights of protein bands were calculated with three independent experiments by Amersham TM Imager 600 Analysis Software Version1.0 (GE Healthcare, Piscataway, NJ).

Western blot was performed as previously described. Briefly, proteins in cell lysates were resolved on SDS-PAGE and were transferred on PVDF membrane after electrophoresis. After blocking in 5% milk in PBS, the membrane was incubated with primary antibody and second antibody sequentially, following band detection by enhanced chemiluminescence (ECL) using Pierce™ ECL Western Blotting Substrate (GE Healthcare Bio-Sciences).

Whole cell lysates were prepared in whole cell extract buffer (50 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, and complete protease inhibitor (Promega)). Equal amounts of protein (30–50 μg) were electrophoresed on a mini protean any KD acrylamide gel (BioRad), transferred to Hybond ECL nitrocellulose (GE Healthcare). Transfer was verified via Ponceau S staining then blot was blocked with 5% nonfat dry milk in TBST for 1 hour at room temperature, followed by primary antibody overnight at 4 °C. After washing extensively, blots were incubated for 1–2 h at room temperature with appropriate HRP-linked secondary antibody (GE Healthcare), washed, developed using Pierce ECL Western Blotting Substrate, and exposed to film for detection. Primary Antibodies used and their concentrations were as follows:

Antibody	Catalog #	Vendor	Dilution
ARNT1	SC-17811	Santa Cruz	1:500
ARNT2	SC-393683	Santa Cruz	1:500
GAPDH	SC-365062	Santa Cruz	1:1000
GLUT1	PA5–16793	Thermo Fisher	1:250
HIF-1α	3716	Cell Signalling	1:1000 (in BSA)
HIF-2α	NB100–122	Novus	1:1000
LDH	SC-133123	Santa Cruz	1:1000
Tubulin	MA1–850	Thermo Fisher	1:1000