Hif 1α antibody

Cells lines were serum-starved for 24 hours and stimulated in fresh medium with 50 ng/mL VEGF-A (Cell Signaling Technology). Cells were then incubated under normoxic conditions, and protein lysates were collected 18 hours later. Western blot analysis was carried out using specific antibodies against EZH2 (AC22), H3K27me3 (C36B11), HIF-1α (HIF-1α antibody), VEGFR-2 (55B11) (Cell Signaling Technology), and E2F3 (ab54945; Abcam).

Western blots were incubated with HIF-1α antibody (1:1000, #3716, Cell Signaling, Danvers, MA), p-αAMPK antibody (1:1000, #2535, Cell Signaling), αAMPK antibody (1:1000, #2603, Cell Signaling) overnight at 4°C. Levels of β-actin (Cell Signaling #4970) (1:1000) or vinculin (SigmaAldrich) (1:4000) were used as the loading control. Densitometry was performed using Image Lab software v5.2.1 (Bio-Rad, Hercules, CA). Values are represented as the ratio of target protein to loading control. For the HIF-1α blot using MDA-MB-435 cell lysate, unrelated lanes between the AG311-treated samples and CoCl₂ control were removed. The image was spliced together at that position.

RNA immunoprecipitation (RIP) assays were performed as previously described ³⁸ . THP1 macrophages (~10 million) were collected and suspended in 1 mL RIPA buffer (50mM Tris pH 7.5, 150mM NaCl, 1mM EDTA, 1% NP-40, 0.5mM DTT, 100 U/mL RNAseOUT, 1X protease inhibitor cocktail) on ice. The cells were centrifuged at 20000g for 15 minutes in 4^oC, and the lysates were washed with Protein A/G magnetic beads (Millipore; 16–663) at 4^oC for 1 hour. 30 uL Protein A/G magnetic beads were incubated with 5 ug of HIF1α antibody (Cell Signaling Technology; 36169) or IgG control in 200 ul of RIPA buffer for 30 minutes at room temperature and were incubated with the pre-cleared lysate for four hours at 4^oC. Samples were then washed at least five times with RIPA buffer and the RNA was isolated with TRIzol and chloroform. Samples were spun at 16000g for 10 minutes at 4^oC. 6 uL linear acrylamide, 60 uL 5 M ammonium acetate and 600 uL 2-propanol were added and vortexed. To precipitate the RNA samples, samples were placed in −80C for at least one hour then centrifuged at 16000g for 10 minutes at 4^oC. The pellet was washed with 80% ethanol, then spun down again, the pellet was air dried and resuspended in water. The isolated RNA was subjected to reverse transcription, and the lincRNA was assessed by qRT-PCR using Applied Biosystems Fast Real Time PCR System.

Cell lysates were prepared in RIPA buffer with protease inhibitors. Protein concentration was measured by Pierce BCA kit. About 30–40 ug protein lysate were reduced and separated by SDS-PAGE electrophoresis and transferred to nitrocellulose membranes. The blots were blocked with 5% nonfat milk in TBS containing 0.1% Tween 20 for one hour and were incubated with antibodies overnight at 4^oC. Primary antibodies against PARP (Cell Signaling Technology; 9542S), Caspase 9 (Cell Signaling Technology; 9502T), Cleaved Caspase 3 (Cell Signaling Technology; 9664S), and HIF1α antibody (Cell Signaling Technology; 36169) were used to probe the membranes. For loading control, β-actin (Cell Signaling Technology; 5125S) was used. The blots were incubated for 2 hours in HRP-conjugated secondary antibody (Thermo Fisher; 31460) at room temperature and bands were imaged with Pierce ECL Western Blotting Substrate (Thermo Fisher; 32209). Western blot was quantified with pooled densitometry analyses and densitometry data was considered a secondary and complementary way to present a “semi-quantitative” sense of results across multiple blots. A commercial ELISA for human NTN1 (LSBio; LS-F5003–1) was used according to the manufacturer’s instructions.