Anti-HIF-1-alpha Rabbit Monoclonal Antibody

Western blot analysis of HIF-1-Alpha using anti-HIF-1-Alpha antibody (M00013-4).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human HepG2 whole cell lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-HIF-1-Alpha antigen affinity purified monoclonal antibody (Catalog # M00013-4) at 1:500 overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:500 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for HIF-1-Alpha at approximately 110 kDa. The expected band size for HIF-1-Alpha is at 110 kDa.

All lanes use the Antibody at 1:1K dilution for 1 hour at room temperature.

(H) The protein expression of α-SMA, COL1α1, vimentin, fibronectin, E-cadherin, 4EBP1, FLAG, and HIF-1α in liver tissues analyzed using western blotting. (I) Immunofluorescence double staining of α-SMA and HIF-1α in the liver sections of mice (×100 magnification). The arrow heads represent colocalization of staining. (J) Relative quantification of α-SMA, HIF-1α, and colocalization of α-SMA and HIF-1α. EV, AAV2/8-scramble; OE, AAV2/8-4ebp1-4A-3xflag. Values are the mean ± SD (unpaired two-sample Student’s t test). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; ns, not significant. α-SMA, alpha-smooth muscle actin; AAV, adeno-associated virus; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H&E, hematoxylin and eosin; HIF-1α, hypoxia inducible factor 1 subunit alpha; IL-6, interleukin 6; RT-qPCR, quantitative reverse transcription polymerase chain reaction; TGF-β1, transforming growth factor beta 1.
Index in iScience under a CC BY license. DOI: 10.1016/j.isci.2025.113412

(F) The protein expression of α-SMA, COL1α1, vimentin, fibronectin, E-cadherin, 4EBP1, FLAG, and HIF-1α in liver tissues was analyzed using western blotting. (G) Immunofluorescence double staining of α-SMA and HIF-1α in the liver sections of mice (×100 magnification). The arrowheads represent colocalization of staining. (H) Relative quantification of α-SMA, HIF-1α, and colocalization of α-SMA and HIF-1α. EV, AAV2/8-scramble; OE, AAV2/8-4ebp1-4A-3xflag. Values are the mean ± SD (unpaired two-sample Student’s t test). ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; ns, not significant. α-SMA, alpha-smooth muscle actin; AAV, adeno-associated virus; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BDL, bile duct ligation; DAPI, 4′,6-diamidino-2-phenylindole; DBIL, direct bilirubin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H&E, hematoxylin-eosin; HIF-1α, hypoxia inducible factor 1 subunit alpha; IL-6, Interleukin 6; TBIL, total bilirubin; TGF-β1, transforming growth factor beta 1.
Index in iScience under a CC BY license. DOI: 10.1016/j.isci.2025.113412

(I) The protein expression of α-SMA, COL1α1, vimentin, fibronectin, E-cadherin, 4EBP1, FLAG, and HIF-1α in LX-2 cells analyzed using western blotting. (J) Immunofluorescence double staining of α-SMA and HIF-1α in vehicle or TGF-β1-stimulated LX-2 cells transducted with LV-empty or LV-4EBP1-4A-3xflag (×400 magnification). (M and N) (M) The apoptosis of vehicle or TGF-β1-stimulated LX-2 cells transducted with LV-empty or LV-4EBP1-4A-3xflag determined using flow cytometry and (N) percentages of apoptotic cells (early and late apoptotic cells). EV, LV-empty; OE, LV-4EBP1-4A-3xflag. Values are the mean ± SD (unpaired two-sample Student’s t test). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; ns, not significant. α-SMA, alpha-smooth muscle actin; DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HIF-1α, hypoxia inducible factor 1 subunit alpha; LV, lentivirus; PI, propidium iodide; RT-qPCR, quantitative reverse transcription polymerase chain reaction; TGF-β1, transforming growth factor beta 1.
Index in iScience under a CC BY license. DOI: 10.1016/j.isci.2025.113412

(H) The protein expression of α-SMA, COL1α1, vimentin, fibronectin, E-cadherin, and HIF-1α in LX-2 cells was analyzed using western blotting. (I) Immunofluorescence double staining of α-SMA and HIF-1α in TGF-β1-stimulated and DMSO or AZD8055-treated LX-2 cells transducted with LV-control-shRNA or LV-4EBP1-shRNA (×400 magnification). (L and M) (L) The apoptosis of LX-2 cells determined using flow cytometry and (M) percentages of apoptotic cells (early and late apoptotic cells). NC, LV-control-shRNA; KD, LV-4EBP1-shRNA. Values are the mean ± SD (unpaired two-sample Student’s t test). ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; ns, not significant. α-SMA, alpha-smooth muscle actin; DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HIF-1α, hypoxia inducible factor 1 subunit alpha; LV, lentivirus; PI, propidium iodide; TGF-β1, transforming growth factor beta 1.
Index in iScience under a CC BY license. DOI: 10.1016/j.isci.2025.113412