HIF1α Immunoprecipitation and Immunoblot

Cell pellets were lysed using a RIPA lysis buffer with Halt proteasome inhibitor (Thermo Scientific), sonicated briefly and centrifuged at 15000 × g at 4°C for 15 minutes. Immunoprecipitation was performed as described previously [55 (link)]. Antibodies against HA-HIF1α were obtained from Covance (Princeton, New Jersey). An indirect immunoprecipitation kit (Millipore) and manufacturer-suggested protocols were used. Immunoblot was performed as described previously [56 (link)]. PVDF Membranes were probed using antibodies to HA-HIF1α (Covance), HO-HIF1α (Cell Signalling Technology, Danvers, Massachusetts), HIF1α (Novus Biologicals, Littleton, Colorado), HIF2α (Novus) or ubiquitin (Abcam, Cambridge, Massachusetts).

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Gao P., Yang C., Nesvick C.L., Feldman M.J., Sizdahkhani S., Liu H., Chu H., Yang F., Tang L., Tian J., Zhao S., Li G., Heiss J.D., Liu Y., Zhuang Z, & Xu G. (2016). Hypotaurine evokes a malignant phenotype in glioma through aberrant hypoxic signaling. Oncotarget, 7(12), 15200-15214.

Publication 2016

HO-HIF1α HIF1α HIF2α ubiquitin

Antibodies Biologicals Buffer Cell Hif1α Immunoblot Immunoprecipitation Membranes Novus Pellets Proteasome inhibitor Pvdf Ripa Ubiquitin

Corresponding Organization :

Other organizations : Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Seventh People's Hospital of Dalian, National Institute of Neurological Disorders and Stroke, National Institutes of Health, National Cancer Institute, Harbin Medical University, First Affiliated Hospital of Harbin Medical University, Dalian Polytechnic University

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Variable analysis

independent variables

RIPA lysis buffer with Halt proteasome inhibitor
Antibodies against HA-HIF1α

dependent variables

Immunoprecipitation
Immunoblot

control variables

Cell pellets
Sonication
Centrifugation at 15000 × g at 4°C for 15 minutes
Indirect immunoprecipitation kit (Millipore)
PVDF Membranes

positive controls

HA-HIF1α
HO-HIF1α
HIF1α
HIF2α
Ubiquitin

negative controls

Not explicitly mentioned

Annotations

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