Immunoblotting and Immunoprecipitation of HIF-1α Pathway

Western blotting and immunoprecipitation were performed using previous methods.^{60 (link)} HIF-1α antibody (1:1 000, 10006421; Cayman), VHL antibody (1:1 000, sc-5575; Santa Cruz), PHD1 antibody (1:1 000, NB100-310; Novus), PHD2 antibody (1:1 000, NB100-2219; Novus), PHD3 antibody (1:1 000, NB100-303; Novus), HSP70 antibody (1:500, ADI-SPA-812; Enzolife), HSP90 antibody (1:500, sc-13119; Santa Cruz) and β-actin antibody (1:2 000, A2066; Sigma) were used for immunoblotting. Protein bands were quantified using ImageJ. For immunoprecipitation, nuclear extracts from cultured B cells were incubated with Dynabeads protein G and 5 μg anti-HIF-1α antibody (H1a67) overnight at 4 °C. Anti-HIF-1α, (1:1 000, 10006421; Cayman) anti-HSP70 (1:500, ADI-SPA-812; Enzolife) and anti-ubiquitin (1:500, sc-166553; Santa Cruz) antibodies were used for immunoblotting. Western blot source data are included in Fig. S9.

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Meng X., Lin Z., Cao S., Janowska I., Sonomoto K., Andreev D., Katharina K., Wen J., Knaup K.X., Wiesener M.S., Krönke G., Rizzi M., Schett G, & Bozec A. (2022). Estrogen-mediated downregulation of HIF-1α signaling in B lymphocytes influences postmenopausal bone loss. Bone Research, 10, 15.

Publication 2022

sc-5575 NB100-310 NB100-2219 NB100-303 sc-13119 A2066 sc-166553

Actin Anti antibody Antibodies Antibody Cayman Cells extracts Hsp70 Hsp90 Immunoprecipitation Novus Phd1 Protein Protein g Ubiquitin Western blot

Corresponding Organization :

Other organizations : Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, University Medical Center Freiburg, University of Freiburg

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Variable analysis

independent variables

Western blotting and immunoprecipitation methods (previous methods.)

dependent variables

HIF-1α protein levels
VHL protein levels
PHD1 protein levels
PHD2 protein levels
PHD3 protein levels
HSP70 protein levels
HSP90 protein levels
β-actin protein levels

control variables

Antibody dilutions used for immunoblotting (HIF-1α 1:1000, VHL 1:1000, PHD1 1:1000, PHD2 1:1000, PHD3 1:1000, HSP70 1:500, HSP90 1:500, β-actin 1:2000)
Protein band quantification using ImageJ
Overnight incubation of nuclear extracts with Dynabeads protein G and 5 μg anti-HIF-1α antibody at 4°C for immunoprecipitation
Antibodies used for immunoblotting after immunoprecipitation (anti-HIF-1α 1:1000, anti-HSP70 1:500, anti-ubiquitin 1:500)

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