Protocol detail

Hypoxia-Inducible Factor-1α Detection

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Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing Protease Inhibitor Cocktail (Calbiochem). Nuclear and cytoplasmic fractions were prepared from the BV2 cells using NE-PER reagents [32 (link)] according to the manufacturer’s protocol. Protein concentrations were determined by BCA assay [32 (link)]. Samples were loaded on 12% Bis-Tris pre-cast polyacrylamide gel (Invitrogen) and transferred to PVDF membrane (BIO-RAD). Membrane was then probed with antibody against HIF-1ɑ (NB100-449, Novus) followed by goat anti-rabbit IgG antibody conjugated with horseradish peroxidase (Santa Cruz) and developed with the Pierce ECL substrate (Thermo Fisher Scientific).

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Bok S., Kim Y.E., Woo Y., Kim S., Kang S.J., Lee Y., Park S.K., Weissman I.L, & Ahn G.O. (2017). Hypoxia-inducible factor-1α regulates microglial functions affecting neuronal survival in the acute phase of ischemic stroke in mice. Oncotarget, 8(67), 111508-111521.

Publication 2017

PVDF membrane NB100-449 goat anti-rabbit IgG antibody conjugated with horseradish peroxidase Pierce ECL substrate

Anti igg Antibody Assay Bis tris Buffer Cast Cells Cytoplasmic Goat Horseradish peroxidase Membrane Novus Polyacrylamide gel Protease inhibitor Protein Pvdf Rabbit Radioimmunoprecipitation assay

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Levels of HIF-1α protein

control variables

RIPA buffer containing Protease Inhibitor Cocktail (Calbiochem)
Protein concentrations determined by BCA assay
Samples loaded on 12% Bis-Tris pre-cast polyacrylamide gel (Invitrogen)
Proteins transferred to PVDF membrane (BIO-RAD)

controls

Positive control: Antibody against HIF-1α (NB100-449, Novus)
Negative control: Not explicitly mentioned

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