Western Blot Assay Protocol

Western blot assay was performed as described previously^{4 (link),36 (link)}. Briefly, cells at 70%–80% confluency were scraped with 1X RIPA cell lysis buffer (1 mM β-glycerophosphate, 20 mM Tris-HCl (pH 7.5), 1 mM Na₂EDTA, 1 mM Na₃VO₄, 150 mM NaCl, 1 mM EGTA, 2.5 mM Na₄P₂O₇, 1 μg/mL leupeptin, 1% Triton, 10% protease inhibitor). Proteins were separated by 10% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes (Millipore). Immune-reactive bands were visualized using a Pierce enhanced chemiluminescence (ECL) detection kit (Thermo Scientific); β-actin was used as a loading control. Information pertaining to antibodies used is detailed in Supplementary Table 1.

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Mittal K., Kaur J., Sharma S., Sharma N., Wei G., Choudhary I., Imhansi-Jacob P., Maganti N., Pawar S., Rida P., Toss M.S., Aleskandarany M., Janssen E.A., Søiland H., Gupta M.V., Reid M.D., Rakha E.A, & Aneja R. (2021). Hypoxia Drives Centrosome Amplification in Cancer Cells via HIF1α-dependent Induction of Polo-Like Kinase 4. Molecular Cancer Research, 20(4), 596-606.

Publication 2021

polyvinylidene fluoride membranes Pierce enhanced chemiluminescence (ECL) detection kit

Actin Antibodies Buffer Cell Chemiluminescence Egta Leupeptin Membranes Na4p2o7 Nacl Polyvinylidene fluoride Protease inhibitor Proteins Ripa Sds page Tris Western blot assay β glycerophosphate

Corresponding Organization : Georgia State University

Other organizations : Nottingham University Hospitals NHS Trust, University of Nottingham, Stavanger University Hospital, Emory University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein levels (visualized by immunoreactive bands)

control variables

Cell confluency (70%–80%)
RIPA cell lysis buffer (1 mM β-glycerophosphate, 20 mM Tris-HCl (pH 7.5), 1 mM Na2EDTA, 1 mM Na3VO4, 150 mM NaCl, 1 mM EGTA, 2.5 mM Na4P2O7, 1 μg/mL leupeptin, 1% Triton, 10% protease inhibitor)
SDS-PAGE (10%)
Polyvinylidene fluoride membranes
β-actin (loading control)

controls

Positive control: None mentioned
Negative control: None mentioned

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