Immunoblotting Analysis of Cell Cycle Markers

Cell lysates were prepared using cold RIPA lysis buffer (150 mM NaCl, 50 mM Tris HCl [pH 8], 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS) plus a protease inhibitor cocktail (Complete, Roche), 1 mM NaOV₄, and 1 mM DTT. Immunoblotting were then performed as reported^{32 (link)} using the following primary antibodies: anti-phospho-histone H2AX-S139 (γH2AX) (EMD Millipore), -Ki67 (Abcam), -cyclin D1 (Calbiochem), -cyclin D2, -cyclin D3 (Cell Signalling), -cyclin E, cyclin A - vinculin (Santa Cruz), -RB1, -p27^Kip1 and -GRB2 (BD Transduction Laboratories).

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Sonego M., Pellizzari I., Dall’Acqua A., Pivetta E., Lorenzon I., Benevol S., Bomben R., Spessotto P., Sorio R., Gattei V., Belletti B., Schiappacassi M, & Baldassarre G. (2017). Common biological phenotypes characterize the acquisition of platinum-resistance in epithelial ovarian cancer cells. Scientific Reports, 7, 7104.

Publication 2017

-cyclin D2 -cyclin D3 -p27Kip1

Antibodies Buffer Cell Cold Cyclin a Cyclin d1 Cyclin d2 Cyclin d3 Cyclin e Grb2 Histone h2ax Nacl P27kip1 Protease inhibitor Ripa Sodium deoxycholate Tris Vinculin

Corresponding Organization :

Other organizations : National Cancer Institute, Centro di Riferimento Oncologico

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Variable analysis

independent variables

Type of primary antibodies used for immunoblotting

dependent variables

Protein expression levels of phospho-histone H2AX-S139 (γH2AX)
Protein expression levels of Ki67
Protein expression levels of cyclin D1
Protein expression levels of cyclin D2
Protein expression levels of cyclin D3
Protein expression levels of cyclin E
Protein expression levels of cyclin A
Protein expression levels of vinculin
Protein expression levels of RB1
Protein expression levels of p27Kip1
Protein expression levels of GRB2

control variables

RIPA lysis buffer composition (150 mM NaCl, 50 mM Tris HCl [pH 8], 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS)
Protease inhibitor cocktail (Complete, Roche)
1 mM NaOV4
1 mM DTT

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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