Subcellular Fractionation and Western Blot Analysis

In all, 3 × 10⁶ cells were used for the extraction of nuclear and cytoplasmic proteins with the subcellular protein fractionation kit for cultured cells (78840, ThermoFisher Scientific) in accordance to the manufacturer’s instructions. In all, 10 µg of the nuclear and cytoplasmic fractions were then analysed by SDS-PAGE^{61 (link)}, and the following primary antibodies were used: WDR26 (ab85961, Abcam, 1:2000), NOLC1 (sc-374033, Santa Cruz, 1:1000), FLAG (A8592, Sigma, 1:1000), H3 (ab1791, Abcam, 1:2000), and HSP90 (ab13495, Abcam, 1:5000).

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Napoli M., Li X., Ackerman H.D., Deshpande A.A., Barannikov I., Pisegna M.A., Bedrosian I., Mitsch J., Quinlan P., Thompson A., Rajapakshe K., Coarfa C., Gunaratne P.H., Marchion D.C., Magliocco A.M., Tsai K.Y, & Flores E.R. (2020). Pan-cancer analysis reveals TAp63-regulated oncogenic lncRNAs that promote cancer progression through AKT activation. Nature Communications, 11, 5156.

Publication 2020

ab85961 sc-374033 A8592 ab1791 ab13495

Antibodies Cells Cultured cells Cytoplasmic Fractionation Hsp90 Protein proteins Wdr26

Corresponding Organization :

Other organizations : Moffitt Cancer Center, The University of Texas MD Anderson Cancer Center, Baylor College of Medicine, University of Houston

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Variable analysis

independent variables

Subcellular protein fractionation kit for cultured cells (78840, ThermoFisher Scientific)

dependent variables

Nuclear and cytoplasmic protein fractions
Protein expression levels of WDR26, NOLC1, FLAG, H3, and HSP90

control variables

Amount of nuclear and cytoplasmic fractions (10 µg) analyzed by SDS-PAGE
Primary antibodies used: WDR26 (ab85961, Abcam, 1:2000), NOLC1 (sc-374033, Santa Cruz, 1:1000), FLAG (A8592, Sigma, 1:1000), H3 (ab1791, Abcam, 1:2000), and HSP90 (ab13495, Abcam, 1:5000)

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

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