FBXO7 Silencing Protocol for Functional Studies

FBXO7 silencing was conducted by transfecting ON-TARGETplus siRNA duplexes (GE Dharmacon) into cells using RNAiMAX (Life Technologies). Four individual siRNA duplexes (siFBXO7-1, -2, -3, -4) targeting unique regions within the FBXO7 coding sequence or a pool (siFBXO7-P) comprised of equimolar amounts of each individual siRNAs and a non-targeting control (siControl) were employed. The two most efficient siRNA duplexes (siFBXO7-2 and -4) were initially identified, and along with the siFBXO7-P were employed in all subsequent experiments. Silencing efficiencies were assessed by western blot 4 days post-transfection as detailed previously (67 (link)) with the antibodies and dilutions indicated in Supplementary Material, Table S12. Relative protein expression levels were determined using semi-quantitative image analysis in which FBXO7 band intensities were first normalized to the corresponding loading control (α-tubulin or cyclophilin B) and are presented relative to siControl (siRNA) or NT-Control (CRISPR/Cas9).

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Palmer M.C., Neudorf N.M., Farrell A.C., Razi T., Lichtensztejn Z, & McManus K.J. (2021). The F-box protein, FBXO7, is required to maintain chromosome stability in humans. Human Molecular Genetics, 31(9), 1471-1486.

Publication 2021

ON-TARGETplus siRNA duplexes RNAiMAX

Antibodies Cells Crispr Cyclophilin Dilutions Protein Sirna Transfection Western blot α tubulin

Corresponding Organization : University of Manitoba

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Variable analysis

independent variables

SiFBXO7-1
SiFBXO7-2
SiFBXO7-3
SiFBXO7-4
SiFBXO7-P
SiControl

dependent variables

FBXO7 protein expression levels

control variables

Loading controls (α-tubulin or cyclophilin B)

positive controls

SiFBXO7-P (pool of 4 individual siRNAs)

negative controls

SiControl (non-targeting control siRNA)

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