Phosphoproteomic analysis of neoadjuvant breast cancer suggests that increased sensitivity to paclitaxel is driven by CDK4 and filamin A - Nature Communications

psm, or simply non-detected , non-detected proteins were transformed to 1 psm. This transformation allows performing ratio calculations . Proteins with fold-change in Log2 > 2 or not identified in IgG control and with at least 3 psm in bait or 1 psm in both technical replicates, were considered as potential candidates.In order to confirm or reject the targets discovered by mass spectrometry analysis, immunohistochemistry stainings were performed when available antibodies were found.

A vector for human CDK4 overexpression, pRc/CMV-CDK4 was kindly provided by M. Malumbres to generate stably MDA-MB-231 cells overexpressing CDK4. Cells were transfected at 60–70% density with Lipofectamine 3000 according to the manufacturer’s instructions. An empty vector was used as control; these cells were named MDA-MB-231 WT along the manuscript’s main text. Transduced cells were selected in 1500 µg/mL neomycin for 2 weeks.

Regarding acetylated tubulin bands, band intensities were quantified using ImageJ software . For this purpose, developed films were scanned and band intensities representing acetylated alpha tubulin and total alpha tubulin expression were quantified. For each sample, the ratio between the amounts of acetylated and total alpha tubulin was calculated. Uncropped scans of blots are supplied in the Source data file.

physorg_com NatureComms Now if they could just mitigate the destruction of the ovaries.

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