Assessment of nascent protein accumulation in different subcellular fractions following protrusion induction
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ABSTRACT: To assess if newly synthesised ribosomal proteins participate in canonical ribosome biogenesis in the nucleus, following their enhanced translation upon protrusion induction, Light (L) SILAC labelled MDA-MB231 breast cancer cells were seeded on top of collagen-I coated 3 micron transwell filters (corning). No media was added to the bottom chamber and cells were allowed to adhere to filter overnight. The next day, the media on top of cells was replaced with either medium (M) (closed pores) or heavy (H) SILAC media to pulse label newly synthesised proteins. H media was also added to the bottom chamber (open pores) in order to allow formation of protrusions in the H condition. Cells were pulsed for 4 hrs before being lysed and H and M pulsed samples were mixed together. Mixed lysates were then subjected to subcellular fractionation by serial solubilization (cytosol, membrane and nucleus) using Pierce subcellular protein fractionation kit (78840).
INSTRUMENT(S): Q Exactive Plus
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Permanent Cell Line Cell, Cell Culture
DISEASE(S): Breast Cancer
SUBMITTER: Maria Dermit Salazar
LAB HEAD: Faraz Mardakheh
PROVIDER: PXD021205 | Pride | 2020-10-20
REPOSITORIES: Pride
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