Labeling and identification of LNCaP cell surface proteins: A pilot study

Abstract

Cell surface biotinylation is an effective technique for identifying membrane proteins in the LNCaP prostate cancer cell line. We have demonstrated the identification of androgen-regulated membrane proteins and their validation in tissue samples.

LYRIC/AEG-1 Is Targeted to Different Subcellular Compartments by Ubiquitinylation and Intrinsic Nuclear Localization Signals

“…All media were supplemented with 10% fetal bovine serum (Labtech). Androgen regulation experiments were done as previously described (20) and media were supplemented with either the dihydrotestosterone analogue R1881 (10 nmol/L; Sigma) or an equal volume of vehicle (ethanol) for 24 h before being harvested. For transfection, cells were grown to 40% confluency.…”

“…All procedures were done as described in ref. (20). Membranes were incubated with primary antibody-anti-RNA polymerase II (1:2,000), anti-GFP (1:5,000), anti-fibrillarin (1:1,000), anti-histone H3 (1:5,000), and anti-actin (1:5,000; all from Abcam); anti -g-adaptin (1:2,000, BD Transduction Laboratories), anti-p65 (1:1,000, Abcam), or anti-LYRIC/ AEG-1 SS or AK at 1:2,000.…”

“…Confocal microscopy. Cells were grown, fixed stained, and mounted as described (20) using an anti-fibrillarin antibody (1:400) and 594-conjugated Alexafluor secondary antibody (1:10,000, Molecular Probes). All scale bars represent 10 Am.…”

Nuclear LYRIC/AEG-1 interacts with PLZF and relieves PLZF-mediated repression

“…When HDAC1 and HDAC4 were immunoprecipitated from COS7 cells cotransfected with PLZF-V5 and GFP-LYRIC/AEG-1, the presence of overexpressed LYRIC/AEG-1 increased b-Galactosidase assay confirms the interaction between PLZF and LYRIC/AEG-1 (left panel) PLZF well circled in black. Endogenous PLZF was immunoprecipitated (IP'd) using an anti-PLZF antibody (Calbiochem, Nottingham, UK) from 1 mg of KG1 protein lysate made as described (Whitaker et al, 2007;central panel). 1%: input; C: IP control; IP: IP.…”

“…Slight differences in PLZF molecular weight occur as a result of different salt concentrations in the samples. HeLa cells were grown and fixed on coverslips as described (Whitaker et al, 2007) following transfection with GFP-LYRIC/AEG-1 and PLZF-V5 using NanoJuice (Novagen, Nottingham, UK; right panel). Cells were probed with an anti-V5 antibody (Abcam, Cambridge, UK) and Alexafluor 594 (Molecular Probes, Paisley, UK) and mounted with 46-diamidino-2-phenylindole (DAPI).…”