Extended Data Fig. 6: Identification of methylated lysine 270 in FOXA1 as an LSD1 substrate.
From: Chromatin binding of FOXA1 is promoted by LSD1-mediated demethylation in prostate cancer
a, FOXA1 immunoprecipitation was performed in LNCaP cells treated with different LSD1 inhibitors (S2101 at 50 μM and C12 at 5 μM), followed by immunoblotting for methyllysine (images cropped from the same blot). b, FOXA1 immunoprecipitation was performed in LN-LSD1WT or LN-LSD1K661A cells, followed by immunoblotting for methyllysine. c, Mass-spectrometry analysis on immunoprecipitated V5-FOXA1 in LNCaP cells stably overexpressing V5-tagged FOXA1. Covered residues are in yellow. Residues with detected post-translational modifications are indicated in green. d, LNCaP stable cells expressing doxycycline-inducible V5-tagged FOXA1-WT (LNCaP-tetFOXA1WT) or FOXA1-K270R (LNCaP-tetFOXA1K270R) were generated. V5-FOXA1 expression induced by doxycycline treatment (0-0.1 μg/ml) was confirmed by immunoblotting. e, ChIP-qPCR for FOXA1-WT or K270R binding (anti-V5) at AR-regulated enhancers in these stable cells (doxycycline supplemented) treated with S2101 (50 μM, 24 h). f, g, CWR22-RV1 cells stably expressing doxycycline-inducible FOXA1-WT (CWR22RV1-tetFOXA1WT) or K270R mutant (CWR22RV1-tetFOXA1K270R) were established. Immunoblotting for LSD1 in those stable cells (doxycycline supplemented) transfected with siRNA against LSD1 (siLSD1) or non-target control (siNTC) (f) and ChIP-qPCR for V5-FOXA1 binding (g) were performed. Note: Experiments described in this figure were all done under hormone-depleted conditions.
