λ protein phosphatase  (New England Biolabs)

Journal: PLOS One

Article Title: Identification of CAND1 as a DNA-dependent protein kinase-regulated coactivator of androgen receptor and the ARv7 splice variant

doi: 10.1371/journal.pone.0349130

Figure Lengend Snippet: (A) After standard ARE pulldowns, the AR-DNA-bound complexes were washed and resuspended in 1x kinase buffer with/without ATP at 30 o C for listed times. Gel mobility shifts in AR were detected as a function of ATP addition. All pulldowns had 100 nM R1881 present. Proteins were analyzed by immunoblotting. Lanes 3 and 4 represent the standard AR-ARE pulldown. (B) To test if the above AR gel mobility shifts were reflective of phosphorylation events, the AR-CoR complexes were treated with ATP or AMP-PNP (non-hydrolyzable analogue) for 30 min at 30 o C. The DNA-PK inhibitor NU7441 or λ protein phosphatase (LPP) were pre-incubated for 5 min before adding ATP. LPP buffer was used for the LPP control reaction. (C) DNA-PK plays a role in phosphorylating AR. ATP treatment of the AR-CoR complexes using HeLa NE where DNA-PKcs was immunodepleted reveals a reduction of AR gel mobility shifts ( left panel) with the loss of DNA-PK. Proteins were assayed by immunoblotting and the level of unphosphorylated AR was quantified by Image J after normalization to the loading control HDAC1 ( right panel). P-value here = 0.00011. (D) DNA-bound AR or ARv7 was incubated with purified heterotrimeric DNA-PK (i.e., DNA-PKcs and Ku70/80) and then treated (-/+) ATP (-/+) NU7441 to demonstrate direct phosphorylation of AR/ARv7 by DNA-PK. Gel mobility shifts were assayed by immunoblotting. (E) ATP treatment (-/+) NU7441 pre-treatment demonstrates that DNA-PK enzymatic activity is needed for stabilizing CAND1 and ANP32A in the AR/ARv7 complexes. Auto-phosphorylation of DNA-PKcs at serine 2056 (pS2056) is a marker of DNA-PK activity and its inhibition by NU7441. Proteins were assayed by immunoblotting. ARv7 was detected here with the N-20 antibody. (F) HeLa NE used in 3xARE-E4 cell-free transcription assays was pre-treated (-/+) NU7441 to determine effects on AR and ARv7 transcriptional activity. E4 mRNA transcript levels were measured by RT-qPCR. In panels A-C and E, HDAC1 was used as a loading control and ARE pulldowns were conducted using HeLa NE as listed. All AR reactions had 100 nM R1881 present. Short exp., Medium exp., and Long exp. denote short, medium, and long exposures during x-ray film development. P-values: for ARv7, p = 0.00015; for AR, p = 0.000035. **** p < 0.0005, ***** p < 0.0001.

Article Snippet: DNA-PK inhibitor NU7441 was purchased from Tocris Bioscience. λ protein phosphatase (LPP) was purchased from New England Biolabs.

Techniques: Western Blot, Phospho-proteomics, Incubation, Control, Purification, Activity Assay, Marker, Inhibition, Quantitative RT-PCR