Rasd1 Modulates the Coactivator Function of NonO HEK293T cells. Similar to (A), induction of cells with forskolin leads to the up-regulation of cAMP target genes – NR4A1, NR4A2 and FOS (compare Bars I with II of each gene). Next, rescue of NONO was performed by transfection of plasmid expressing NonO in NONO-knockdown cells. The transcripts of NR4A1, NR4A2 and FOS were up-regulated, confirming the involvement of NonO in the regulation of their transcription (compare Bars III with IV of each gene). In NONO-knockdown cells with over-expression of Rasd1, the transcripts levels of NR4A1, NR4A2 and FOS were comparable to NONO- knockdown cells (compare bars III and V of each gene). This suggests that Rasd1 requires NONO to repress the transcription of the CREB-target genes. Repression of target genes, NR4A1 and NR4A2, was observed in NONO-knockdown cells that were co-transfected with Rasd1 and NonO (compare Bars IV with VI of each gene). The expression of FOS transcript remained unaffected by the presence of Rasd1 (compare Bars IV with VI of FOS), similar to that in Bar IV of (A). b-actin was used as an internal control for normalization. On the right are representative western blots showing expression of NonO and Rasd1in HEK293T cells. Actin serves as a loading control. NonO and endogenous NONO are detected by anti-NONO; Rasd1 is detected with anti-GST; and actin is detected with anti-actin. (C) ChIP was performed using forskolin-treated (20 mM for 15 minutes) HEK293T cell lysates transfected with either pGST-Rasd1 or pGST (negative control) and incubated with either no antibody control (No AB) or anti-NONO (NONO). Primers targeting the CRE-site of FOS and NR4A2 promoters were used for subsequent PCR study. The results indicated that in the presence of Rasd1, more NONO was bound to the NR4A2 promoter (compare Lanes 2 and 3). The amount of NONO bound to the FOS promoter displayed no significant difference with or without transfection of pGST-Rasd1 (Lanes 5 and 6). I, input; and E, elute. (D) Next ChIP was performed similar to (C) using forskolin-treated HEK293T cells transfected with His-Rasd1 (Rasd1), empty vector (Vec), or His-Del-NLS (Del-NLS; mutant Rasd1 that does not interact with NonO). The sonicated lysates were subsequently incubated with Anti-Xpress, and primers targeting the CRE-sites on the FOS and NR4A2 promoters were used for PCR study. Results indicate that neither Rasd1 nor mutant Rasd1 binds to the FOS promoter (Lanes 5 and 6). On the other hand, Rasd1 but not its mutant, Del-NLS, specifically binds to the CRE-site of the NR4A2 promoter, suggesting that interaction of Rasd1 and NonO is required to suppress the transcription of NR4A2 (compare Lanes 2 and 3). I, input; and E, elute. doi:10.1371/journal.pone.0024401.g005 after seeding of 36105 cells into each well of a 6-well plate unless stated otherwise. COS-7 cells were transfected using Lipofectami- neTM 2000 (Invitrogen, USA, CA) according to manufacturer’s instructions. Calcium phosphate method was employed for transfection of plasmids into HEK293T cells. In vitro pulldown assay and mass spectrometry In vitro affinity pulldown assay using mammalian cell lysates was conducted to identify novel interacting partners of Rasd1. COS-7 cells seeded on 10cm plates were transfected the next day with His- Rasd1 (24 mg) or pcDNA4/HisMax B (24 mg) (negative control) at 90% confluence. Cells were harvested as previously stated [84]. 20 ml of Ni-NTA magnetic agarose beads (Qiagen, USA, CA) were added to purify His-Rasd1 for 1 hour at 4uC. Beads were washed according to manufacturer’s instructions. This was fol- lowed by incubation of His-Rasd1-bound beads with PC-12 lysates overnight (O/N) at 4uC. After incubation, beads were washed and bound-proteins were eluted by heating in Laemmli buffer at 95uC for 10 minutes. Samples were fractionated on 12% SDS-PAGE acrylamide gel and subsequently stained with Coomassie blue. Bands of interest were excised by scalpels and cut into 1mm3 cubes. The cubes were then destained with 50% methanol at room temperature before digestion was carried out with 10 ng/ml Trypsin (Promega, USA, WI) O/N at 37uC. Subsequently, 50% ACN/5% Trifluoroacetic acid was used to extract peptides. The peptides were then dried under vacuum and cleaned with ZipTipH C18 (Merck Millipore, USA, MA) according to manufacturer’s instruction. MALDI-TOF/MS was used to elucidate the identity of the unknown bands. SwissProt database was used for the analysis of the peptide spectra obtained. The MALDI/TOF-MS score obtained for NonO was 199. GST pulldown in vivo interaction studies COS-7 cells were co-transfected with plasmids expressing NonO (2 mg) and Rasd1 (2 mg). Cells were harvested as previously stated [84]. Interaction studies were performed using Mag- neGSTTM particles (Promega, USA, WI) according to manufac- turer’s instructions. Bound proteins were eluted by heating in Laemmli buffer followed by SDS-PAGE and Western blotting. with NP40 lysis buffer (1% NP40, 150 mM NaCl, 50 mM Tris-Cl, pH 8.0, 1% deoxycholic acid, 0.1% SDS, protease inhibitor (Roche, Switzerland, Basel)) and incubated at 4uC for 20 minutes. The crude lysate was cleared by centrifugation at 13,000 rpm for 20 minutes at 4uC. Experiment was performed with rProtG agarose beads (Invitrogen, USA, CA) as previously described [84]. The pre-cleared lysates were incubated with rabbit polyclonal anti- NMT55/p54NRB IgG (Abcam, UK, Cambridge) or rabbit control IgG (Abcam, UK, Cambridge; negative control). Co-IP using mouse brain lysates was performed using goat polyclonal anti-Rasd1 (Abcam, UK, Cambridge) as described elsewhere [84]. NonO was detected using goat anti-NONO (Abcam, UK, Cambridge) or rabbit anti-NONO (Santa Cruz, USA, CA). Reporter gene assay Effects of Rasd1 and NonO in the CREB signaling pathway were investigated using PathDetect CREB trans-Reporting System (Stratagene, USA, CA). PathDetect CREB trans-Reporting System is a GAL4-dependent reporter gene assay. Factors influencing the phosphorylation of CREB protein (fused to GAL4-DNA-binding domain) will be monitored effectively by similar changes in the luciferase activity. HEK293T cells were transfected with pSV-b-Gal (b-Galactosidase; internal reporter), pFR-Luc (reporter plasmid with 5xGAL4 binding site and TATA box as minimal promoter), and pFA2-CREB (CREB (1–280) fused to GAL4-DNA binding domain (dbd) trans-activator plasmid) plus different combinations of pHis-Rasd1, and pNonO-V5 as indicated in Figure 2. pcDNA3.1/ V5-His B and pcDNA4/HisMax B served as negative controls for pNonO-V5 and pHis-Rasd1, respectively. Cells were induced with 20 mM forskolin (Sigma-Aldrich, USA, MO) for 4 hours before harvesting. Luciferase assay was performed using Luciferase Assay System (Promega, USA, WI) according to manufacturer’s protocol. 20/20n Luminometer (Promega, USA, WI) was used to measure luciferase levels. b-gal levels were measured using b-Gal Enzyme Assay System (Promega, USA, WI) according to manufacturer’s protocol. Normalisation of data was performed using luciferase values of wells transfected with pFC2-dbd (negative control) construct containing only GAL4-dbd and with no trans-activator function. All values of empty vectors were set as 1. Statistical analyses were performed using two-tailed unpaired student’s t-test. Co-immunoprecipitation (co-IP) assay Co-IP of endogenous proteins was performed by scraping one 10 cm plate of HEK293T cells in 1.5 ml of PBS, followed by centrifugation at 13,000 rpm for 1 minute. The cells were lysed Indirect immunofluorescence HEK293T and COS-7 cells were transfected as indicated in Figures 3 and 4, respectively. Experiment was performed as described previously [19]. Primary antibody incubation was PLoS ONE | www.plosone.org 12 September 2011 | Volume 6 | Issue 9 | e24401