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. 2011 Sep 7;6(9):e24401. doi: 10.1371/journal.pone.0024401

Figure 1. NonO is identified as a novel interacting partner of Rasd1 via affinity pull-down assay.

Figure 1

(A) Coomassie blue stained SDS-PAGE gel of affinity pulldown assay. Ni-NTA magnetic beads were incubated either with lysate from pHis-Rasd1 transfected COS-7 cells or with lysate of empty vector transfected cells. Next, washes were conducted to remove non-specific binding proteins. His-Rasd1 bound to the magnetic beads was then incubated with PC-12 lysate. The beads were boiled to separate the protein complexes for fractionation on SDS-PAGE (12%). The 55 kDa, 50 kDa and 30 kDa bands were observed in the elute lane of His-Rasd1 (Lane 5) but not in the elute lane of the negative control (Lane 1). Protein bands were excised for further analysis using mass spectrometry. The proteins identified were NonO, Tubulin beta 5 and Rasd1, respectively. E, elute; W, wash; and I, input. (B) Co-precipitation assay was performed to study in vivo interaction between Rasd1 and NonO. COS-7 cells were co-transfected with plasmids expressing HA-Rasd1 and either GST-NonO or GST. The lysates were then incubated with GSH-linked magnetic beads to precipitate GST-tagged proteins. HA-Rasd1 was observed to co-precipitate specifically with GST-NonO but not GST (compare Lanes 1 with 2). (C) A similar interaction assay was performed for NonO-V5 and GST-Rasd1 proteins. In this experiment, COS-7 cells were co-transfected with pNonO-V5 and either pGST-Rasd1 or pXJGST. GSH-linked magnetic beads were added to the cell lysates to pull-down GST-tagged proteins. NonO-V5 was observed to be co-precipitated with GST-Rasd1 but not with GST (compare Lanes 1 with 2). (D) To study the in vivo interaction of endogenous Rasd1 and NonO, co-IP was performed on HEK293T cell lysates incubated with either rabbit anti-NonO or rabbit control IgG. Detection of the blot with anti-Rasd1 showed that RASD1 was co-IP specifically by NONO (Lane 2). (E) A similar Co-IP was conducted using mouse brain lysate incubated with either anti-Rasd1 or goat control IgG. NonO was only co-precipitated by lysate incubated with anti-Rasd1 (Lane 2). NonO-V5 is detected with anti-V5 (Invitrogen, USA, CA); GST-tagged proteins are detected with anti-GST (Santa Cruz, USA, CA); HA-Rasd1 is detected with anti-Xpress (Invitrogen, USA, CA); endogenous NonO is detected with goat anti-NONO; and endogenous Rasd1 is detected with goat anti-Rasd1.