Volume 8 issue 6 June 2007
Nuclear functions of the Arf guanine nucleotide exchange factor BRAG2
Jillian L. Dunphy, Keqiang Ye and James E. Casanova

Supplemental Figure 1: Detection of nucleolar BRAG2a HeLa cells were transiently transfected with BRAG2a-GFP (B and E), HA-BRAG2a (C, F-G and J-K), or untagged BRAG2a (D and H), with (A and E-H) or without (B-D) myc-PIKE-S. Asterisks indicate untransfected cells.  The position of nuclei was determined by DAPI staining (shown in Merge).  Nuclear staining was observed for BRAG2a, regardless of N-terminal HA-tag, C-terminal GFP tag, or the absence of a tag (B-D).  PIKE-S staining is usually nuclear (A) with occasional cytoplasmic tubule staining (A, arrow).  Co-transfection with PIKE induces BRAG2 nuclear patches, which are more pronounced for GFP than HA tagged or un-tagged BRAG2 (E-H). This appears to be at least partly due to poor antibody penetration. In particular, nucleolar HA-BRAG2a appears to be more easily detected when using the anti-HA antibody than the anti-BRAG2a antibody (compare F to G).  Bar is 1µm. Images are representative of at least three separate experiments.

Figure 1 (jpg)

Supplemental Figure 2: Nucleolar BRAG2 alters the localization of endogenous fibrillarin HeLa cells were transfected with GFP-fibrillarin (A) or stained for endogenous fibrillarin after mock transfection (B), or transfection with BRAG2a-GFP and myc-PIKE-S (C).  Endogenous fibrillarin is also shown in a PC12 cell after transient transfection with BRAG2a-GFP and myc-PIKE-S (D). PIKE is not shown in triple transfections and the PC12 image is deconvolved. Both GFP-fibrillarin (A) and endogenous fibrillarin (B) normally show a punctate pattern of staining that is evenly distributed throughout nucleoli. However, after co-transfection with BRAG2a-GFP and myc-PIKE-S endogenous fibrillarin in both HeLa (C) and PC12 cells (D) becomes restricted to the periphery of nucleoli. Each image is representative of at least three separate experiments. Bar is 1µm.

Figure 2 (jpg)

Supplemental Figure 3: Expression of the N-terminus of PIKE induces concentration of coilin in nucleoli HeLa cells were transiently transfected with GFP-coilin (A-G), and either full-length PIKE-S (B-C) or the partial PIKE constructs, myc-PIKE 1-384 (D), 1-669 (E), 268-753 (F), and 670-1186 (G).  Where triple-transfection with HA-BRAG2a was performed (C) PIKE is not shown.  GFP-coilin alone (A) localizes to the nucleoplasm, CB (B, inset) and faintly to nucleoli.  PIKE expression promotes nucleolar accumulation of coilin (B) where coilin co-localizes with BRAG2 (C).  The N-terminus of PIKE alone is sufficient (D) and required (F-G) to induce this localization of coilin.   Bars are 1µm. Images are representative of at least three separate experiments.  Asterisks indicate non-transfected cells.

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Supplemental Figure 4: PIKE, BRAG2 & SMN co-localize on cytoplasmic tubules HeLa cells were transiently transfected with myc-PIKE-L (A), PIKE–S (C-D) or PIKE 1-384 (B), and BRAG2a-GFP (A), HA-BRAG2a 1-250 (B), or GFP-SMN (C).  BRAG2a and PIKE-L were often found to co-localize on cytoplasmic tubules (A, plus inset).  A similar phenomenon was observed with the N-termini of PIKE (1-384) and BRAG2a (1-250; B plus inset).  Expression of PIKE-S did not obviously alter the nuclear localization of SMN (C).  However the presence of cytoplasmic PIKE-S appeared to induce co-localization of SMN with PIKE on cytoplasmic tubules (C, plus inset).  Staining for endogenous tubulin-a suggests that the cytoplasmic PIKE tubules are aligned along microtubules (deconvolved images shown in D, plus inset).  Bars are 1mm. Each image is representative of at least three separate experiments. The GFP-SMN construct was obtained from Angus Lamond (University of Dundee, [30]).

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