JCS009092 Supplementary Material

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• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. Alignment of Homo sapiens (hs) RCC1 exon 6′ with the corresponding Mus musculus (mm) genomic sequence. The predicted splice donor and acceptor sites are indicated in bold. The dashed line indicates the splice site that generates RCC1γ.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. Densitometry to quantify relative RCC1 isoform protein levels in U2OS and HeLa cells. (A) SDS lysates of asynchronously growing (A), hydroxyurea-treated (HU), and Nocodazole-treated (Noc) cells that were either adherent (G2) or rounded-up (M), were analysed by SDS-PAGE and immunoblotting. ECL signal was detected using a Fuji LAS-3000 mini luminescence analysis system. Areas of equal volume (shown in blue) were selected manually around the bands of interest, and the signal intensity quantified using AIDA software (Raytest). Several background readings were taken as indicated. (B) Calculated ratios of RCC1γ/RCC1α, normalised to background.

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. The effect of importins on guanine nucleotide exchange activity of recombinant RCC1 isoforms. Untagged recombinant RCC1α, RCC1γ or GST were used to catalyse exchange of 8-H³GDP on Ran, in the presence or absence of GST-importin β and His6-importin α3. Values are means of four experiments ± s.d.

• Supplemental Figure S4 (Adobe PDF) -

  Fig. S4. Phosphorylation of exogenous RCC1 isoforms expressed in U2OS cells. Phosphorylation at serine 11 of RCC1α-GFP, RCC1γ-GFP (with a C-terminal GFP tag) or pEGFP.N1 as a control, transiently expressed in cells. Cells were arrested in mitosis (M) or allowed to grow asynchronously (A). Similar results were observed with fusions containing N-terminal GFP.

• Supplemental Figure S5 (Adobe PDF) -

  Fig. S5. Phosphorylation of recombinant RCC1 proteins in extracts. (A) Phosphorylation at serine 11 of GST-RCC1α, GST-RCC1γ or GST as a control, incubated in mitotic (M) or asynchronous (A) HeLa extracts. Two different exposures are shown for each blot. (B) Phosphorylation at serine 11 of untagged RCC1α, RCC1γ or GST as a control, incubated in mitotic (M) or asynchronous (A) HeLa extracts. Two different exposures of the anti-RCC1 blots are shown to allow both endogenous and exogenous proteins to be compared.

• Supplemental Figure S6 (Adobe PDF) -

  Fig. S6. Endogenous and exogenous RCC1 expression in lysates of tsBN2 cells from a cell proliferation assay as shown in Fig. 6. The days spent at the restrictive temperature (39.7°C) are indicated. pEGFP.C3 vector only was used as a control (c). As control samples had too few cells for analysis by immunoblotting after 1 day at the restrictive temperature, the ‘0 days’ control was used for comparison on all blots.