JCS031591 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. UbcH10 is degraded during anaphase in HeLa cells. Asynchronous HeLa cells were fixed in methanol:acetone, and stained for immunofluorescence with anti-UbcH10 (left) and anti-cyclin B1 (middle) antibodies, and with Hoechst 33342 to visualise the DNA (right). Merged image on the far right. Scale bar, 30 µm.

• Supplemental Figure S2 -

  Fig. S2. UbcH10-Venus is catalytically active but the R114C mutant is not. Nocodazole-arrested HeLa cells expressing or not expressing various forms of UbcH10 were solubilised and immunoblotted with the indicated antibodies before (left and middle panels) or after (right panel) incubating with ubiquitin and an ATP regenerating system. Arrowheads show shift in molecular mass due to binding ubiquitin. Lane 1, untransfected; lane 2, venus alone; lane 3, venus-UbcH10; lane 4, venus-UbcH10^R129A; lane 5, venus-UbcH10-ΔN27; lane 6, venus-UbcH10^C114A. Cleavage products are marked with an asterisk.

• Supplemental Figure S3 -

  Fig. S3. The catalytically inactive mutant of UbcH10 is not degraded in mitosis and a mutation in the putative destruction box destabilizes the protein. G2 phase HeLa cells were microinjected with expression constructs encoding wild type venus-UbcH10 (n=18) or venus-UbcH10^R129A (n=23) (top) or catalytically inactive venus-UbcH10^C114A (n=14) (bottom) and followed by time-lapse fluorescence and DIC microscopy. Cells in the top panel remained in G2 phase throughout filming. Images were taken every 3 minutes, and the total cell fluorescence minus background for each cell measured and plotted against time.

• Supplemental Figure S4 -

  Fig. S4. UbcH10 is overexpressed in transfected cells. HeLa cells were electroporated with a construct encoding UbcH10 with GFP under an IRES (lanes 2), or with the empty IRES-GFP vector (lanes 1). 24 hours after electoporation cells were lysed in SDS sample buffer and immunoblotted with anti-GFP (left panel) or anti-UbcH10 (right panel) antibodies. Results are representative of two independent experiments.

• Supplemental Figure S5 -

  Fig. S5. siRNA oligonucleotides reduce UbcH10 levels by more than 90%. HeLa cells were transfected with 50 nM siRNA oligonucleotides against UbcH10 and samples taken after 36 hours. Samples were immunoblotted with the indicated antibodies and revealed by ECL or by fluorescence and quantified using a CCD camera (Li-COR system, panel i, right). The oligos shown in panel ii were used for the experiments in Fig. 4 and the timing of mitosis in RPE cells.