DEV053801 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Recruitment of ubiquitin to Cx43 gap junction plaques in response to TPA treatment. IAR20 cells were treated with TPA (100 ng/ml) for 15 minutes (A,B), or incubated with TPA (100 ng/ml) and PD98059 (50 µM) for 15 minutes (C). Cells were double-stained with anti-Cx43 antibodies and FK2 anti-ubiquitin antibodies as indicated. Cells were visualized using confocal immunofluorescence microscopy. Merged images are shown in the right panels, with yellow indicating colocalization. Scale bars: 5 µm.

• Supplemental Figure S2 -

  Fig. S2. Ultrastructural localization of Cx43 by immunoelectron microscopy. IAR20 cells were transfected with siRNA against Hrs and Tsg101. After 48 hours of transfection, cells were treated with TPA (100 ng/ml) and cycloheximide (chx, 10 µM) for 1.5 hours. Cells were fixed for immunoelectron microscopy and cryosectioned. Cx43 was detected using 10 nm immunogold particles. PM, plasma membrane; N, nucleus. Scale bars: 200 nm.

• Supplemental Figure S3 -

  Fig. S3. Recovery of gap junctions during prolonged TPA treatment in cells depleted of Hrs and/or Tsg101. IAR20 cells were transfected with control siRNA or with siRNA against Hrs or Tsg101 alone or simultaneously, as indicated. After 48 hours of transfection, cells were treated with TPA (100 ng/ml) and cycloheximide (chx, 10 µM) for 4.5 hours. Cells were stained with anti-Cx43 antibodies and visualized using immunofluorescence microscopy.

• Supplemental Figure S4 -

  Fig. S4. Depletion of Hrs and/or Tsg101 does not affect TPA-induced inhibition of gap junctional communication. IAR20 cells were transfected with control siRNA or with siRNA against Hrs or Tsg101 as indicated. After 48 hours of transfection, cells were treated with TPA (100 ng/ml) and cycloheximide (chx, 10 µM) for 1.5 hours and the level of gap junction communication was determined. Values shown are the mean ± s.e.m. of three independent experiments.