JCS075705 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Trichoplein is also localized on desmosomes and keratin IFs. (A,B) Localization of trichoplein (Tricho.; green) and desmoplakin (DP) 1 (red) in mouse small intestine. Nuclei were also stained with DAPI (D; blue). (C) Calcium switch experiments. Cells were treated as described in the legend to Fig. 1D. Treated cells were subjected to immunocytochemistry with anti-Odf2 and anti-desmoplakin (DP) 1/2 at the indicated time after switching. Arrowheads indicate the centrosome position. (D) Trichoplein localization in HeLa cells. HeLa cells were stained with antibodies to pan-keratin or trichoplein. High magnification views are also indicated in insets. Arrowhead indicates a centrosome. Scale bar: 10 µm.

• Supplemental Figure S2 -

  Fig. S2. Complex formation between His-tagged trichoplein and GFP-tagged Odf2β/ninein in COS7 cells. COS7 cells transfected with vectors carrying indicated proteins were subjected to immunoprecipitation with anti-GFP (A), or affinity chromatography with Ni-NTA-agarose (B).

• Supplemental Figure S3 -

  Fig. S3. Interaction between trichoplein and Odf2β/ninein. (A) Yeast two-hybrid interaction of trichoplein with keratin-18 (K18), ninein, or Odf2β. (B) GST pull-down assays. 1, MBP + GST-ninein; 2 and 5, MBP-trichoplein + GST; 3, MBP-trichoplein + GST-ninein; 4, MBP + GST-Odf2β; 6, MBP-trichoplein + GST-Odf2β.

• Supplemental Figure S4 -

  Fig. S4. Effects of trichoplein depletion on cell cycle profile. HeLa cells were treated with control (Cont.) or human trichoplein-specific siRNA (Sq. 4 or 2) for 48 hours. Treated cells were subjected to immunoblotting (left) or FACS analysis (right). As a loading control, cell lysates were also immunoblotted with anti-p38 MAPK (left). For FACS analysis showing the DNA content in each group, about one million treated cells were collected by trypsinization, resuspended in buffer solution (CycleTEST^TM PLUS kit; Becton-Dickinson, San Diego, CA) and stored at −80°C. Then, we treated cells according to the manufacturer�s protocol (CycleTEST^TM PLUS kit) and analyzed them using a Becton-Dickinson FacsScan and CellQuest software. The percentage of G0−G1, S, or G2−M phase in each group is indicated in the box. We obtained a similar FACS profile with Sq. 4 (data not shown).