JCS003251 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Phosphorylation of Ser283 within tropomyosin-1 promotes formation of actin stress fibers in MDA MB 231 cells. Parental MDA MB 231 cells (A,B) or MDA MB 231 cells stably expressing tropomyosin-1 S283A (C,D) or tropomyosin-1 S283E (E,F) mutant proteins were left untreated (A,C,E) or treated with H₂O₂ (250 μM) for 30 minutes (B,D,F). Thereafter, cells were fixed and stained for F-actin using FITC-phalloidin. (G) Quantification of the above experiments. Cells expressing trans-cytoplasmic actin stress fibers or membrane blebs were counted and the ratio calculated in comparison with the total number of counted cells. (H) Extracts of the cells from the above experiment were made and were processed for immunodetection of tropomyosin (upper panel) and actin (lower panel) using specific antibodies: C, parental MDA MB 231 cells; A, MDA MB 231 cells stably expressing tropomyosin-1 S283A; E, MDA MB 231 cells stably expressing tropomyosin-1 S283E.

• Supplemental Figure S2 -

  Fig. S2. Phosphorylation of tropomyosin-1 impairs DAP-kinase-mediated apoptosis. (A) HeLa cells were left un-transfected or were co-transfected with plasmids expressing constitutively activated FLAG-tagged DAP kinase together with empty vector or plasmids expressing wild-type tropomyosin-1 (wt TM) or mutant tropomyosin-1 S283A (A) or S283E (E). Six days later, floating and adherent cells were pooled and the proteins were extracted. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was processed for immunodetection of cleaved caspase-3 (upper panel), FLAG-DAP kinase (second panel) and actin as a loading control (lower panel).