JCS03076 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Palladin staining of shRNAi-transfected cells. A7r5 cells transfected with pSuper RNAi targeting palladin were plated on fibronectin overnight and serum-starved for 2 hours prior to PDGF treatment. Cells were fixed, permeabilized and stained with polyclonal anti-palladin antibody. Transfected cells (green fluorescence) were detected by the presence of GFP encoded in the pSuper vector.

• Supplemental Figure 2 -

  Fig. S2. Western blot analysis from cytosolic extracts of Eps8 null mouse embryonic fibroblasts (MEF Eps8 −/−) and rescued MEF (MEF Eps8 −/− + myc-Eps8). Blots were probed with anti-Eps8, anti-tubulin and anti-actin antibodies.

• Supplemental Figure 3 -

  Fig. S3. Abi-1 binding to Sos-1 and Eps8 is unaffected by palladin overexpression or palladin knockdown. Purified GST or GST fusion protein containing the region of Abi-1 that binds to Eps8 and Sos-1 (GST-E2) (aa 33-480) were incubated with lysates from palladin-overexpressing (myc-palladin) and palladin-knockdown (KD) HeLa cells. Myc empty vector and control siRNA were used as negative controls, respectively. Protein complexes, recovered by agarose-bound glutathione, were analyzed by immunoblotting with anti-Sos-1 antibody (upper panel), anti-eps8 antibody (middle panel) or anti-GST antibody (lower panel). (A) Total cell lysates. (B) Pull down assays.

• Movie 1 -

  Movie 1. Dynamics of GFP-palladin in A7r5 cells after 5 minutes of PDGF stimulation.

• Movie 2 -

  Movie 2. Phase-contrast movie of GFP-palladin transfected A7r5 cells showed in Movie 1.