JCS027482 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. CD spectra of the wild type and FLF mutant. The unit of mean residue ellipticity is deg ^. cm^{2 .} dmol^{-1 .} residue^-1. The protein concentration was 20 µM. The spectra were acquired in 0.1-cm-pathlength cells. The conditions were 50 mM sodium phosphate, pH 7 at 25°C.

• Supplemental Figure S2 -

  Fig. S2. HSQC spectra of the wild-type SAM and FLF mutant. The ¹⁵N-¹H correlation peaks are shown in black and red for the wild-type SAM and FLF mutant, respectively. The same buffer was used for the two samples. Representative residues distant in sequence and space from the mutated region are highlighted with blue circles (T26, G27, D55, A58, C63, R65 and K76). Mutation does not change the chemical shifts of these residues, indicating that the global structures of the mutant and wild-type protein are the same. Several representative residues close to the mutated region in both sequence and space are highlighted with green rectangles (Y35, E36, I42 and I44). Representative residues distant in sequence but close in space to the mutated region (Q30, L66, N70 and A73) are highlighted with red triangles.

• Supplemental Figure S3 -

  Fig. S3. SAM domain of DLC1 is not required for DLC1 induction of cell protrusions. HeLa cells were transfected for 20 hours with GFP expression plasmids for full-length DLC, DLC SAM, and DLC ΔSAM. Morphological changes and cytoskeletal rearrangements were revealed by direct staining with rhodamine-conjugated phalloidin for actin filaments. GFP-fusion proteins were directly detected by green fluorescence and GFP vector was used as negative control.

• Supplemental Figure S4 -

  Fig. S4. EF1A1 knockdown sensitizes DLC1-mediated changes in cellular morphology. (A) HEK293T cells were transfected with different constructs of pSilencer2.1-U6 hygro plasmids for EF1A1 (see below), and screened for clones that either failed to knockdown (sh-22 and sh-32) or conferring successful knockdown (sh-42) by analyzing the expression of Flag-tagged EF1A1 as the target. (B) HeLa cells were co-transfected for 20 hours with 0.5 µg Flag-tagged full-length DLC1 or DLC1-R677E, together with 1 µg pSilencer2.1-U6 hygro plasmids for EF1A1 and 0.3 µg GFP vector as the tracer/marker to indicate transfected cells. Morphological changes and cytoskeletal rearrangements were revealed by direct staining with rhodamine-conjugated phalloidin for actin filaments and cell borders. The three short hairpin RNA (shRNA) oligonucleotides against human EF1A1 were designed using the siRNA target finder (Ambion), synthesized (Operon) and cloned into the pSilencer 2.1-U6 hygro vector (Ambion). The shRNA sequences are: EF1A1seq2: 5′-GATCCATGCGGTGGCATCGACAAACTCGAGTTTGTCGATGCCACCGCATTTTTTTGGAAA-3′; EF1A1seq3: 5′-GATCCGTATGCCTTGGTTCAAGGGACTCGAGTCCCTTGAACCAAGGCATATTTTTTGGAAA-3′; EF1A1seq4: 5′-GATCCGCGTGTCTGTCAAAGATGTCCTCGAGGACATCTTTGACAGACACGTTTTTTGGAAA-3′. Constructs were sequenced to confirm sequence fidelity and knockdown efficiency was determined by co-transfection with Flag-EF1A1 plasmid at a 10:1 ratio. Lysates were analyzed 72 hours after transfection.