DEV02494 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Alignment of nematode FOZI-1 proteins. (A) Overall comparison of nematode FOZI-1 proteins. Ce, C. elegans; Cb, C. briggsae; Cr, C. remanei. Homologs were identified by BLAST searches. (B) Sequence alignment of nematode FOZI-1 proteins. Bars indicate degree of sequence identity.

• Supplemental Figure 2 -

  Fig. S2. FOZI-1 dimerizes but does not cap barbed end of actin filaments. (A) FH2^FOZI-1 does not cap the barbed end of actin filaments. The capping assay was performed as described previously (Copeland et al., 2004). FH2^FOZI-1 (0.4 μM) or GST (0.4 μM) was added to the indicated concentrations of pre-polymerized actin filaments in separate reactions. The reactions were allowed to reach equilibrium and fluorescence was measured to determine total F-actin. The presence of FH2^FOZI-1 does not affect the critical concentration (∼0.1 μM), indicating that it does not cap the barbed end. Fluorescence (excitation 365 nm/emission 407 nm) was measured in a Spectromax M2 (Molecular Devices). (B) FH2^FOZI-1 forms an apparent dimer. FH2^FOZI-1 was subjected to gel filtration chromatography. FH2^FOZI-1 (41 kDa) eluted at 12.76 ml, while BSA (66 kDa) eluted at 14.53 ml. The relative mobility of FH2^FOZI-1 in comparison with the BSA standard suggests that FH2^FOZI-1 exists primarily as a homodimer. Methods are as follows: 1 ml BSA (1.0 mg/ml) (Sigma, A6003) or 1ml isolated FH2^FOZI-1 (1.0 mg/ml) were run on an S200 10/300 gel filtration column in 20 mM Tris (pH 7.0), 150 mM NaCl, 1 mM EDTA, 1 mM DTT at a flow-rate of 0.5 ml/minute using an AKTA Purifier FPLC system (GE Biosciences). The elution profile was followed by OD280. Identity of the eluted proteins was confirmed by SDS-PAGE.