JCS02711 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Expression levels of Sprouty2/4 in 293T cells transfected with varying amounts of Sprouty2/4 expression plasmid, and in Swiss 3T3 cells stimulated with FGF-2 for 3 hours. 293T cells were transfected with various amounts of expression plasmid encoding Flag-tagged Sprouty2 or Flag-tagged Sprouty4 as indicated. Swiss 3T3 cells were serum-starved for 24 hours and then stimulated with 20 ng/ml of FGF-2 for 3 hours. Total cell lysates (50 mg protein) were subjected to immunoblot analysis with anti-Sprouty2/4 antibody or anti-actin antibody (loading control). The relative intensity of Sprouty2/4 band, as compared with that of the respective actin signal, was determined by using the Multi Gauge software and normalized to 1.00 for FGF-2-stimulated Swiss 3T3 cells.

• Supplemental Figure 2 -

  Fig. S2. Expression of four Sprouty isoforms in various human tumor cells. Semi-quantitative RT-PCR (25 cycles) was performed with ThermoScript^TM RT-PCR system (Invitrogen) using 2 mg of total RNA isolated from OVCAR3 (ovarian carcinoma), Colo201 (colon carcinoma), HT1080 (fibrosarcoma), KMP4 (pancreatic carcinoma) or T98G (glioblastoma) cells. Sets of primers used were 5¢-TGCTCCCAGGACCCTACC-3¢ and 5¢-TACCCTGACCCCGGGAGG-3¢, Sprouty1 (372 bp); 5¢-GCTCAGAGTGGCAACGGG-3¢ and 5¢-CACCTGGCTTGAGCTCAG-3¢, Sprouty2 (472 bp); 5¢-GATCAAAGGCTCTTGGCC-3¢ and 5¢-AGTGGAGCAGTGGTAGAA-3¢, Sprouty3 (315 bp); 5¢-CAGCCTGATGCTCAGCCC-3¢and 5¢-CGCCCGCTGAAGGAGATC-3¢, Sprouty4 (360 bp) and 5¢-CCACCCATGGCAAATTCCATGGCA-3¢and 5¢-TCTAGACGGCAGGTCAGGTCCACC-3¢, GAPDH (598 bp) (an internal control). The amplified products were separated on 2% agarose gels.

• Supplemental Figure 3 -

  Fig. S3. siRNA knockdown of Sprouty4 in Swiss 3T3 cells. Swiss 3T3 cells were transfected with Sprouty1 siRNA (1), Sprouty4 siRNA (4), Sprouty1 siRNA and Sprouty4 siRNA in combination (1+4), or matched scrambled RNAs to Sprouty1 siRNA and Sprouty4 siRNA in combination (C). After 24 hours, cells were serum-starved for 24 hours and then stimulated with FGF-2 (20 ng/ml) for 3 hours. Total cell lysates (50 mg protein) were subjected to immunoblot analysis with anti-Sprouty4 antibody or anti-actin antibody (loading control). The relative intensity of Sprouty4 band, as compared with that of the respective actin signal, was determined by using the Multi Gauge software and normalized to 1.00 for scramble RNA-transfected control cells.

• Supplemental Figure 4 -

  Fig. S4. Proposed model of Sprouty1/4 homo-/hetero-oligomer interaction with Grb2/Sos1. In this model, Sprouty proteins are assumed to form homo-/heterodimers through their C-terminal domains. Sprouty1 homodimer binds to and sequesters free Grb2 and Grb2-Sos1 complex, and Sprouty4 homodimer binds to and sequesters free Sos1 and Grb2-Sos1 complex. These interactions suppress the reciprocal association of Grb2 and Sos1 and further considerably reduce the association of Grb2-Sos1 complex with other signaling molecules such as FRS2. On the contrary, the heterodimer formed between Sprouty1 and Sprouty4 binds to and sequesters free Grb2, free Sos1 and Grb2-Sos1 complex, which would result in a more efficient blockade of the interaction between Grb2-Sos1 complex and FRS2. This model also explains why anti-Grb2 antibody failed to immunoprecipitate Sprouty4, and anti-Sos1 antibody failed to immunoprecipitate Sprouty1 (Fig. 2A): because of the topological orientation of each protein, epitope(s) recognized by the anti-Sos1 antibody or anti-Grb2 antibody may be masked in the Grb2-Sos1-Sprouty1 complex or the Sos1-Grb2-Sprouty4 complex, respectively.