JCS042325 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Amino acid sequences of murine EFA6A and EFA6As and schematic representation of the EFA6A gene. (A) Amino acid sequence of murine EFA6A; the putative proline-rich, Sec7, PH and coiled-coil domains are underlined. The 327 amino acids identical in EFA6As are indicated in bold. (B) Amino acid sequence of the N-terminal 66 amino acids of EFA6As. (C) Schematic representation of the intron-exon structure of EFA6A; the first exon of EFA6As (9′), encoding the N-terminal 66 amino acids, is contained in a large intron, of 3.4 kb, between exons 9 and 10 of EFA6A; the internal promoter is indicated by the arrow.

• Supplemental Figure S2 -

  Fig. S2. Morphology of cortical neurons transfected with the indicated constructs. Primary cortical neurons were transfected with the indicated constructs, and analyzed by immunofluorescence as described in the Materials and Methods. In cotransfected cells (EFA6A/ARF6T27N, EFA6A/Cdc42N17, EFA6A/Rac1N17, EFA6A/RhoAV14) the label in parenthesis indicates the fluorescence signal visualized in the picture. Antibodies: anti-FLAG (EFA6A, 375, DC, EFA6As, GEF); anti-Myc (ARF6Q67L, ARF6T27N), MAP2. EGFP-tagged Cdc42N17, Rac1N17 and RhoAV14 are visualized by EGFP fluorescence. Scale bar: 20 µm.

• Supplemental Figure S3 -

  Fig. S3. Morphology of cortical neurons cotransfected with EFA6As and Cdc42N17, Rac1N17, RhoAV14. Primary cortical neurons were cotransfected with EFA6As and EGFP or EGFP-tagged Cdc42N17, Rac1N17 and RhoAV14, and analyzed by immunofluorescence as described in the Materials and Methods. EFA6As: anti-EFA6As polyclonal antibody; EGFP: anti-GFP monoclonal antibody. Scale bar: 20 µm.

• Supplemental Figure S4 -

  Fig. S4. Morphology of cortical neurons transfected with siRNAs specific for EFA6A or EFA6As. Primary cortical neurons were cotransfected with EGFP and the indicated siRNAs as described in the Materials and Methods. Transfected neurons were identified using anti-GFP polyclonal antibodies and anti-MAP2 antibodies. Scale bar: 20 µm.

• Supplemental Figure S5 -

  Fig. S5. Morphology of cortical neurons cotransfected with EFA6A and EFA6As. Primary cortical neurons were cotransfected with Myc-tagged EFA6A and FLAG-tagged EFA6As, and analyzed by immunofluorescence as described in the Materials and Methods, using anti-EFA6As polyclonal antibody and anti-myc monoclonal antibody. Scale bar: 20 µm.

• Supplemental Figure S6 -

  Fig. S6. Expression of EFA6A and EFA6As polypeptides in mouse brain. Protein extracts were loaded on 8% SDS-polyacrylamide gels, and transferred to PVDF membrane. The membrane was incubated with polyclonal anti-EFA6A (raised against a 13 amino acid sequence of the C-term region, identical in EFA6A and EFA6As) at 0.5 µg/ml, followed by ECL^TM anti-rabbit IgG Horseradish Peroxidase linked (Amersham) at 1:6000 dilution. Liver, Kidney, Brain: protein extracts from 6-week-old CD-1 mouse tissues; EGFP: EGFP-transfected HeLa cells; EFA6A: EFA6A-transfected HeLa cells; EFA6As: EFA6As-transfected HeLa cells. Arrow, EFA6A; arrowhead, EFA6As. Using these primary and secondary antibody dilutions, the high molecular mass bands detected in kidney and in liver (see Fig. 1D) are not revealed.