JCS0121774 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. Genomic organization of H. sapiens BNIPXL at chromosome 9q21.2. Dotted lines indicate exons spliced to form BNIPXLα (upper) and BNIPXLβ (lower), respectively. Nucleotide numbers and the corresponding exon numbers are indicated.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. Phylogenetic relationships of the BNIP2 family BCH domains and the yeast Sec14p lipid-binding domain. The similarity between H. sapiens BNIPXL, BNIP2, BNIP-S, BNIP-H, BPGAP1 and the S. cerevisiae Sec14p lipid-binding domain were analysed by ClustalW alignment using the PHYLIP algorithms. A distance bar is shown.

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. Expression profile of BNIPXL isoforms. Full-length (first panel) and BCH domain (second panel) was amplified with appropriate primers in (1) adult human brain, kidney and liver cDNA libraries and (2) different human cell lines, as indicated. BNIPXL plasmid controls and GAPDH expression was analyzed for size comparison and sample normalization, respectively. Results are representative of at least two separate determinations.

• Supplemental Figure S4 (Adobe PDF) -

  Fig. S4. Multiple sequence alignments of BNIPXL with the BNIP-S Rho-binding domain and the Class I Rho-binding motifs from RhoA effectors. Identical residues in all members are shaded black, residues that are conserved in most of the members are shaded dark gray whereas the significant but least-conserved residues are light gray.

• Supplemental Figure S5 (Adobe PDF) -

  Fig. S5. BNIPXL deletion mutants retain cytosolic distribution. HeLa cells expressing FLAG-BNIPXL full-length and deletions were processed and visualized by confocal fluorescence microscopy. Scale bars: 50 µm.

• Supplemental Figure S6 (Adobe PDF) -

  Fig. S6. BNIPXL and BMCC1 binds to proto-Lbc. Lysates expressing Myc-proto-Lbc with FLAG-BMCC1 or WBCH were immunoprecipitated and bound proteins detected with anti-Myc (first panel) and anti-FLAG (second panel) to show the amounts of precipitated proteins. Expression of Lbc (third panel) and BNIPXL (fourth panel) was verified. Asterisks indicate the expected bands. Arrows indicate the non-specific bands arising from probing with anti-Myc.

• Supplemental Figure S7 (Adobe PDF) -

  Fig. S7. BNIPXL also interacts with p115-RhoGEF, another RhoA-specific GEF bearing the DH-PH domain. Lysates expressing Myc-p115-RhoGEF and FLAG-BNIPXL full-length, WBCH or CBCH were subjected to immunoprecipitation and bound proteins detected with anti-Myc (first panel) and anti-Flag (second panel) to show the amount of precipitated protein. Expression of Lbc (third panel) and BNIPXL (fourth panel) was verified. Asterisks indicate the bands of interest.

• Supplemental Figure S8 (Adobe PDF) -

  Fig. S8. The BNIP2 family BCH domains and the Sec14p-like domains of selective Dbl family GEFs share conserved regions. Multiple sequence alignments of the BNIPXL and BNIP2 BCH domains, the S. cerevisiae Sec14p lipid-binding domain and the Sec14p-like domains from Dbl, Ost and Dbs RhoGEFs using ClustalW with default parameters. Residues that are identical are shaded black, whereas those that are similar or conserved are light gray.