JCS016964 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 - Adobe PDF

• Supplemental Table S2 - Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. Immunostaining of human tissue specimens with ORP3 antibodies. Immunohistochemical staining of (A) kidney, (B) kidney stained with anti-ORP3 preincubated with the immunizing peptide, (C) colon and (D) testis. (E) Visualization of cholesterol crystals (white structures) and ORP3 in a coronary artery lesion. (F) Immunofluorescence staining of coronary artery lesion for ORP3 (green) and CD68⁺ (red), an overlay in which colocalization of ORP3 and CD68 is visualized in yellow. Human tissue samples were fixed in 10% neutral-buffered formalin immediately after excision, dehydrated, embedded in paraffin and sectioned to 4 µm sections. For light microscopy the primary antibodies were detected using avidin-biotin complex system (Vectastain ABC Elite kit, Vector Laboratories). 3,3′-diamino-benzidine (Sigma-Aldrich) was used as chromogen and sections were counterstained with Mayer’s hematoxylin. For immunofluorescence, ORP3 was stained with rabbit anti-ORP3 and CD68 with a monoclonal antibody. The primary antibodies were visualized with fluorochrome-conjugated goat anti-rabbit and goat anti-mouse antibodies (Molecular Probes). Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma). Specificity of the immunostainings was ensured by replacing the primary antibodies with equivalent concentration of an isotype-matched nonimmune immunoglobulin, by pre-incubation of primary antibody with the immunizing peptide, or by replacing the primary antibody with PBS. Stained sections were photographed with a digital camera (Spot RT color operated with Spot advanced software, version 4.1, Diagnostic instruments, Sterling Heights) attached to Nikon Eclipse E600 microscope.

• Supplemental Figure S2 -

  Fig. S2. The human ORP3 cDNA constructs. Pleckstrin-homology (PH) and OSBP-related ligand-binding domains are indicated with blue and red, respectively. The mutated PH domain mPH2 (K59I, K60A, K70A, R71I) is denoted with yellow stars. Wild-type FFAT-motif (EFFDA), located at amino acids 449-453, is shown as a black bar. The mutated FFAT-motif (EVVVA) is indicated by a green diamond. The numbers indicate amino acid positions.

• Supplemental Figure S3 -

  Fig. S3. Effects of PMA and okadaic acid treatments of HEK293 cells. HEK293 cells were cultured on fibronectin-coated coverslips in normal medium for 20 hours. Adherent cells were treated with 100 nM phorbol myristate acetate (PMA) or 50 nM okadaic acid (OKA) for 1 hour. After fixing, the cells were stained with anti-ORP3 and anti-cortactin (lamellipodium marker) antibodies. ORP3 and cortactin-positive end of protrusion (arrows). ORP3-negative, but cortactin-positive lamellipodium (arrowheads). For each condition, high- and low-magnification views are shown. NT, non-treated cells. Scale bars: 20 µm.

• Supplemental Figure S4 -

  Fig. S4. Treatments of HEK293 cells with EGTA or the anti-E-cadherin antibody. Confluent HEK293 cells in normal growth medium were treated for 30 minutes with 5 mM EGTA (EGTA) or the anti-E-cadherin antibody (DECMA-1; 50 µg IgG/ml). Cells were stained using an antibody against β-catenin (β-CAT). NT, non-treated cells. Scale bars: 20 µm.