Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - Specificity of the anti-MAYP antibody used for affinity purification and immunofluorescence staining. (A). Proteins from subcellular fractions (Yeung and Stanley, 1990) (Cy, cytosol, 110μg/lane; M, NP-40-soluble membrane, 50μg/lane; Csk, cytoskeleton (NP-40-insoluble membrane), 25μg/lane; N, nuclei, 20μg/lane) of BAC1.2F5 macrophages incubated with (+) or without (-) CSF-1 for 1min at 37°C were resolved by 5-15% gradient SDS-PAGE, transferred to PVDF and immunoblotted with α –MAYP-C. Arrows indicate the MAYP band. (B). After stripping, the membrane was reprobed with α –MAYP-C that had been absorbed with solubilized nuclear extract (Nα –MAYP-C). (C). Immunofluorescence staining of MAYP in BAC1.2F5 macrophages using α –MAYP-C (C, 1), Nα –MAYP-C (C, 2) and Nα –MAYP-C pre-absorbed with 250 fold molar excess of the peptide antigen (C, 3). Arrows indicate nucleolar staining (Bar, 10 μm).
• Supplemental Figure 2 - Quantitation of the extent of membrane ruffling. Using Image J, the perimeter of each cell was traced to obtain the cell area (C, blue line), together with the perimeters of each ruffle (magenta lines) to obtain the ruffled area per cell (R=sum of areas of ruffles). The percent ruffling for each cell was calculated using the formula (Rx100)/(R+C).
• Supplemental Figure 3 - Effects of GST-MAYP on F-actin organization in vitro. (A). Western Blot of purified, bacterially expressed GST-MAYP probed with antibodies to Nα –MAYP-C?n(left panel) and SDS-PAGE of purified GST-MAYP stained with Coomassie Blue (right panel). (B). In vitro bundling assay. Actin (3μM) was polymerized alone (panels B1 and B4) or with either GST-MAYP (1μM) (panels B2 and B5) or GST (1μM) (panels B3 and B6). The organization of actin fibers was evaluated by electron microscopy at 40,000x magnification. The lower panels (B4-B6) represent a 3x magnification of the boxed areas. Bars, 200nm.
• Supplemental Figure 4 - Distribution of MAYP and EF-1α on actin bundles generated by co-polymerization in vitro. Actin (3μM) was polymerized with either mycMAYP (1μM) (left panel) or EF-1α (1μM) (right panel). The actin fibers were fixed on carbon and Formvar-coated copper grids, stained with anti-MAYP or anti-EF-1?? antibodies and the distribution of MAYP and EF-1α on the actin bundles was evaluated by electron microscopy at 40,000x magnification. Bar, 200 nm.
• Supplemental Figure 5 - Controls for immunodetection of cellular MAYP. (A). Upper panels, staining of BAC1.2F5 macrophages with Nα –MAYP-C and primary (preimmune IgG) and secondary (anti-Rabbit Cy5) antibody controls. Lower panels, phase contrast images fields shown in the corresponding upper panel. (B). Colocalization of myc and MAYP immunofluorescence signals in BAC1.2F5 cells overexpressing myc-tagged MAYP. (C). Immunodetection of MAYP in BAC1.2F5 cells expressing reduced (left upper panel), normal (middle upper panel) and increased (right upper panel) levels of MAYP. Lower panels, phase contrast images of fields shown in the corresponding upper panel.
• Supplemental Figure 6 - Association of mycMAYP with filopodial actin in macrophages. Macrophages overexpressing myc-tagged MAYP were grown on glass coverslips, gently extracted with Triton X-100, fixed and stained with anti-MAYP antibodies and 10 nm gold-conjugated secondary antibodies, rotary shadowed and examined by transmission electron microscopy at 27,000x magnification. Bar, 200 nm.
• Supplemental Figure 7 - Association of mycMAYP with the actin cytoskeleton in macrophages. Macrophages overexpressing myc-tagged MAYP were grown, extracted and fixed as described in the legend to Figure S6 and stained with anti-myc antibodies and 6 nm gold-conjugated secondary antibodies, rotary shadowed and examined by transmission electron microscopy at 27,000x magnification. Bar, 200 nm. (A) secondary antibody control; (B-C), MAYP staining.

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