Supplemental Materials

This article contains the following supporting material:

• Figure S1 - Effect of ARNO and ARNO(E156K) on GTPγS binding to ARL4D and ARF6. The assays were carried out in a rapid filtration system as described in Materials and Methods. (A) Samples of recombinant ARL4D (1 µg) and ARF6 (1 µg) were incubated at 30°C for 40 min without or with 1 µg each of ARNO or ARNO(E156K) in the presence of 0.2 µg/µl phosphatidylserine, 1 mM EDTA, 2 mM MgCl₂, 4 µM GTPγS (2x10⁶ cpm of ³⁵SGTPγS). (B) Time course of GTPγS binding to ARL4D and ARF6. One µg ARF6 or ARL4D was incubated for the indicated time at 30°C with 1 µg ARNO. Data are the means of values from triplicate assays plus/minus one-half the range. Data are representative of at least two separate experiments with different protein preparations. Error bars smaller than symbols are not shown.
• Figure S2 - Specificity of ARL4D antibody. (A) Alignment of deduced amino acid sequences of human ARL4A, ARL4C, ARL4D, ARF1 and ARF6. Accession numbers are provided from a previous report (Kahn et al., 2006). The name ARL4B was originally given to a pseudogene and therefore is not used for the nomenclature of this family. Underlines indicate peptides immunogens used to generate ARL4D-specific antibodies. Peptide N and peptide B correspond to a.a. 2-18 (GNHLTEMAPTASSFLPC) and a.a. 139-155 (QPGALSAAEVEKRLAVR) of ARL4D, respectively. Alignments were performed with Clustal_W 1.83. Symbol "*" indicates the residues are identical; ":" and "." conserved and semi-conserved substitutions. (B) Specificity of antibody against ARL4D. ARL4D antibodies were generated by immunization of rabbits with ARL4D-B peptide. Approximately 50-100 ng of the indicated purified recombinant His-tagged ARFs/ARLs were subjected to immunoblot analysis with anti-ARL4D (upper panel) or anti-His tag (lower panel) antibodies. (C) Detection of endogenous ARL4D protein by immunoblot analysis using ARL4D antibody. Cell lysate of ARL4D transfected HeLa cells was used as a positive control. α-Tubulin expression in different cell lysates was also analyzed to ensure that an equal amount of protein from each cell line was used.
• Figure S3 - Subcellular localization of ARL4D and its mutants in COS-7 Cells. COS-7 cells were transiently transfected with plasmids encoding ARL4D or its mutants, fixed, stained with anti-ARL4D-B antibody and visualized by confocal microscopy. The Z stack images of several focal planes are shown. Bar, 10 μm.
• Figure S4 - ARL4D(Q80L) promoted ARNO translocation. COS-7 cells were transiently transfected with ARNO only (lane 1) or with ARNO and ARL4D(Q80L) (lane 2). Cells were collected and treated with DSP. Subcellular fractionation was performed using a CNM compartment protein extraction kit and cytosolic fraction (C) and membrane fraction (M) were analyzed for the amount of FLAG-ARNO by immunoblotting.
• Figure S5 - ARL4D(Q80L) did not induce ARNOΔCT to redistribute to the plasma membrane. (A) COS-7 cells were transiently transfected with plasmid encoding ARNO lacking the C-terminal PH domain and polybasic sequence (FLAG-ARNOΔCT) alone or cotransfected with the ARL4D(Q80L), fixed and stained with ARL4 and FLAG antibodies and visualized by confocal microscopy. (B) ARL4D(Q80L) induces redistribution of ARNO C-terminus (ARNOCT) to the plasma membrane. COS-7 cells were transiently transfected with plasmid encoding FLAG-ARNOCT alone or cotransfected with the active form of ARL4 and FLAG-ARNOCT and then subjected to immunostaining. (C) ARL4D(Q80L) induces redistribution of ARNOC7A to the plasma membrane. Bars, 10 μm. (D) The effect of ARL4D(Q80L) to recruit ARNOPH to plasma membrane was decrease. (E) Quantification of translocation of ARNO mutants to plasma membrane in ARL4D(Q80L)-expressing cells. The method for quantifying the ARNO plasma membrane localization is described in Materials and Methods. Results are the means ± SEM of two independent experiments. Bars, 10 μm.
• Figure S6 - ARL4D(Q80L) induces redistribution of ARNO(E156K) to the plasma membrane. COS-7 cells were transiently transfected with FLAG-ARNO(E156K) alone (A) or co-transfected with ARL4D(Q80L) (B). Cells were fixed, permeabilized, and labeled with anti-FLAG and anti-ARL4D. Bars, 10 μm.
• Figure S7 - Active form of ARL4D induces redistribution of other members of the cytohesin family to the plasma membrane. COS-7 cells were transfected with FLAG-tagged cytohesin family member alone or co-transfected with the ARL4D(Q80L) (A, cytohesin-1; B, cytohesin-3; C, cytohesin-4). Cytohesin and ARL4D were detected with FLAG M2 and ARL4D antibodies, respectively. Bars, 10 μm.
• Figure S8 - Active forms of ARL4A or ARL4C induce redistribution of ARNO to the plasma membrane. COS-7 cells were transfected with ARL4A and its mutants (A) or myc-tagged ARL4C and its mutants (B) alone, or co-transfected with FLAG-tagged ARNO. ARNO and ARL4A or ARL4C were detected with FLAG M2 and ARL4A(Lin et al., 2000) or myc antibodies, respectively. Cell images were visualized by fluorescence microscopy. Bars, 10 μm.