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Results: 7

1.
FIGURE 7.

FIGURE 7. From: The Connecdenn Family, Rab35 Guanine Nucleotide Exchange Factors Interfacing with the Clathrin Machinery.

Interaction model of the distinct mechanisms by which the connecdenn family couple Rab35 activation and binding to AP-2.

Andrea L. Marat, et al. J Biol Chem. 2010 April 2;285(14):10627-10637.
2.
FIGURE 1.

FIGURE 1. From: The Connecdenn Family, Rab35 Guanine Nucleotide Exchange Factors Interfacing with the Clathrin Machinery.

The connecdenn (CD) family. A, alignment of human connecdenn 1/DENND1A (gi55749779), connecdenn 2/DENND1B (gi218563726), and connecdenn 3/DENND1C (gi74750652). Identical amino acids are boxed. The uDENN, DENN, and dDENN modules of the DENN domain are overscored with solid, dotted, and dashed lines, respectively. The outlined gray box indicates conserved AP-2 α-ear platform binding FXDXF motifs. Dark gray shading indicates clathrin-binding boxes and DLL motifs, and light gray shading indicates AP-2 α-ear-binding DPF and WXXF-acidic motifs. B, equal protein extracts (200 μg) of rat tissues blotted with antibodies against connecdenn 1, 2, and 3. Molecular masses are indicated in kilodaltons.

Andrea L. Marat, et al. J Biol Chem. 2010 April 2;285(14):10627-10637.
3.
FIGURE 6.

FIGURE 6. From: The Connecdenn Family, Rab35 Guanine Nucleotide Exchange Factors Interfacing with the Clathrin Machinery.

Knockdown of connecdenn 2 causes an enlargement and redistribution of early endosomes. A, equal lysates from COS-7 cells transfected with a non-targeting siRNA or siRNA targeting connecdenn 2 (siRNA CD2 #1 and #2) were analyzed by Western blot for expression levels of connecdenn 2 (CD2), clathrin heavy chain (CHC), and early endosomal antigen 1 (EEA1). B, COS-7 control and knockdown cells were processed for immunofluorescence to examine the morphology of early endosomes using the marker EEA1, and transferrin uptake at 16 °C, which causes a block in the endocytosis of transferrin at the early endosomes. Staining for AP-2 and the trans-Golgi marker TGN46 reveals no changes to those compartments. Scale bar, 10 μm.

Andrea L. Marat, et al. J Biol Chem. 2010 April 2;285(14):10627-10637.
4.
FIGURE 5.

FIGURE 5. From: The Connecdenn Family, Rab35 Guanine Nucleotide Exchange Factors Interfacing with the Clathrin Machinery.

Connecdenn 2 co-immunoprecipitation and co-localization with AP-2. A, soluble rat brain extract was incubated with protein G-Sepharose alone or protein G-Sepharose precoupled to a monoclonal antibody to α-adaptin (AP.6). Immunoprecipitated proteins were processed for Western blot with the indicated antibodies. Aliquots of soluble brain extract (starting material) equivalent to 1/10th that added to the immunoprecipitation were analyzed in parallel. B, COS-7 cells were transfected at low levels with FLAG-tagged full-length connecdenn 2 and processed for immunofluorescence with a polyclonal antibody against the FLAG-tag (red) and monoclonal antibody against AP-2 (AP.6, green). Arrowheads on higher magnification panels indicate examples of co-localizing punctae. Scale bars: lower magnification, 10 μm; higher magnification, 2.5 μm.

Andrea L. Marat, et al. J Biol Chem. 2010 April 2;285(14):10627-10637.
5.
FIGURE 2.

FIGURE 2. From: The Connecdenn Family, Rab35 Guanine Nucleotide Exchange Factors Interfacing with the Clathrin Machinery.

Generalized GEF activity of connecdenn DENN domains. A, GST, GST-Rab35, GST-Rab3, and GST-Rab6, maintained in the nucleotide-free state with 5 mm EDTA or pre-loaded with GTPγS, were used to affinity-purify FLAG-tagged DENN domain of connecdenn (CD) 1–3 from HEK-293T cell lysates. Specifically bound proteins were detected by anti-FLAG antibody. Cell lysate (starting material) equivalent to 1/10th that added to beads was analyzed in parallel. B, GEF activity of 150 nm purified DENN domains of CD 1–3 or basal exchange activity of Rab35 alone measured as the relative incorporation of GTP-[γ-35S] onto 1.25 μm GDP-loaded Rab35 as a function of time. C, rate of GDP to GTP exchange activity per minute of 150 nm purified DENN domains of CD1 (square), CD2 (triangle), and CD3 (inverted triangle), or DH-PH domains of intersectin long (circle) as a function of concentration of GDP-loaded Rab35.

Andrea L. Marat, et al. J Biol Chem. 2010 April 2;285(14):10627-10637.
6.
FIGURE 3.

FIGURE 3. From: The Connecdenn Family, Rab35 Guanine Nucleotide Exchange Factors Interfacing with the Clathrin Machinery.

Connecdenn 1, 2, and 3 are CCV-associated proteins with unique AP-2-binding properties. A, equal protein aliquots of rat brain subcellular fractionations leading to highly enriched CCVs blotted with the indicated antibodies (homogenate (H), pellet (P), supernatant (S), and sucrose gradient (SG)). B, CCVs were stripped of their coats by two successive rounds of incubation with 0.5 m Tris, stripped CCVs were centrifuged, and the supernatant (S) and pellet (P) fractions were analyzed. C, soluble lysates of FLAG-CD1, FLAG-CD2, and green fluorescent protein (GFP)-CD3 from transfected HEK-293T cells were incubated with equimolar amounts of GST or GST fused to wild-type α- and β2-ear and the indicated α-ear point mutants, precoupled to glutathione-Sepharose beads. Western blotting against the appropriate tag revealed specifically bound proteins. Aliquots of cell lysate (starting material) equivalent to 1/10th that added to the beads were analyzed in parallel.

Andrea L. Marat, et al. J Biol Chem. 2010 April 2;285(14):10627-10637.
7.
FIGURE 4.

FIGURE 4. From: The Connecdenn Family, Rab35 Guanine Nucleotide Exchange Factors Interfacing with the Clathrin Machinery.

Connecdenn 2 binds directly to the β2-ear platform in a manner distinct from ARH. A, soluble lysates of HEK-293T cells expressing FLAG-β2-ear were incubated with equimolar amounts of GST or the indicated regions of connecdenn 2 fused to GST, precoupled to glutathione-Sepharose beads. B, alignment of the regions of ARH and connecdenn 2 that bind the β2-ear ear. Areas of similarity are boxed, critical residues in ARH required for β2 binding are indicated by asterisks. C, soluble lysates of HEK-293T cells expressing FLAG-β2-ear were incubated with equimolar amounts of GST or GST fusion proteins encoding the indicated regions of ARH and connecdenn (CD) 2, either in the wild-type (WT) form or containing the indicated point mutations, precoupled to glutathione-Sepharose beads. D, purified MBP-CD2 467–550-FLAG or soluble rat brain extract were incubated with GST or equimolar amounts of GST fused to the β2-ear WT and β2-ear with the indicated platform point mutations, precoupled to glutathione-Sepharose beads. E, soluble rat brain extract was incubated with GST or GST fused to the β2-ear WT, precoupled to glutathione-Sepharose beads in the presence of indicated molar ratios of purified MPB-ARH 248–270. The Ponceau-stained transfer reveals binding of MBP-ARH to GST-β2-ear WT. F, soluble lysates of HEK-293T cells expressing FLAG-β2-ear were incubated with GST or equimolar amounts of GST fusion proteins encoding either WT connecdenn 2 487–511 or the same construct containing the indicated point mutations, precoupled to glutathione-Sepharose. The Ponceau-stained transfer of the GST fusion proteins reveals equal loading. For A, C, D, and F, an aliquot of the lysate (starting material) equivalent to 1/10th that added to the beads was analyzed in parallel. Western blotting against the FLAG epitope or endogenous CD2 revealed specifically bound protein.

Andrea L. Marat, et al. J Biol Chem. 2010 April 2;285(14):10627-10637.

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