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goat polyclonal anti arfgap3 antibodies  (Novus Biologicals)
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Novus Biologicals goat polyclonal anti arfgap3 antibodies
Figure1. RIBEYE(B)andRIBEYE(AB)interactwithArfGAP3intheYTHsystem.SummaryplatesofYTHanalysesobtainedwiththeindicatedbaitandpreyplasmids.Forconvenience,experimental bait–preypairsareunderlayeredincolor(greenincaseofinteractingbait–preypairs;controlmatingsarenoncolored).RIBEYE(B)andalsofull-lengthRIBEYE[RIBEYE(AB)]interactwithArfGAP3in the YTH system (matings 1 and 2). Mating 11 denotes an unrelated positive control (Magupalli et al., 2008). pSE1111 is an irrelevant prey vector and pSE1112 is an irrelevant bait vector (Tai et al., 1999; Magupalli et al., 2008). Negative control matings of the <t>ArfGAP3</t> prey clone with empty bait clones (mating 7) or irrelevant bait clones (mating 8) demonstrate that the ArfGAP3 clone is not autoactivating.TheothermatingsrepresentnegativecontrolmatingsfortheRIBEYEbaitclones(matings3–6)orRIBEYEpreyclones(matings9and10),demonstratingthattheseconstructsare alsonotautoactivatingintheYTHsystem.RE(AB),full-lengthRIBEYE,containingbothRIBEYE(A)-andRIBEYE(B)-domain;RE(A),RIBEYE(A)-domain;RE(B),RIBEYE(B)-domain;AGD,ArfGAP-domain of ArfGAP3.
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Figure1. RIBEYE(B)andRIBEYE(AB)interactwithArfGAP3intheYTHsystem.SummaryplatesofYTHanalysesobtainedwiththeindicatedbaitandpreyplasmids.Forconvenience,experimental bait–preypairsareunderlayeredincolor(greenincaseofinteractingbait–preypairs;controlmatingsarenoncolored).RIBEYE(B)andalsofull-lengthRIBEYE[RIBEYE(AB)]interactwithArfGAP3in the YTH system (matings 1 and 2). Mating 11 denotes an unrelated positive control (Magupalli et al., 2008). pSE1111 is an irrelevant prey vector and pSE1112 is an irrelevant bait vector (Tai et al., 1999; Magupalli et al., 2008). Negative control matings of the ArfGAP3 prey clone with empty bait clones (mating 7) or irrelevant bait clones (mating 8) demonstrate that the ArfGAP3 clone is not autoactivating.TheothermatingsrepresentnegativecontrolmatingsfortheRIBEYEbaitclones(matings3–6)orRIBEYEpreyclones(matings9and10),demonstratingthattheseconstructsare alsonotautoactivatingintheYTHsystem.RE(AB),full-lengthRIBEYE,containingbothRIBEYE(A)-andRIBEYE(B)-domain;RE(A),RIBEYE(A)-domain;RE(B),RIBEYE(B)-domain;AGD,ArfGAP-domain of ArfGAP3.

Journal: Journal of Neuroscience

Article Title: ArfGAP3 Is a Component of the Photoreceptor Synaptic Ribbon Complex and Forms an NAD(H)-Regulated, Redox-Sensitive Complex with RIBEYE That Is Important for Endocytosis

doi: 10.1523/jneurosci.3837-13.2014

Figure Lengend Snippet: Figure1. RIBEYE(B)andRIBEYE(AB)interactwithArfGAP3intheYTHsystem.SummaryplatesofYTHanalysesobtainedwiththeindicatedbaitandpreyplasmids.Forconvenience,experimental bait–preypairsareunderlayeredincolor(greenincaseofinteractingbait–preypairs;controlmatingsarenoncolored).RIBEYE(B)andalsofull-lengthRIBEYE[RIBEYE(AB)]interactwithArfGAP3in the YTH system (matings 1 and 2). Mating 11 denotes an unrelated positive control (Magupalli et al., 2008). pSE1111 is an irrelevant prey vector and pSE1112 is an irrelevant bait vector (Tai et al., 1999; Magupalli et al., 2008). Negative control matings of the ArfGAP3 prey clone with empty bait clones (mating 7) or irrelevant bait clones (mating 8) demonstrate that the ArfGAP3 clone is not autoactivating.TheothermatingsrepresentnegativecontrolmatingsfortheRIBEYEbaitclones(matings3–6)orRIBEYEpreyclones(matings9and10),demonstratingthattheseconstructsare alsonotautoactivatingintheYTHsystem.RE(AB),full-lengthRIBEYE,containingbothRIBEYE(A)-andRIBEYE(B)-domain;RE(A),RIBEYE(A)-domain;RE(B),RIBEYE(B)-domain;AGD,ArfGAP-domain of ArfGAP3.

Article Snippet: Also the following commercially available antibodies did not work in postembedding immunogold electron microscopy: rabbit and goat polyclonal anti-ArfGAP3 antibodies (AAS68618C and ASA34060; Antibody Verify), rabbit polyclonal anti-ArfGAP3 (NBP118921; Novus Biologicals), rabbit polyclonal anti-ArfGAP3 (HPA000638; Sigma), and rabbit polyclonal anti-ArfGAP3 (A302-032A; Biomol).

Techniques: Positive Control, Plasmid Preparation, Negative Control, Clone Assay

Figure 2. Summary tables of YTH matings. A, RIBEYE interacts with the ArfGAP-domain (AGD) of ArfGAP3. RIBEYE(B) and also full-length RIBEYE [RIBEYE(AB)] interact with the ArfGAP-domain (AGD) of ArfGAP3 in the YTH system (matings 1 and 2). Mating 9denotesanunrelatedpositivecontrol(Magupallietal.,2008).Theothermatingsareauto-activationcontrols.Noneoftheyeast constructs is auto activating. B, The NAD(H)-binding subdomain of RIBEYE(B) interacts with ArfGAP3. The NAD(H)-binding sub- domainofRIBEYE(B),theNBD,interactswithArfGAP3(mating1),butnotthesubstrate-bindingsubdomainofRIBEYE(B),theSBD (mating 5). Matings 2–4 are positive control matings–(Bassoon for SBD-RE(B); tom Dieck et al., 2005), Munc119 for NBD-RE(B) (Alpadietal.,2008),RE(A)forRE(B)(Magupallietal.,2008)–andmatings6–11arenegativecontrols(auto-activationcontrols). C, Schematic domain structures of ArfGAP3. D, An NAD(H)-binding-deficient mutant of RIBEYE(B), RE(B)G730A, does not interact with ArfGAP3 (mating 1), while wild-type RIBEYE does (mating 6). Mating 7 is a positive control mating for RE(B)G730A. E, Arf1 interactswithArfGAP3(mating1).Allconstructsarenon-autoactivatingasdemonstratedbythenegativecontrolmatings(2–5); mating 6 is a positive control mating. RE(A), RIBEYE(A)-domain; RE(B), RIBEYE(B)-domain; RE(B)G730A, RIBEYE(B)G730A; GAP- dom, GAP-domain (AGD) of ArfGAP3; NBD, NAD(H)-binding subdomain of RIBEYE(B); SBD, substrate-binding subdomain of RIBEYE(B).

Journal: Journal of Neuroscience

Article Title: ArfGAP3 Is a Component of the Photoreceptor Synaptic Ribbon Complex and Forms an NAD(H)-Regulated, Redox-Sensitive Complex with RIBEYE That Is Important for Endocytosis

doi: 10.1523/jneurosci.3837-13.2014

Figure Lengend Snippet: Figure 2. Summary tables of YTH matings. A, RIBEYE interacts with the ArfGAP-domain (AGD) of ArfGAP3. RIBEYE(B) and also full-length RIBEYE [RIBEYE(AB)] interact with the ArfGAP-domain (AGD) of ArfGAP3 in the YTH system (matings 1 and 2). Mating 9denotesanunrelatedpositivecontrol(Magupallietal.,2008).Theothermatingsareauto-activationcontrols.Noneoftheyeast constructs is auto activating. B, The NAD(H)-binding subdomain of RIBEYE(B) interacts with ArfGAP3. The NAD(H)-binding sub- domainofRIBEYE(B),theNBD,interactswithArfGAP3(mating1),butnotthesubstrate-bindingsubdomainofRIBEYE(B),theSBD (mating 5). Matings 2–4 are positive control matings–(Bassoon for SBD-RE(B); tom Dieck et al., 2005), Munc119 for NBD-RE(B) (Alpadietal.,2008),RE(A)forRE(B)(Magupallietal.,2008)–andmatings6–11arenegativecontrols(auto-activationcontrols). C, Schematic domain structures of ArfGAP3. D, An NAD(H)-binding-deficient mutant of RIBEYE(B), RE(B)G730A, does not interact with ArfGAP3 (mating 1), while wild-type RIBEYE does (mating 6). Mating 7 is a positive control mating for RE(B)G730A. E, Arf1 interactswithArfGAP3(mating1).Allconstructsarenon-autoactivatingasdemonstratedbythenegativecontrolmatings(2–5); mating 6 is a positive control mating. RE(A), RIBEYE(A)-domain; RE(B), RIBEYE(B)-domain; RE(B)G730A, RIBEYE(B)G730A; GAP- dom, GAP-domain (AGD) of ArfGAP3; NBD, NAD(H)-binding subdomain of RIBEYE(B); SBD, substrate-binding subdomain of RIBEYE(B).

Article Snippet: Also the following commercially available antibodies did not work in postembedding immunogold electron microscopy: rabbit and goat polyclonal anti-ArfGAP3 antibodies (AAS68618C and ASA34060; Antibody Verify), rabbit polyclonal anti-ArfGAP3 (NBP118921; Novus Biologicals), rabbit polyclonal anti-ArfGAP3 (HPA000638; Sigma), and rabbit polyclonal anti-ArfGAP3 (A302-032A; Biomol).

Techniques: Construct, Binding Assay, Positive Control, Mutagenesis

Figure 3. RIBEYE(B) specifically interacts with ArfGAP3 in fusion protein pull-down assays (SDS-PAGE analyses). Pull-down analyses of RIBEYE(B)/ArfGAP3 complexes were analyzed by Coomassie blue-stained polyacrylamide gel after SDS-PAGE. Lanes 1–4showtheindicatedpurifiedfusionproteins(inputfractions).Allinputlanesrepresent30%oftheinputfraction.Inputproteins wereloadedinseparatelanestodemonstratethattheinputfusionproteinsdisplayonlyasingle,mainproteinband.Inlanes5–8, 100% of the pull-down reactions were loaded. MBP-tagged fusion proteins were used as immobilized bait proteins and GST- taggedproteinsassolublepreyproteins.OnlyArfGAP3-MBPpulleddownRE(B)-GST(lane5,arrowhead)butnotMBPalone(lane 7). Neither MBP alone nor ArfGAP3-MBP pulled down GST alone (lanes 6 and 8). SDS-PAGE demonstrated that ArfGAP3-MBP specifically pulled down RIBEYE(B)-GST, demonstrating interaction of the two proteins in this assay system.

Journal: Journal of Neuroscience

Article Title: ArfGAP3 Is a Component of the Photoreceptor Synaptic Ribbon Complex and Forms an NAD(H)-Regulated, Redox-Sensitive Complex with RIBEYE That Is Important for Endocytosis

doi: 10.1523/jneurosci.3837-13.2014

Figure Lengend Snippet: Figure 3. RIBEYE(B) specifically interacts with ArfGAP3 in fusion protein pull-down assays (SDS-PAGE analyses). Pull-down analyses of RIBEYE(B)/ArfGAP3 complexes were analyzed by Coomassie blue-stained polyacrylamide gel after SDS-PAGE. Lanes 1–4showtheindicatedpurifiedfusionproteins(inputfractions).Allinputlanesrepresent30%oftheinputfraction.Inputproteins wereloadedinseparatelanestodemonstratethattheinputfusionproteinsdisplayonlyasingle,mainproteinband.Inlanes5–8, 100% of the pull-down reactions were loaded. MBP-tagged fusion proteins were used as immobilized bait proteins and GST- taggedproteinsassolublepreyproteins.OnlyArfGAP3-MBPpulleddownRE(B)-GST(lane5,arrowhead)butnotMBPalone(lane 7). Neither MBP alone nor ArfGAP3-MBP pulled down GST alone (lanes 6 and 8). SDS-PAGE demonstrated that ArfGAP3-MBP specifically pulled down RIBEYE(B)-GST, demonstrating interaction of the two proteins in this assay system.

Article Snippet: Also the following commercially available antibodies did not work in postembedding immunogold electron microscopy: rabbit and goat polyclonal anti-ArfGAP3 antibodies (AAS68618C and ASA34060; Antibody Verify), rabbit polyclonal anti-ArfGAP3 (NBP118921; Novus Biologicals), rabbit polyclonal anti-ArfGAP3 (HPA000638; Sigma), and rabbit polyclonal anti-ArfGAP3 (A302-032A; Biomol).

Techniques: SDS Page, Staining

Figure4. RIBEYE(B)specificallyinteractswithArfGAP3infusionproteinpull-downassays(Westernblotanalyses).Toexclude thatthetaghasanimportanceforthepull-downresultsandtofurtherexcludethatanypreyproteinisunspecificallypulleddown bybait-GST,wealsoanalyzedtheresultsofthepull-downassaysbyWesternblottingwithanti-MBPandanti-GSTantibodies.The reaction buffer used for these experiments contained 1 mM ME. GST-tagged fusion proteins were used as immobilized bait proteins and eluted MBP-tagged proteins as soluble prey proteins. Similar to the experiments described in Figure 3, only RIBEYE(B)-MBP (lane 5) and not MBP alone (lane 6) is pulled down by the ArfGAP3(AGD)-GST. GST alone (lane 7) and MBP alone (lane8)donotpulldownRIBEYE(B)-MBP,asshownbyWesternblottingwithantibodiesagainstMBP(Fig.4A),demonstratingthe specificity of the interaction. The Western blot data fully confirm the results shown in Figure 3 that were obtained by SDS-PAGE analyses.InFigure4B,thesameblotasanalyzedinFigure4Awasreprobed(afterstrippingoftheblot)withantibodiesagainstGST to show equal loading of the bait proteins. RE(B)-MBP, RIBEYE(B)-MBP; ArfGAP3(AGD)-MBP, MBP-tagged ArfGAP-domain (AGD) of ArfGAP3.

Journal: Journal of Neuroscience

Article Title: ArfGAP3 Is a Component of the Photoreceptor Synaptic Ribbon Complex and Forms an NAD(H)-Regulated, Redox-Sensitive Complex with RIBEYE That Is Important for Endocytosis

doi: 10.1523/jneurosci.3837-13.2014

Figure Lengend Snippet: Figure4. RIBEYE(B)specificallyinteractswithArfGAP3infusionproteinpull-downassays(Westernblotanalyses).Toexclude thatthetaghasanimportanceforthepull-downresultsandtofurtherexcludethatanypreyproteinisunspecificallypulleddown bybait-GST,wealsoanalyzedtheresultsofthepull-downassaysbyWesternblottingwithanti-MBPandanti-GSTantibodies.The reaction buffer used for these experiments contained 1 mM ME. GST-tagged fusion proteins were used as immobilized bait proteins and eluted MBP-tagged proteins as soluble prey proteins. Similar to the experiments described in Figure 3, only RIBEYE(B)-MBP (lane 5) and not MBP alone (lane 6) is pulled down by the ArfGAP3(AGD)-GST. GST alone (lane 7) and MBP alone (lane8)donotpulldownRIBEYE(B)-MBP,asshownbyWesternblottingwithantibodiesagainstMBP(Fig.4A),demonstratingthe specificity of the interaction. The Western blot data fully confirm the results shown in Figure 3 that were obtained by SDS-PAGE analyses.InFigure4B,thesameblotasanalyzedinFigure4Awasreprobed(afterstrippingoftheblot)withantibodiesagainstGST to show equal loading of the bait proteins. RE(B)-MBP, RIBEYE(B)-MBP; ArfGAP3(AGD)-MBP, MBP-tagged ArfGAP-domain (AGD) of ArfGAP3.

Article Snippet: Also the following commercially available antibodies did not work in postembedding immunogold electron microscopy: rabbit and goat polyclonal anti-ArfGAP3 antibodies (AAS68618C and ASA34060; Antibody Verify), rabbit polyclonal anti-ArfGAP3 (NBP118921; Novus Biologicals), rabbit polyclonal anti-ArfGAP3 (HPA000638; Sigma), and rabbit polyclonal anti-ArfGAP3 (A302-032A; Biomol).

Techniques: Western Blot, SDS Page

Figure 5. RIBEYE(B) is recruited by ArfGAP3 into a Golgi-like distribution in transfected COS cells. COS7 cells were transfected with the indicated mcherry-tagged ArfGAP3 or EGFP-tagged RIBEYE(B)constructs.Transfectedcellswereanalyzedfortheintracellulardistributionoftherespectiveproteinsviadirectepifluorescencemicroscopy.CellstransfectedwithArfGAP3-mcherryalone show the typical enrichment at the Golgi apparatus in a perinuclear localization (A, B), as already previously shown (Dogic et al., 1999; Eugster et al., 2000; Lewis et al., 2004; Watson et al., 2004; Frigerio et al., 2007; Kartberg et al., 2010; Yu et al., 2012). In contrast, RIBEYE(B) is diffusely distributed in single-transfected cells (C; Schmitz et al., 2000). If RIBEYE(B)-EGFP is cotransfected with ArfGAP3-mcherry,RIBEYE(B)virtuallycompletelyredistributedfromadiffusedistributionintotheGolgi-like,perinuclearlocalizationindicatinginteractionbetweenRIBEYE(B)andArfGAP3(D).n, nucleus. The arrow in D points to the Golgi-like localization to which the RIBEYE(B)-EGFP signal is translocated in ArfGAP3-mcherry-transfected cells. Scale bars: A–D, 10 m.

Journal: Journal of Neuroscience

Article Title: ArfGAP3 Is a Component of the Photoreceptor Synaptic Ribbon Complex and Forms an NAD(H)-Regulated, Redox-Sensitive Complex with RIBEYE That Is Important for Endocytosis

doi: 10.1523/jneurosci.3837-13.2014

Figure Lengend Snippet: Figure 5. RIBEYE(B) is recruited by ArfGAP3 into a Golgi-like distribution in transfected COS cells. COS7 cells were transfected with the indicated mcherry-tagged ArfGAP3 or EGFP-tagged RIBEYE(B)constructs.Transfectedcellswereanalyzedfortheintracellulardistributionoftherespectiveproteinsviadirectepifluorescencemicroscopy.CellstransfectedwithArfGAP3-mcherryalone show the typical enrichment at the Golgi apparatus in a perinuclear localization (A, B), as already previously shown (Dogic et al., 1999; Eugster et al., 2000; Lewis et al., 2004; Watson et al., 2004; Frigerio et al., 2007; Kartberg et al., 2010; Yu et al., 2012). In contrast, RIBEYE(B) is diffusely distributed in single-transfected cells (C; Schmitz et al., 2000). If RIBEYE(B)-EGFP is cotransfected with ArfGAP3-mcherry,RIBEYE(B)virtuallycompletelyredistributedfromadiffusedistributionintotheGolgi-like,perinuclearlocalizationindicatinginteractionbetweenRIBEYE(B)andArfGAP3(D).n, nucleus. The arrow in D points to the Golgi-like localization to which the RIBEYE(B)-EGFP signal is translocated in ArfGAP3-mcherry-transfected cells. Scale bars: A–D, 10 m.

Article Snippet: Also the following commercially available antibodies did not work in postembedding immunogold electron microscopy: rabbit and goat polyclonal anti-ArfGAP3 antibodies (AAS68618C and ASA34060; Antibody Verify), rabbit polyclonal anti-ArfGAP3 (NBP118921; Novus Biologicals), rabbit polyclonal anti-ArfGAP3 (HPA000638; Sigma), and rabbit polyclonal anti-ArfGAP3 (A302-032A; Biomol).

Techniques: Transfection, Construct

Figure 6. The binding between RIBEYE(B)-domain and the ArfGAP-domain (AGD) of ArfGAP3 is stimulated by NAD(H) in a redox-sensitive manner. A, B, Pull-down experiments were performed as in Figure 3 both in the absence (lanes 3—10; A, B) or presence(lane11;A,B)of1mMME.RIBEYEbindstotheArfGAP-domain(AGD)ofArfGAP3onlyinthepresenceof1mMME(lane 11; A, B) but not in the absence of 1 mM ME (lane 3; A, B). Lanes 1 and 2 show the respective input fractions (30% input). We testedwhetheradditionofNADH(A)orNAD (B)couldsubstituteforthepresenceof1mMMEinpromotingArfGAP3/RIBEYE(B) interaction. As a matter of fact, increasing concentrations of both NADH (A) as well as NAD (B) could promote binding of RIBEYE(B)-GSTtotheAGDofArfGAP3evenintheabsenceof1mMME(lanes4—10;A,B).Thereducedformofthedinucleotide, NADH (A), was more effective than the oxidized form, NAD (B), to promote RIBEYE(B)/ArfGAP3 interaction. Semiquantitative evaluation of the binding experiments (n 4) demonstrated that 450 nM NADH and 700 nM NAD are promoting half- maximal binding of ArfGAP3 to RIBEYE(B) in the pull-down assays.

Journal: Journal of Neuroscience

Article Title: ArfGAP3 Is a Component of the Photoreceptor Synaptic Ribbon Complex and Forms an NAD(H)-Regulated, Redox-Sensitive Complex with RIBEYE That Is Important for Endocytosis

doi: 10.1523/jneurosci.3837-13.2014

Figure Lengend Snippet: Figure 6. The binding between RIBEYE(B)-domain and the ArfGAP-domain (AGD) of ArfGAP3 is stimulated by NAD(H) in a redox-sensitive manner. A, B, Pull-down experiments were performed as in Figure 3 both in the absence (lanes 3—10; A, B) or presence(lane11;A,B)of1mMME.RIBEYEbindstotheArfGAP-domain(AGD)ofArfGAP3onlyinthepresenceof1mMME(lane 11; A, B) but not in the absence of 1 mM ME (lane 3; A, B). Lanes 1 and 2 show the respective input fractions (30% input). We testedwhetheradditionofNADH(A)orNAD (B)couldsubstituteforthepresenceof1mMMEinpromotingArfGAP3/RIBEYE(B) interaction. As a matter of fact, increasing concentrations of both NADH (A) as well as NAD (B) could promote binding of RIBEYE(B)-GSTtotheAGDofArfGAP3evenintheabsenceof1mMME(lanes4—10;A,B).Thereducedformofthedinucleotide, NADH (A), was more effective than the oxidized form, NAD (B), to promote RIBEYE(B)/ArfGAP3 interaction. Semiquantitative evaluation of the binding experiments (n 4) demonstrated that 450 nM NADH and 700 nM NAD are promoting half- maximal binding of ArfGAP3 to RIBEYE(B) in the pull-down assays.

Article Snippet: Also the following commercially available antibodies did not work in postembedding immunogold electron microscopy: rabbit and goat polyclonal anti-ArfGAP3 antibodies (AAS68618C and ASA34060; Antibody Verify), rabbit polyclonal anti-ArfGAP3 (NBP118921; Novus Biologicals), rabbit polyclonal anti-ArfGAP3 (HPA000638; Sigma), and rabbit polyclonal anti-ArfGAP3 (A302-032A; Biomol).

Techniques: Binding Assay

Figure 7. Western blot analyses of two antibodies that were generated against the C terminus of ArfGAP3. A, Schematic drawing denotes the areas against which the two polyclonal ArfGAP3 antibodies(Cterm2andCterm3)weregenerated.B–D,InWesternblotanalyses,bothantibodies(Cterm2andCterm3)detectedasinglebandattheexpectedrunningpositionofArfGAP3at55 kDa.ThisArfGAP3Westernblotbandcouldbeblockedbypre-absorptionoftheantibodywiththerespectiveArfGAP3-GSTfusionprotein(lane2;C;datanotshown)butnotbypre-incubationwith GSTalone(C,lane3;datanotshown).Cb,Loadingcontrol(immunoblottingofthesameblotasshowninCa)afterstrippingoftheblotandreprobingwithanantibodyagainstGCAP2(Venkatesan et al., 2010), demonstrating equal protein loading.

Journal: Journal of Neuroscience

Article Title: ArfGAP3 Is a Component of the Photoreceptor Synaptic Ribbon Complex and Forms an NAD(H)-Regulated, Redox-Sensitive Complex with RIBEYE That Is Important for Endocytosis

doi: 10.1523/jneurosci.3837-13.2014

Figure Lengend Snippet: Figure 7. Western blot analyses of two antibodies that were generated against the C terminus of ArfGAP3. A, Schematic drawing denotes the areas against which the two polyclonal ArfGAP3 antibodies(Cterm2andCterm3)weregenerated.B–D,InWesternblotanalyses,bothantibodies(Cterm2andCterm3)detectedasinglebandattheexpectedrunningpositionofArfGAP3at55 kDa.ThisArfGAP3Westernblotbandcouldbeblockedbypre-absorptionoftheantibodywiththerespectiveArfGAP3-GSTfusionprotein(lane2;C;datanotshown)butnotbypre-incubationwith GSTalone(C,lane3;datanotshown).Cb,Loadingcontrol(immunoblottingofthesameblotasshowninCa)afterstrippingoftheblotandreprobingwithanantibodyagainstGCAP2(Venkatesan et al., 2010), demonstrating equal protein loading.

Article Snippet: Also the following commercially available antibodies did not work in postembedding immunogold electron microscopy: rabbit and goat polyclonal anti-ArfGAP3 antibodies (AAS68618C and ASA34060; Antibody Verify), rabbit polyclonal anti-ArfGAP3 (NBP118921; Novus Biologicals), rabbit polyclonal anti-ArfGAP3 (HPA000638; Sigma), and rabbit polyclonal anti-ArfGAP3 (A302-032A; Biomol).

Techniques: Western Blot, Generated

Figure 8. Coimmunoprecipitation of RIBEYE and ArfGAP3 from the bovine retina (Western blot analyses). A, ArfGAP3 immune serum (lane 3) and ArfGAP3 pre-immune serum (lane 2) were tested for their capability to coimmunoprecipitate RIBEYE from the bovine retina. Lane 1 shows the input fraction (1% of total input). RIBEYE is coimmunoprecipitated by ArfGAP3 im- mune serum (Cterm3 antiserum; lane 3) but not by ArfGAP3 pre-immune serum (lane 2). B, Shows the same blot as in A but reprobed with anti-ArfGAP3 antibodies (after stripping of the blot).ThisblotshowsthatArfGAP3wassuccessfullyimmunoprecipitatedbytheimmuneserum (lane3)butnotbythecontrolpre-immuneserum(lane2).HCandLCindicatetheIgheavyand light chains, respectively.

Journal: Journal of Neuroscience

Article Title: ArfGAP3 Is a Component of the Photoreceptor Synaptic Ribbon Complex and Forms an NAD(H)-Regulated, Redox-Sensitive Complex with RIBEYE That Is Important for Endocytosis

doi: 10.1523/jneurosci.3837-13.2014

Figure Lengend Snippet: Figure 8. Coimmunoprecipitation of RIBEYE and ArfGAP3 from the bovine retina (Western blot analyses). A, ArfGAP3 immune serum (lane 3) and ArfGAP3 pre-immune serum (lane 2) were tested for their capability to coimmunoprecipitate RIBEYE from the bovine retina. Lane 1 shows the input fraction (1% of total input). RIBEYE is coimmunoprecipitated by ArfGAP3 im- mune serum (Cterm3 antiserum; lane 3) but not by ArfGAP3 pre-immune serum (lane 2). B, Shows the same blot as in A but reprobed with anti-ArfGAP3 antibodies (after stripping of the blot).ThisblotshowsthatArfGAP3wassuccessfullyimmunoprecipitatedbytheimmuneserum (lane3)butnotbythecontrolpre-immuneserum(lane2).HCandLCindicatetheIgheavyand light chains, respectively.

Article Snippet: Also the following commercially available antibodies did not work in postembedding immunogold electron microscopy: rabbit and goat polyclonal anti-ArfGAP3 antibodies (AAS68618C and ASA34060; Antibody Verify), rabbit polyclonal anti-ArfGAP3 (NBP118921; Novus Biologicals), rabbit polyclonal anti-ArfGAP3 (HPA000638; Sigma), and rabbit polyclonal anti-ArfGAP3 (A302-032A; Biomol).

Techniques: Western Blot, Stripping Membranes

Figure9. ArfGAP3isstronglyenrichedatsynapticribbonsofphotoreceptorsynapsesinsitu(conventionalimaging).ArfGAP3colocalizeswithsynapticribbons.The0.5-m-thickretinalsections aredoubleimmunolabeledwithantibodiesagainstArfGAP3andmonoclonalantibodiesagainstRIBEYE(B)/CtBP2(A,B).ArfGAP3Cterm3-antibodywasusedinA,andArfGAP3Cterm2wasusedfor immunolabeling of ArfGAP3 in B. Strong ArfGAP3 immunosignals were found in an identical manner with both ArfGAP3 antibodies at the RIBEYE-immunolabeled synaptic ribbons and in close vicinity to synaptic ribbons. The dashed circles denote single immunolabeled photoreceptor presynaptic terminals. Arrows in B and C point to single immunolabeled synaptic ribbons. No immunosignalswereobservedinthepresynapticterminalsifpre-immuneserumwasused(datanotshown).AandBwereobtainedbyconventionalimaging.OPL,Outerplexiformlayer.Scalebars: A, 1 m; B, 5 m.

Journal: Journal of Neuroscience

Article Title: ArfGAP3 Is a Component of the Photoreceptor Synaptic Ribbon Complex and Forms an NAD(H)-Regulated, Redox-Sensitive Complex with RIBEYE That Is Important for Endocytosis

doi: 10.1523/jneurosci.3837-13.2014

Figure Lengend Snippet: Figure9. ArfGAP3isstronglyenrichedatsynapticribbonsofphotoreceptorsynapsesinsitu(conventionalimaging).ArfGAP3colocalizeswithsynapticribbons.The0.5-m-thickretinalsections aredoubleimmunolabeledwithantibodiesagainstArfGAP3andmonoclonalantibodiesagainstRIBEYE(B)/CtBP2(A,B).ArfGAP3Cterm3-antibodywasusedinA,andArfGAP3Cterm2wasusedfor immunolabeling of ArfGAP3 in B. Strong ArfGAP3 immunosignals were found in an identical manner with both ArfGAP3 antibodies at the RIBEYE-immunolabeled synaptic ribbons and in close vicinity to synaptic ribbons. The dashed circles denote single immunolabeled photoreceptor presynaptic terminals. Arrows in B and C point to single immunolabeled synaptic ribbons. No immunosignalswereobservedinthepresynapticterminalsifpre-immuneserumwasused(datanotshown).AandBwereobtainedbyconventionalimaging.OPL,Outerplexiformlayer.Scalebars: A, 1 m; B, 5 m.

Article Snippet: Also the following commercially available antibodies did not work in postembedding immunogold electron microscopy: rabbit and goat polyclonal anti-ArfGAP3 antibodies (AAS68618C and ASA34060; Antibody Verify), rabbit polyclonal anti-ArfGAP3 (NBP118921; Novus Biologicals), rabbit polyclonal anti-ArfGAP3 (HPA000638; Sigma), and rabbit polyclonal anti-ArfGAP3 (A302-032A; Biomol).

Techniques: Immunolabeling

Figure 10. A, B, Pre-absorption control experiments for the immunolabeling analyses shown in Figure 9A. Double immunolabeling of 0.5-m-thick mouse retinal sections with ArfGAP3 (Cterm3)antibodypre-absorbedeitherwithArfGAP3-GST-fusionprotein(B)orwithGSTalone(A).Tovisualizeribbonsynapses,sectionswerecoimmunolabeledwithmousemonoclonalantibodies against RIBEYE(B)-domain/CtBP2. Pre-absorption with ArfGAP3Cterm3-GST fusion protein completely blocked the ArfGAP3 immunosignals at the synaptic ribbon (B), whereas GST alone had no influence on the ArfGAP3 immunosignals (A), demonstrating the specificity of the previous immunolabeling results. C, D, Pre-absorption control experiments for the immunolabeling analyses showninFigure9B.Doubleimmunolabelingof0.5-m-thickmouseretinalsectionswithArfGAP3(Cterm2)antibodypre-absorbedwitheitherArfGAP3Cterm2-GST-fusionprotein(D)orwithGST alone (C). Synaptic ribbons were coimmunolabeled with mouse monoclonal antibodies against RIBEYE(B)-domain/CtBP2. Pre-absorption with the specific fusion protein completely blocked the ArfGAP3 immunosignals at the synaptic ribbon (D), whereas GST alone had no influence on the ArfGAP3 immunosignals (C), demonstrating the specificity of the previous immunolabeling results. A–D were obtained by conventional imaging. OPL, Outer plexiform layer; INL, inner nuclear layer. Scale bars: A–D, 5 m.

Journal: Journal of Neuroscience

Article Title: ArfGAP3 Is a Component of the Photoreceptor Synaptic Ribbon Complex and Forms an NAD(H)-Regulated, Redox-Sensitive Complex with RIBEYE That Is Important for Endocytosis

doi: 10.1523/jneurosci.3837-13.2014

Figure Lengend Snippet: Figure 10. A, B, Pre-absorption control experiments for the immunolabeling analyses shown in Figure 9A. Double immunolabeling of 0.5-m-thick mouse retinal sections with ArfGAP3 (Cterm3)antibodypre-absorbedeitherwithArfGAP3-GST-fusionprotein(B)orwithGSTalone(A).Tovisualizeribbonsynapses,sectionswerecoimmunolabeledwithmousemonoclonalantibodies against RIBEYE(B)-domain/CtBP2. Pre-absorption with ArfGAP3Cterm3-GST fusion protein completely blocked the ArfGAP3 immunosignals at the synaptic ribbon (B), whereas GST alone had no influence on the ArfGAP3 immunosignals (A), demonstrating the specificity of the previous immunolabeling results. C, D, Pre-absorption control experiments for the immunolabeling analyses showninFigure9B.Doubleimmunolabelingof0.5-m-thickmouseretinalsectionswithArfGAP3(Cterm2)antibodypre-absorbedwitheitherArfGAP3Cterm2-GST-fusionprotein(D)orwithGST alone (C). Synaptic ribbons were coimmunolabeled with mouse monoclonal antibodies against RIBEYE(B)-domain/CtBP2. Pre-absorption with the specific fusion protein completely blocked the ArfGAP3 immunosignals at the synaptic ribbon (D), whereas GST alone had no influence on the ArfGAP3 immunosignals (C), demonstrating the specificity of the previous immunolabeling results. A–D were obtained by conventional imaging. OPL, Outer plexiform layer; INL, inner nuclear layer. Scale bars: A–D, 5 m.

Article Snippet: Also the following commercially available antibodies did not work in postembedding immunogold electron microscopy: rabbit and goat polyclonal anti-ArfGAP3 antibodies (AAS68618C and ASA34060; Antibody Verify), rabbit polyclonal anti-ArfGAP3 (NBP118921; Novus Biologicals), rabbit polyclonal anti-ArfGAP3 (HPA000638; Sigma), and rabbit polyclonal anti-ArfGAP3 (A302-032A; Biomol).

Techniques: Control, Immunolabeling, Bioprocessing, Imaging

Figure 11. ArfGAP3 is strongly enriched at the synaptic ribbon of photoreceptor synapses in situ (SR-SIM imaging with ArfGAP3 Cterm3 antibody). A, 0.5-m-thick retinal sections double immunolabeled with affinity-purified rabbit antibodies against ArfGAP3 (ArfGAP3 Cterm3 antibody) and mouse monoclonal antibodies against RIBEYE(B)/CtBP2. B, Shows 0.5-m-thick retinal sectionsdoubleimmunolabeledwithaffinity-purifiedrabbitantibodiesagainstArfGAP3(Cterm3antibody)andmousemonoclonalantibodiesagainsttheactivezoneproteinbassoon.ArrowsinA andBpointtosingleimmunolabeledsynapticribbons.ArrowheadsdenoteArfGAP3immunoreactivityatthesynapticribbon.AandBwereobtainedbySR-SIMimaging.ONL,Outernuclearlayer; OPL, outer plexiform layer; INL, inner nuclear layer. Scale bars: A, B, 1 m.

Journal: Journal of Neuroscience

Article Title: ArfGAP3 Is a Component of the Photoreceptor Synaptic Ribbon Complex and Forms an NAD(H)-Regulated, Redox-Sensitive Complex with RIBEYE That Is Important for Endocytosis

doi: 10.1523/jneurosci.3837-13.2014

Figure Lengend Snippet: Figure 11. ArfGAP3 is strongly enriched at the synaptic ribbon of photoreceptor synapses in situ (SR-SIM imaging with ArfGAP3 Cterm3 antibody). A, 0.5-m-thick retinal sections double immunolabeled with affinity-purified rabbit antibodies against ArfGAP3 (ArfGAP3 Cterm3 antibody) and mouse monoclonal antibodies against RIBEYE(B)/CtBP2. B, Shows 0.5-m-thick retinal sectionsdoubleimmunolabeledwithaffinity-purifiedrabbitantibodiesagainstArfGAP3(Cterm3antibody)andmousemonoclonalantibodiesagainsttheactivezoneproteinbassoon.ArrowsinA andBpointtosingleimmunolabeledsynapticribbons.ArrowheadsdenoteArfGAP3immunoreactivityatthesynapticribbon.AandBwereobtainedbySR-SIMimaging.ONL,Outernuclearlayer; OPL, outer plexiform layer; INL, inner nuclear layer. Scale bars: A, B, 1 m.

Article Snippet: Also the following commercially available antibodies did not work in postembedding immunogold electron microscopy: rabbit and goat polyclonal anti-ArfGAP3 antibodies (AAS68618C and ASA34060; Antibody Verify), rabbit polyclonal anti-ArfGAP3 (NBP118921; Novus Biologicals), rabbit polyclonal anti-ArfGAP3 (HPA000638; Sigma), and rabbit polyclonal anti-ArfGAP3 (A302-032A; Biomol).

Techniques: In Situ, Imaging, Immunolabeling, Affinity Purification, Bioprocessing

Figure 12. Localization of ArfGAP3 in the presynaptic photoreceptor terminal in relation to other presynaptic proteins. The 0.5-m-thick retinal sections were triple immunolabeled with affinity-purified rabbit polyclonal antibodies against ArfGAP3 (Cterm3), mouse monoclonal antibodies against dynamin (hudy-1), and DyLight 650 directly labeled primary antibodies against RIBEYE(B)/CtBP2.ArfGAP3andRIBEYEarelocatedveryclosetoeachother(A,B).TheArfGAP3immunosignalislocatedwithinthering-likedynaminimmunosignalthatdemarcatesthepresynaptic plasmamembraneoftheperiactivezonethatsurroundsthesynapticribbon(Wahletal.,2013).Awasobtainedbyconventionalimaging;BisamicrographobtainedbySR-SIMimaging.Arrowsin A and B point to immunolabeled single synaptic ribbons. Arrowheads indicate ArfGAP3 immunoreactivity at the synaptic ribbon. OPL, Outer plexiform layer. Scale bars: A, B, 1 m.

Journal: Journal of Neuroscience

Article Title: ArfGAP3 Is a Component of the Photoreceptor Synaptic Ribbon Complex and Forms an NAD(H)-Regulated, Redox-Sensitive Complex with RIBEYE That Is Important for Endocytosis

doi: 10.1523/jneurosci.3837-13.2014

Figure Lengend Snippet: Figure 12. Localization of ArfGAP3 in the presynaptic photoreceptor terminal in relation to other presynaptic proteins. The 0.5-m-thick retinal sections were triple immunolabeled with affinity-purified rabbit polyclonal antibodies against ArfGAP3 (Cterm3), mouse monoclonal antibodies against dynamin (hudy-1), and DyLight 650 directly labeled primary antibodies against RIBEYE(B)/CtBP2.ArfGAP3andRIBEYEarelocatedveryclosetoeachother(A,B).TheArfGAP3immunosignalislocatedwithinthering-likedynaminimmunosignalthatdemarcatesthepresynaptic plasmamembraneoftheperiactivezonethatsurroundsthesynapticribbon(Wahletal.,2013).Awasobtainedbyconventionalimaging;BisamicrographobtainedbySR-SIMimaging.Arrowsin A and B point to immunolabeled single synaptic ribbons. Arrowheads indicate ArfGAP3 immunoreactivity at the synaptic ribbon. OPL, Outer plexiform layer. Scale bars: A, B, 1 m.

Article Snippet: Also the following commercially available antibodies did not work in postembedding immunogold electron microscopy: rabbit and goat polyclonal anti-ArfGAP3 antibodies (AAS68618C and ASA34060; Antibody Verify), rabbit polyclonal anti-ArfGAP3 (NBP118921; Novus Biologicals), rabbit polyclonal anti-ArfGAP3 (HPA000638; Sigma), and rabbit polyclonal anti-ArfGAP3 (A302-032A; Biomol).

Techniques: Immunolabeling, Affinity Purification, Bioprocessing, Labeling

Figure 13. The ArfGAP3 effector Arf1 is enriched at the synaptic ribbon complex of photoreceptor synapses. A, B, Double immunolabeling experiments of 0.5-m-thick retinal sections with mousemonoclonalantibodiesagainstArf1andrabbitpolyclonalantibodiesagainstRIBEYE(U2656)demonstratedacloseenrichmentofArf1aroundthesynapticribboncomplex.ONL,Outernuclear layer; OPL, outer plexiform layer. Scale bars: A, B, 1 m. (C) Western blot analyses demonstrated that Arf1 is strongly expressed in the mouse retina.

Journal: Journal of Neuroscience

Article Title: ArfGAP3 Is a Component of the Photoreceptor Synaptic Ribbon Complex and Forms an NAD(H)-Regulated, Redox-Sensitive Complex with RIBEYE That Is Important for Endocytosis

doi: 10.1523/jneurosci.3837-13.2014

Figure Lengend Snippet: Figure 13. The ArfGAP3 effector Arf1 is enriched at the synaptic ribbon complex of photoreceptor synapses. A, B, Double immunolabeling experiments of 0.5-m-thick retinal sections with mousemonoclonalantibodiesagainstArf1andrabbitpolyclonalantibodiesagainstRIBEYE(U2656)demonstratedacloseenrichmentofArf1aroundthesynapticribboncomplex.ONL,Outernuclear layer; OPL, outer plexiform layer. Scale bars: A, B, 1 m. (C) Western blot analyses demonstrated that Arf1 is strongly expressed in the mouse retina.

Article Snippet: Also the following commercially available antibodies did not work in postembedding immunogold electron microscopy: rabbit and goat polyclonal anti-ArfGAP3 antibodies (AAS68618C and ASA34060; Antibody Verify), rabbit polyclonal anti-ArfGAP3 (NBP118921; Novus Biologicals), rabbit polyclonal anti-ArfGAP3 (HPA000638; Sigma), and rabbit polyclonal anti-ArfGAP3 (A302-032A; Biomol).

Techniques: Immunolabeling, Western Blot

Figure 14. RIBEYE(B) and Arf1 compete with each other for binding to ArfGAP3. We tested in fusion protein pull-down experiments whether Arf1 and RIBEYE(B) can bind simultaneously to ArfGAP3 or whether they compete with each other for ArfGAP3-binding. A, B, Show representative Westerns blots incubated with the indicated antibodies to test for the binding of the respective fusion proteins. After detection of the GST-tagged protein (A1, B1), blots were stripped and re-incubated with antibodies against MBP (A2, B2). A3, B3, SNAP-tagged immobilized ArfGAP3 bait protein was visualized with SNAP-Vista Green (NEB). A, We tested whether increasing concentrations of Arf1 added to a fixed concentration of immobilized ArfGAP3 would inhibit binding of RIBEYE(B) to ArfGAP3. RIBEYE(B) was kept at a constant concentration in these experiments. B, We tested whether increasing concentrations of RIBEYE(B) added to a fixed concentration of immobilizedArfGAP3wouldinhibitbindingofArf1toArfGAP3.Arf1waskeptataconstantconcentrationintheselatterexperiments.Inbothsetsofexperiments,weobservedacompetitivebehavior betweenArf1andRIBEYE(B)inbindingtoArfGAP3.ThesedatademonstratethatRIBEYE(B)competeswithArf1forbindingtoArfGAP3.indicatingthatbindingofRIBEYE(B)andArf1toArfGAP3is mutually exclusive. Abbreviations: AGD*, extended ArfGAP-domain of ArfGAP3.

Journal: Journal of Neuroscience

Article Title: ArfGAP3 Is a Component of the Photoreceptor Synaptic Ribbon Complex and Forms an NAD(H)-Regulated, Redox-Sensitive Complex with RIBEYE That Is Important for Endocytosis

doi: 10.1523/jneurosci.3837-13.2014

Figure Lengend Snippet: Figure 14. RIBEYE(B) and Arf1 compete with each other for binding to ArfGAP3. We tested in fusion protein pull-down experiments whether Arf1 and RIBEYE(B) can bind simultaneously to ArfGAP3 or whether they compete with each other for ArfGAP3-binding. A, B, Show representative Westerns blots incubated with the indicated antibodies to test for the binding of the respective fusion proteins. After detection of the GST-tagged protein (A1, B1), blots were stripped and re-incubated with antibodies against MBP (A2, B2). A3, B3, SNAP-tagged immobilized ArfGAP3 bait protein was visualized with SNAP-Vista Green (NEB). A, We tested whether increasing concentrations of Arf1 added to a fixed concentration of immobilized ArfGAP3 would inhibit binding of RIBEYE(B) to ArfGAP3. RIBEYE(B) was kept at a constant concentration in these experiments. B, We tested whether increasing concentrations of RIBEYE(B) added to a fixed concentration of immobilizedArfGAP3wouldinhibitbindingofArf1toArfGAP3.Arf1waskeptataconstantconcentrationintheselatterexperiments.Inbothsetsofexperiments,weobservedacompetitivebehavior betweenArf1andRIBEYE(B)inbindingtoArfGAP3.ThesedatademonstratethatRIBEYE(B)competeswithArf1forbindingtoArfGAP3.indicatingthatbindingofRIBEYE(B)andArf1toArfGAP3is mutually exclusive. Abbreviations: AGD*, extended ArfGAP-domain of ArfGAP3.

Article Snippet: Also the following commercially available antibodies did not work in postembedding immunogold electron microscopy: rabbit and goat polyclonal anti-ArfGAP3 antibodies (AAS68618C and ASA34060; Antibody Verify), rabbit polyclonal anti-ArfGAP3 (NBP118921; Novus Biologicals), rabbit polyclonal anti-ArfGAP3 (HPA000638; Sigma), and rabbit polyclonal anti-ArfGAP3 (A302-032A; Biomol).

Techniques: Binding Assay, Incubation, Concentration Assay

Figure 15. Overexpression of ArfGAP3 in mouse photoreceptors inhibits endocytic uptake of FM1–43. FM1–43 was used to compare endocytic uptake in photoreceptors that were either electroporatedwithmcherryalone(A,B)orArfGAP3-mcherry(C,D).Inmcherry-electroporatedphotoreceptors,therewasanintenseuptakeofFM1–43inthesynapticterminals(A,B).Theuptake of FM1–43 in mcherry-transfected photoreceptors was similar to the FM1–43 uptake in nontransfected photoreceptors (data not shown). In contrast to mcherry-transfected photoreceptors, ArfGAP3-mcherry-overexpressingphotoreceptorsshowedastronginhibitionofFM1–43uptakeinthesynapticterminals(C,D),indicatingthatArfGAP3isessentialinvolvedinendocytosisatthe photoreceptor synapse. OS, Outer segment; IS, inner segment; Scale bars: A–C, 1 m; D, 0.75 m.

Journal: Journal of Neuroscience

Article Title: ArfGAP3 Is a Component of the Photoreceptor Synaptic Ribbon Complex and Forms an NAD(H)-Regulated, Redox-Sensitive Complex with RIBEYE That Is Important for Endocytosis

doi: 10.1523/jneurosci.3837-13.2014

Figure Lengend Snippet: Figure 15. Overexpression of ArfGAP3 in mouse photoreceptors inhibits endocytic uptake of FM1–43. FM1–43 was used to compare endocytic uptake in photoreceptors that were either electroporatedwithmcherryalone(A,B)orArfGAP3-mcherry(C,D).Inmcherry-electroporatedphotoreceptors,therewasanintenseuptakeofFM1–43inthesynapticterminals(A,B).Theuptake of FM1–43 in mcherry-transfected photoreceptors was similar to the FM1–43 uptake in nontransfected photoreceptors (data not shown). In contrast to mcherry-transfected photoreceptors, ArfGAP3-mcherry-overexpressingphotoreceptorsshowedastronginhibitionofFM1–43uptakeinthesynapticterminals(C,D),indicatingthatArfGAP3isessentialinvolvedinendocytosisatthe photoreceptor synapse. OS, Outer segment; IS, inner segment; Scale bars: A–C, 1 m; D, 0.75 m.

Article Snippet: Also the following commercially available antibodies did not work in postembedding immunogold electron microscopy: rabbit and goat polyclonal anti-ArfGAP3 antibodies (AAS68618C and ASA34060; Antibody Verify), rabbit polyclonal anti-ArfGAP3 (NBP118921; Novus Biologicals), rabbit polyclonal anti-ArfGAP3 (HPA000638; Sigma), and rabbit polyclonal anti-ArfGAP3 (A302-032A; Biomol).

Techniques: Over Expression, Transfection