Journal: eLife
Article Title: Analysis of SUMO1-conjugation at synapses
doi: 10.7554/eLife.26338
Figure Lengend Snippet: ( A ) SDS-PAGE (4–12%) followed by Western blot analysis using anti-RIM1 antibody of input and HA peptide eluate fractions from anti-HA immunoprecipitation in the presence of 20 mM NEM from WT and His 6 -HA-SUMO1 KI brains. The presence of RIM1 in both WT and KI eluates indicates non-specific binding of RIM1 to the affinity matrix ( B ) Representative SDS-PAGE (8%) followed by Western blot analysis of input and eluate fractions of anti-RIM1 and anti-IgG immunopurifications in the presence of 20 mM NEM from WT and His 6 -HA-SUMO1 KI brains. Western blot analysis using anti-RIM1 confirms the enrichment of RIM1 in both WT and KI samples solely when anti-RIM1 antibody is used (upper panel). However, no SUMO1-RIM1 band is apparent (lower panel). ( C ) SDS-PAGE (4–12%) followed by anti-syntaxin1α Western blot analysis of input and HA peptide eluate fractions from anti-HA immunoprecipitation in the presence of 20 mM NEM from WT and His 6 -HA-SUMO1 KI brains. The presence of syntaxin1a in both WT and KI eluates indicates non-specific binding of syntaxin1a to the affinity matrix ( D ) Representative SDS-PAGE (12%) followed by Western blot analysis of input and eluate fractions of anti-syntaxin1α and anti-IgG immunopurifications in the presence or absence of 20 mM NEM from WT and His 6 -HA-SUMO1 KI brains. Western blot analysis using anti-syntaxin1a confirms the enrichment of syntaxin1a in both WT and KI samples solely when anti-syntaxin1a antibody is used (upper panel). However, no SUMO1-syntaxin1a band is apparent (lower panel). ( E ) SDS-PAGE (4–12%) followed by anti-synaptotagmin1 and mGluR7 Western blot of input and anti-HA peptide eluate fractions from anti-HA immunoprecipitation in the presence of 20 mM NEM from WT and His 6 -HA-SUMO1 KI brains. The presence of synaptotagmin1 in both WT and KI eluates indicates non-specific binding of synaptotagmin1 to the affinity matrix, while mGluR7 is not enriched in either case. Images are representatives of at least three independent experiments. DOI: http://dx.doi.org/10.7554/eLife.26338.008
Article Snippet: Primary antibodies used in IPs, Western blotting (WB) and immunocytochemistry (ICC) were as follows: mouse anti-SUMO1 21C7 (Hybridoma Bank, Iowa, WB: 1/1000, ICC: 1/50, RRID: AB_2198257 ), mouse anti-HA (Biolegend, 901515, WB: 1/1000, ICC: 1/100, RRID: AB_2565334 ) mouse anti-synapsin1 (Synaptic Systems, 106021, WB: 1/1000, RRID: AB_2617072 ), mouse anti-syntaxin1a (Synaptic Systems, 110111, WB: 1/1000, RRID: AB_887848 ), mouse anti-synaptotagmin (Synaptic System, 105001, WB: 1/1000, RRID: AB_887831 ), mouse anti-gephyrin (Synaptic Systems, 147111, WB: 1/1000, RRID: AB_887719 ), rabbit anti-RIM1 (Synapsin Systems, 140003, WB: 1/1000, RRID: AB_887774 ), rabbit anti-GluK2 (Millipore, 04–921, WB: 1/1000, RRID: AB_1587072 ), rabbit anti-mGlur7 (Upstate, 07–239, WB: 1/1000, RRID: AB_310459 ), mouse anti-GluN1 (Synaptic Systems, 114011, WB: 1/1000, RRID: AB_887750 ), mouse anti-synaptophysin (Synaptic Systems, 101011, WB: 1/1000, RRID: AB_887824 ), rabbit anti-synapsin1/2 (Synaptic Systems, 106002, ICC: 1/2000, RRID: AB_887804 ), rabbit anti-shank2 (Synaptic Systems, 162202, ICC: 1/1000, RRID: AB_2619860 ), chicken anti-Map2 (Novus, NB300213, ICC: 1/1000, RRID: AB_2138178 ), mouse anti-actin (Sigma-Aldrich, clone AC-40, WB: 1/5000, RRID: AB_476730 ).
Techniques: SDS Page, Western Blot, Immunoprecipitation, Binding Assay