DOI: 10.5061/dryad.pg4f4qrp5
title: Quantitative Multiplex Immunoprecipitation (QMI) analysis of protein interaction network rearrangements during homeostatic plasticity

description: Neurons maintain constant levels of excitability using homeostatic
 synaptic scaling, which adjusts the strength of all of a neuron’s
 postsynaptic inputs to compensate for large changes in overall activity.
 Here, we asked how prolonged changes in activity affect network-level
 protein interactions at the synapse. We assessed a glutamatergic synapse
 protein interaction network (PIN) composed of 380 binary associations
 among 21 protein members to identify protein complexes altered by activity
 manipulation in vitro or sensory deprivation in vivo. In cultured cortical
 neurons, we observed widespread bidirectional PIN alterations during up-
 and downscaling that reflected rapid rearrangements of glutamate receptor
 co-associations via synaptic scaffold remodeling. Sensory
 deprivation of the barrel cortex caused unique PIN rearrangements,
 including changes in co-associations between the glutamate
 receptor mGluR5 and the kinase Fyn, consistent with emerging models of
 experience dependent plasticity involving multiple types of homeostatic
 responses. Mice lacking Homer1 or Shank3B did not undergo normal PIN
 rearrangements, suggesting that these Autism Spectrum Disorder
 (ASD)-linked proteins serve as structural hubs for synaptic homeostasis.
 This dataset contains the raw XML and PBX (bioplex protocol) files
 necessary for performing adaptive non-parametric analysis with an emprical
 alpha cutoff (ANC) to identify high-confidence IP_Probe pairs,
 here called "PiSCES" for proteins in shared complexes detected
 by surface epitopes, that are signifianctly differently
 associated between two conditions.
description: <i>Quantitative
 multiplex immunoprecipitation: </i>QMI was performed as described
 previously using previously validated antibodies (PMCID: PMC6150823). Briefly, a master mix
 containing equal numbers of each antibody-coupled Luminex bead was
 prepared and distributed to lysates containing equal amounts of protein
 and incubated overnight on a rotator at 4°C. The next day, each sample was
 washed twice in cold Fly-P buffer (50mM tris pH7.4, 100mM NaCl, 1% bovine
 serum albumin, and 0.02% sodium azide) and distributed into twice as many
 wells of a 96-well plate as there were probe antibodies (for technical
 duplicates). Biotinylated detection (probe) antibodies were added to the
 appropriate wells and incubated at 4°C with gentle agitation for 1 hour.
 The resulting bead-probe complexes were washed 3 times with Fly-P buffer,
 incubated for 30 minutes with streptavidin-PE on ice, washed another 3
 times, resuspended in 125ml ice cold Fly-P buffer, and processed for
 fluorescence using a customized refrigerated Bio-Plex 200. Antibody clone
 names, catalog numbers, and lot numbers are the same as in (PMCID: PMC6150823).
description: The XML files serve as input for ANC. The PBX files, which are
 readable by the Bio-Plex Manager Software, contain information pertaining
 to the layout of each 96-well plate used in the study, including the order
 of the samples, the IP bead regions, and the order of the probes on each
 plate. This information is necessary metadata for performing
 ANC.   <br> For experiments with 2 plates (_P1
 and _P2), the order of the probes on plate P1 are Fyn, PSD95, NMDAR1,
 NMDAR2A, NMDAR2B, mGluR5, Shank3, Homer1, Homer1a, and PI3K; the order of
 the probes on plate P2 are GluA1, GluA2, Syngap, Sap97, NL3, Ube3a,
 CaMKIIa, SAPAP, panShank, Shank1, and PIKE. The exception is the set of
 HomeostaticScaling_48hr_ samples #5-8, which have Fyb at the end of plate
 P1. <br> For experiments with 3 plates (_P1, _P2,
 and _P3), the order of the probes on plate P1 are Fyn, PSD95, NMDAR1,
 NMDAR2A, NMDAR2B, mGluR5, Shank3; the order of the probes on plate P2 are
 Homer1, Homer1a, and PI3K, GluA1, GluA2, Syngap, Sap97; the order of the
 probes on plate P3 are NL3, Ube3a, CaMKIIa, SAPAP, panShank, Shank1, and
 PIKE.