On StACRThr(P)Tyr(P) pulldown because it contains proteins that interact with APP within a Thr(P)668- and Tyr(P)682-dependent manner. The reaction was immunoprecipitated using the -FLAG M2-agarose beads, and proteins had been eluted from the M2-agarose beads employing a competing three FLAG peptide. To identify the lysine residues ubiquitinated (K-ub) in vitro, the eluted material was subjected for the ubiquitin remnant motif (K- -GG) assay (UbiScan). In parallel, we performed UbiScan experiments on total mouse brain lysates to figure out whether or not K-ubs identified in the in vitro assay are also ubiquitinated in vivo. In addition, we used as additional reference the Cell Signaling Information Bank collection of UbiScan outcomes from rodent, human, and chicken tissue, which is often discovered on line. As reported in Table six, various brain-derived proteins related using the ACR bait had been ubiquitinated. These proteins can be separated into 4 groups as follows: (a) group 1, proteins from the ubiquitin-conjugating system; (b) group two, presynaptic proteins; (c) group three, other proteins implicated in AD; and (d) group 4, phosphorylation-dependent ACR interactors. Group 1: constant with all the Western blot displaying ubiquitination of Crbn (Fig. 5B), we identified six K-ub from Crbn, 4 of which happen to be located in vivo. Two other components ofFIGURE 5. ACR plus the AL2CR bind an active CRL4CRBN. A, St, St-ACR, St-AL1CR, and St-AL2CR mouse brain pulldowns have been incubated with ( ) or without ( ) ubiquitin-FLAG (Ub-FLAG), E2, and/or E1. Soon after the reaction, these in vitro ubiquitination assays have been probed on WB with an anti-FLAG antibody. E2 promotes ubiquitination in St-ACR and St-AL2CR pulldowns. The WB shown is representative of 5 independent experiments. B, WB with an -CRBN antibody indicates the in vitro ubiquitination assays of St-ACR brain pulldowns where Crbn is poly-ubiquitinated, indicating that St-ACR isolates from brain an active CRL4CRBN. The WB shown is representative of two independent experiments.17216 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 291 Number 33 AUGUST 12,Modulation of E3 Ligases by APPTABLE six In vitro ubiquitination of proteins present in the St-ACRTyr(P)Thr(P) pulldowns1st column lists a number of the proteins ubiquitinated in vitro in an ACRTyr(P)Thr(P)-dependent manner. 2nd column lists all the lysine residues located ubiquitinated in vitro. The K-ub found in our UbiScans from mouse brains are indicated with (m*) and in prior UbiScan experiments from mouse (m), human (h), rat (r) and chicken (c) tissues, which are reported on the internet.147969-86-6 structure 3rd column reports database accession numbers.Price of 3-Acetyl-4-methoxybenzonitrile Ubiquitin conjugating program Cul4a Cul4b Crbn Chip Arih1 Mib2 Nedd4 Cbl-b Ube3a Ube1 Ube2n Ube2o Presynaptic proteins Scamp1 Sv2a Sv2b Vamp2 Atp6v0a1 Vglu1 Syt1 Syt2 Rab14 Rab3a Snap-25 Syn2 Syn1 Notable targets ApoE Tau Phosphorylation-dependent interactors Grb2 Pin1 eight, 23, h33, 42, 46, 65, 71, 97, m/h104, 133, 134, 191, 330, 404, 411, 416, m465, 473, 708, 724, 728, 734, 749 m112, 247, m315, 398, 460, 495, m676, 684, 931, 939 h/c46, h119, h/m303, 395, 410, c416 126 457 344, 369, 426, 435 m*445, m/h453 37, m*491, h516, 615 28 m/h802 m/h/r82, m/h/r92, m/h/r94 m/h812, m/h816 h/m/r334 m*32 m*62, m65, m341 m/h/r52, m/h/r59, m/h83, m*/h/r85 m262, m/h295 m283 r98, m/r104, m*190, r196, m200, m369 m*191 m/h/r193 m12 m/r103 280 m256, m281, r299, m311, m593 m105 m*455, m*613 h69, m*/h76 h/r65, 97, h99, m/h119, h134 Q3TCH7 A2A432 Q8C7D2 Q9WUD1 Q9Z1K5 Q8R516 P46935 Q3TTA7 O08759 Q02053 P61089 Q6ZPJ3 Q8K021 Q9.PMID:22664133
