Bind to St-Ccas peptides (NSAF for Stub1-derived peptides was 0, 0, 0, 0, and 0.0213 in St negative handle, St-CcasThr(P), St-CcasTyr(P), StCcas, and St-JCasp pulldowns, respectively). Cul4a, Cul4b, Ddb1, and Crbn were the four most abundant UPS-linked proteins interacting with Ccas; their binding was not impacted by phosphorylation, and they didn’t bind St-JCasp (NSAF for St adverse control, St-CcasThr(P), St-CcasTyr(P), St-Ccas, and StJCasp pulldowns have been as follows: for Ddb1, 0.0005, 0.0478, 0.0273, 0.0375, and 0.0001; for Cul4a, 0, 0.0047, 0.003, 0.0026, and 0; for Cul4b, 0, 0.0011, 0.0006, 0.0008, and 0; for Crbn, 0, 0.0252, 0.0167, 0.0213, and 0). As noted above, Cul4a- or Cul4bDdb1 and Crbn type the CRL4CRBN E3 ligase complicated; the proof that all of the CRL4CRBN E3 ligase complicated components bind towards the same domain with the ACR, Ccas, further supports the concept that they bind as a complicated and not independently. To confirm the accuracy with the proteins detected inside the proteomic analysis, we performed Western blotting evaluation with pulldown lysates. As shown in Fig. 1B, Crbn, Cul4a, and Ddb1 were readily detected in St-CcasThr(P), St-CcasTyr(P), and StCcas but not in St and St-JCasp pulldowns.ATG4A, Human (His) On the contrary, Stub1 was discovered exclusively in St-JCasp pulldowns. Grb2, whose interaction with APP demands phosphorylation of TyrVOLUME 291 Number 33 AUGUST 12,17212 JOURNAL OF BIOLOGICAL CHEMISTRYModulation of E3 Ligases by APPTABLE 3 Ddb1, Crbn, Cul4a, and Cul4b bind to Ccas and Stub1 binds to JCaspPhosphorylation of Thr668 and/or Tyr682 does not substantially alter binding of Ddb1, Crbn, Cul4a, and Cul4b, whereas binding of Pin1 and Grb2 is dependent on phosphorylation of Thr668 and Tyr682, respectively.PDGF-BB Protein Formulation Quite a few in the other UPS-related proteins which can be bound for the ACR bind to either Ccas or JCasp or both. UniProtKB/Swiss-Prot E1 and E2 Uba1 Ube2o Ube2r2 E3 Arih1 Nedd4 Park7 Ubr4 Stub1 Cul4a Cul4b Ddb1 Crbn E3 regulators Cand1 Cops1 Cops2 Cops3 Cops4 Cops5 Cops6 Cops7a Cops7b Cops8 DUB Usp5 Otub1 Proteasome Psmd11 Psmd12 Psmd13 Ubiquilin Ubqln2 Phosphorylation-dependent Grb2 Pin1 Molecular masskDaStCcasThr(P)CcasTyr(P)CcasJCaspQ02053 Q6ZPJ3 Q6ZWZ2 Q9Z1K5 P46935 Q99LX0 A2AN08 Q9WUD1 Q3TCH7 A2A432 Q3U1J4 Q8C7D2 Q6ZQ38 Q99LD4 P61202 O88543 O88544 O35864 O88545 Q9CZ04 Q8BV13 Q8VBV7 P56399 Q7TQI3 Q8BG32 Q9D8W5 Q9WVJ2 Q9QZM0 Q60631 Q9QUR118 141 27 64 103 20 572 35 88 111 127 51 136 53 52 48 46 38 36 30 30 23 96 31 47 53 43 67 250 0 0 0 0 0.PMID:23357584 0012 0 0 0 0 0.0005 0 0.0002 0 0 0 0 0 0 0 0 0 0 0 0.0005 0 0 0 0.00620 0 0 0 0 0.0017 0 0 0.0047 0.0011 0.0478 0.0252 0 0 0 0 0 0 0 0 0 0 0.0001 0.0004 0 0.0004 0 0.0002 0.0008 0.0 0 0 0 0 0.0004 0 0 0.003 0.0006 0.0273 0.0167 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.0001 0.1780 0 0 0 0 0.0022 0 0.0004 0.0026 0.0008 0.0375 0.0213 0 0 0 0 0.0003 0 0 0 0 0 0.0001 0.0004 0.0003 0.0002 0.0003 0.0005 0.00050.0001 0.0001 0.0016 0.001 0.0003 0.002 0.0002 0.0213 0 0 0.0012 0 0.0001 0.0018 0.0022 0.001 0.0024 0.0011 0.0015 0.0022 0.0004 0.0005 0.0019 0.0007 0 0 0 0.0002 0(55, 59, 83), and Pin1, which interacts with APP in a Thr668 phosphorylation-dependent manner (84), have been only detected in St-CcasTyr(P) and St-CcasThr(P) pulldowns, respectively, further validating the proteomic method. General, the data suggest that of all the UPS-linked proteins pulled down with St-ACR, the CRL4CRBN E3 ligase complex elements and E3 ligase Stub1 will be the probably to interact directly with APP in vivo. Incidentally, an interaction bet.