0.15 P=0.04 P=0.43 siLUC UPF1 Phosphorylation levels (fold induction) 4 3.5 3 two.5 2 1.5 1 0.5 0 0 2 Time (h) 4 P

P value is comparing siLUC to siZFP36/L1/L2 situations at 1 h following serum induction working with the paired two-tailed Student's t-test.NATURE COMMUNICATIONS | 7:12434 | DOI: ten.1038/ncomms12434 | www.nature.com/naturecommunicationsNATURE COMMUNICATIONS | DOI: 10.1038/ncommsARTICLEUPFCa1 two 34 5 S42 T44 S97 S10* T28*CHHelicase6 7 8 S1031 S1038 S9 ten 11 S1046 S1050 T12 13 14 S1062 S1065 S15 16 17 18 S1073* S1078* S1089 S1096*19 S1116* 1397 nts 818 nts 1397 nts 818 nts 1397 nts 818 ntsb ExogenousUPF1: Chase (min): WT GAP 0 None 120 240 360 0 UPF1-C126S 120 240 360 0 UPF1-wt 120 240 360UPF1 [S/T] 7,8,19 A 120 240UPF1 [S/T] 7,eight,9,10,11,19 A 0 120 24039 t1/2 (min) Exogenous UPF1 : Chase (min): WT GAP 0 610' ?4 UPF1 [S/T] 15,16 A 120 240 360 0 592' ?9 UPF1 [S/T] 1,two A 120 240 360 0 137' ?9 UPF1 [S/T] 1,two,15,16 A 120 240 360 0 148' ?6 UPF1 [S/T] 17,18 A 120 240 360 0 165' ?two UPF1 [S/T] 7,eight,9,ten,11, 17,18,19 A 120 24039 t1/2 (min) 156' ?four 117' ?1 155' ? UPF1 [S/T] 1,2,9,10,11 15,16,17,18 A 0 120 240 360 260' ?1 UPF1 [S/T] 7,eight,9,10,11 15,16,17,18,19 A 0 120 240 360 368' ?five UPF1 [S/T] 1,2,7,8,9,10,11, 15,16,17,18,19 A 0 120 240Exogenous UPF1 : Chase (min): WT GAPUPF1 [S/T] 15,16,17,18 A 120 240UPF1 [S/T] 1,two,15,16,17,18 A 0 120 24039 t1/2 (min) 492' ?8 585' ?3 658' ?9 619' ?7 631' ?c39 mRNA half life (min)800 700 600 500 400 300 200 100 UPF1-wt UPF1-[S/T] 7,8,19 A UPF1-[S/T] 7,eight,9,10,11,19 A UPF1-[S/T] 15,16 A UPF1-[S/T] 1,two,15,16 A UPF1-[S/T] 17,18 A UPF1-[S/T] 15,16, 17,18 A UPF1-[S/T] 1,2, 15,16,17,18,A UPF1-[S/T] 1,2,9,ten,11 15,16,17,18, A UPF1-[S/T] 7,8,9,10,11 15,16,17,18,19 A UPF1-[S/T] 1,two A UPF1-[S/T] 7,8,9,10,11, 17,18,19 A UPF1-[S/T] 1,two,7,eight,9,10 11,15,16,17,18,19 A UPF1-C126S 17470919.2015.1029593 None* ** * ******0 Exogenous UPF1:Figure four | Multiple UPF1 [S/T]Q motifs contribute to NMD. (a) Schematic representation of human UPF1 protein showing the amino acids mutated within this study (black); the position with the helicase domain plus the cysteine/histidine-rich domain (CH) of UPF1 are indicated (to not scale). Numbers following amino-acid symbols correspond for the position within the UPF1 key sequence (GenBank #NP_002902). Asterisk indicates confirmed phosphorylated websites. [S/T]Q motifs not investigated within this function are shown in grey. (b) Northern blots displaying the decay of b39 mRNA in HeLa Tet-Off cells depleted for endogenous UPF1 and expressing order U0126-EtOH myc-tagged variants of UPF1 as indicated.