0.15 P=0.04 P=0.43 siLUC UPF1 Phosphorylation levels (fold induction) 4 3.5 3 two.five 2 1.5 1 0.five 0 0 2 Time (h) four P
Numbers above PD98059 site panels refer to time soon after serum induction; lower panel shows uninduced cells. P worth is comparing siLUC to siZFP36/L1/L2 conditions at 1 h just after serum induction using 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 2 34 5 S42 T44 S97 S10* T28*CHHelicase6 7 eight S1031 S1038 S9 10 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] 7,8,9,10,11,19 A 0 120 24039 t1/2 (min) Exogenous UPF1 : Chase (min): WT GAP 0 610' ?four 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' ?six UPF1 [S/T] 17,18 A 120 240 360 0 165' ?two UPF1 [S/T] 7,8,9,ten,11, 17,18,19 A 120 24039 t1/2 (min) 156' ?four 117' ?1 155' ? UPF1 [S/T] 1,2,9,ten,11 15,16,17,18 A 0 120 240 360 260' ?1 UPF1 [S/T] 7,eight,9,ten,11 15,16,17,18,19 A 0 120 240 360 368' ?five UPF1 [S/T] 1,two,7,eight,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 one hundred UPF1-wt UPF1-[S/T] 7,eight,19 A UPF1-[S/T] 7,8,9,ten,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,eight,9,10,11 15,16,17,18,19 A UPF1-[S/T] 1,2 A UPF1-[S/T] 7,eight,9,10,11, 17,18,19 A UPF1-[S/T] 1,2,7,eight,9,10 11,15,16,17,18,19 A UPF1-C126S 17470919.2015.1029593 None* ** * ******0 Exogenous UPF1:Figure four | Several UPF1 [S/T]Q motifs contribute to NMD.0.15 P=0.04 P=0.43 siLUC UPF1 Phosphorylation levels (fold induction) four 3.five three two.5 2 1.five 1 0.five 0 0 2 Time (h) four P=0.06 UPF1 No induction P-UPFNo induction siLUC siZFP36/L1/LFigure three | Transient increase in UPF1 phosphorylation upon activation of ARE-mediated decay. (a) Western blots showing endogenous UPF1 phosphorylation levels in RAW 264.7 cells treated with LPS and monitored as in Fig. 1a. Numbers refer to hours following LPS addition. Graph displaying UPF1 phosphorylation levels quantified as in Fig. 1 is shown beneath. Indicated values are normalized for the zero jmir.6472 time point .e.m. from three independent experiments. P values are relative to time zero and were calculated employing paired two-tailed Student's t-test. (b) Western blots showing UPF1 phosphorylation levels in NIH 3T3 cells transfected with siRNAs targeting either Firefly Luciferase (LUC) or ZFP36, ZFP36L1 and ZFP36L2 (ZFP36/L1/L2). Numbers above panels refer to time just after serum induction; lower panel shows uninduced cells.
