Figure 5

Effects of puromycin and Upf1 knockdown on the constructs cloned in a pMT21 vector. (a) The structure of the pMT21 vector. SV40ori: simian virus 40 (SV40) origin of replication and enhancer element; AdMLP: adenovirus major late promoter; TPL: the tripartite leader; IVS: intervening sequence; DHFR: murine dihydrofolate reductase coding region; poly(A), polyadenylation signal. (b) The cDNAs of wild-type βc and βc79 were stably expressed in Ba/F3 cells using a pMT21 vector. These transfectants and 5C cells that expressed the wild-type βc in pIREShyg2 were incubated with or without 100 μg/mL puromycin for 4.5 hours. The values for βc/GAPDH in puromycin-treated cells were divided by the values in untreated cells, which are presented as fold increases of βc/GAPDH. Data shown are the average values ± S.E.M. (error bars) (pIREShyg2: n = 8, pMT21: n = 3). **: significantly smaller than the value in the wild-type βc cloned into pIREShyg2 (p < 0.01). (c) Upf1 was knocked down by siRNA in the cells expressing wild-type βc in pIREShyg2 (5C) and pMT21. The amounts of Upf1 and βc transcripts relative to GAPDH from three independent experiments are presented. *: significantly different (p < 0.05).