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. 2013 Jun 27;8(6):e68291. doi: 10.1371/journal.pone.0068291

Figure 6. MARVELD1 represses the recruitment of SMG5 by inhibiting serine phosphorylation of UPF1.

Figure 6

(A) HeLa cells transiently transfected with two independent siRNAs against MARVELD1 or the negative control siRNA and HEK293/MARVELD1 or the control cells HEK293/Vector were analyzed by coimmunoprecipitation using anti-UPF1 or rabbit (r) IgG in the presence of RNase A. The analysis of the intensity of SMG1 and importin β1 immunoprecipitated by anti-UPF1 is displayed as histograms on the right. The coIPs of SMG1 and importin β1 were both normalized to the amount of UPF1 immunoprecipitated by anti-UPF1. (B) Lysates of HeLa cells transiently depleted of MARVELD1 and HEK293/MARVELD1 were transiently transfected with Flag-UPF1 and immunoprecipitated by anti-Flag or mouse (m) IgG. Western blotting was performed using anti-phosphoserine or anti-SMG5. The intensities of phosphoserine level and the SMG5 immunoprecipitated by anti-Flag were normalized to the total Flag-UPF1 and are displayed as histograms on the right. (C) The effects of siRNA-mediated knockdown of MARVELD1 and the overexpression of MARVELD1-V5 were assessed by western blotting. GAPDH was used as an internal control. The average ± S.E. (error bars) is displayed.