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. Author manuscript; available in PMC: 2021 Jul 12.
Published in final edited form as: Nat Cell Biol. 2021 Jan 8;23(1):40–48. doi: 10.1038/s41556-020-00618-1

Fig. 2 ∣. Evidence that FMRP binding to NMD targets is promoted by direct binding to UPF1.

Fig. 2 ∣

a, Western blots of lysates of HEK293T cells, with or without okadaic acid (OA), before (−) or after immunoprecipitation (IP) (±RNase I using anti-UPF1 or normal rabbit serum (NRS)). The results represent three biological replicates. b, As in a, but the immunoprecipitations used anti-p-UPF1 or rabbit IgG (rIgG). c, Pull-down of FLAG-UPF1 (Extended Data Fig. 3c) or FLAG-tagged bacterial alkaline phosphatase (FLAG-BAP) in the presence of FMRP (Extended Data Fig. 3c). The results represent three biological replicates. d, Western blots of lysates of siRNA-transfected HEK293T cells. The results represent three biological replicates. e, Quantitations of mRNAs, normalized to their pre-mRNA, using the lysates from d. The data represent means with s.d. (n = 3 biological replicates). P values pertain to comparisons with control siRNA samples and were determined by two-sided t-test. f, As in d, but transfections included plasmid expressing FLAG or FLAG-FMRP WTR, where R denotes siRNA resistance. g, As in e, but using the lysates from f. P values pertain to comparisons with control siRNA samples and were determined by two-sided t-test. h, Quantitations of RNA, normalized to β-actin mRNA, using the lysates from Extended Data Fig. 3g. The data represent means with s.d. (n = 3 biological replicates). P values are relative to control siRNA samples after anti-p-UPF1 immunoprecipitation (two-sided t-test). i, FLAG-Gl-MS2bs reporter mRNAs harbouring six MS2 coat protein (MS2CP; orange circle) binding sites (MS2bs), alone or fused to FMRP. j, Western blots of lysates of HEK293T cells transfected with plasmid expressing MS2CP or MS2CP-FMRP, pcDNA-EGFP and a pFLAG-Gl-MS2bs reporter. The results represent three biological replicates. k, Quantitations of FLAG-Gl-MS2bs Ter mRNA normalized to the level of FLAG-Gl-MS2bs Norm mRNA. The data represent means with s.d. (n = 3 biological replicates). The P values compare MS2CP-FMRP- versus MS2CP-transfected samples (two-sided t-test). l, FLAG-Gl-MS2bs Norm reporter as in i, but with MS2CP-UPF1. m, As in j, but before or after immunoprecipitation using anti-FMRP or mouse IgG (mIgG). n, As in k, but using the lysates from m. The data represent means with s.d. (n = 3 biological replicates). The P values compare MS2CP-UPF1- versus MS2CP-transfected samples (two-sided t-test). In all panels, *P < 0.05, **P < 0.01 and ***P < 0.001. Statistical source data and unprocessed blots are provided online.