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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

Extended Data Fig. 3 ∣. Liquid chromatography-tandem mass spectrometry, p-UPF1 preferentially co-immunoprecipitates with NMD targets, and a ~3-fold increased SMG1 level typifying FMRP-KD cells recapitulates hyperactivated NMD.

Extended Data Fig. 3 ∣

a, Silver-stained polyacrylamide gel of lysates of HEK293T cells eluted from beads alone (beads-bound), or beads bound by rIgG- or anti-p-UPF1 (α-p-UPF1) used in LC-MS/MS. b, Proteins in anti-p-UFP1 IP identified using LC-MS/MS and ranked (x-axis) by the number of unique mass spectra (y-axis). FMRP and its FXR paralogs, red; proteins that function in NMD, blue. See also Supplementary Table 2. c, Coomassie Blue staining of human FMRP purified from E. coli (left) or FLAG-tagged human UPF1 purified from baculovirus (right). d, Quantitations of mRNAs, normalized to the level of ß-actin mRNA. Means with S.D., n = 3 independent biological replicates. (*)P < 0.05, (**)P < 0.01 or (***)P < 0.001 pertains to comparisons to Ctl-siRNA samples (two-sided t-test). e, Western blots of lysates of HEK293T cells transiently transfected with the denoted siRNA, eluted before (−) or after α-UPF1 or NRS IP. f, Quantitations of the specified RNA, normalized to the level of ß-actin mRNA, using lysates from e. Means with S.D., n = 3 independent biological replicates. P values compare Ctl siRNA- vs. specified siRNA-treated samples (two-sided t-test). g, Pertaining to Fig. 2h, western blots of lysates of HEK293T cells transfected with the specified siRNA, before or after α-p-UPF1 or rIgG IP. h, Western blot of lysates of HEK293T cells transfected with pcDNA-HA or pcDNA-HA-SMG1, the latter to approximate the increased level of SMG1 in FMRP siRNA-transfected cells. i, Quantitations of mRNAs, normalized to the level of their pre-mRNA, using lysates from h. Means with S.D., n = 3 independent biological replicates. P values compare HA-SMG1- vs. HA-transfected samples (two-sided t-test). j, Western blot of lysates of HEK293T cells transfected with the specified siRNA eluted before or after α-UPF1 or NRS IP. Results in a, e, g, h, j are representative of 3 independent biological replicates. Statistical source data and unprocessed blots are provided in Source Data Extended Data Fig. 3.