Fig. 2: NONO O-GlcNAcylation site was identified as Thr440. | Cell Death Discovery

Fig. 2: NONO O-GlcNAcylation site was identified as Thr440.

From: O-GlcNAcylation of NONO regulates paraspeckle component assembly and contributes to colon cancer cell proliferation

Fig. 2

A sWGA precipitation was performed to evaluate immunoprecipitated NONO level upon with or without GlcNAc competition. Immunoprecipitated NONO protein level was tested by western blot analysis. B Validation of O-GlcNAcylation with endogenously-expressed NONO in HCT116 cells. The NONO O-GlcNAcylation in HCT116 was higher in cells transfected with OGT. C The NONO O-GlcNAcylation in HCT116 was lower in cells transfected with OGA. D A co-IP assay was performed to evaluate the interactions between NONO with OGT. HCT116 WCLs were subjected to IP with normal rabbit IgG of anti-NONO antibody. E Mass spectrometry (MS) analysis identifies the O-GlcNAcylation site in NONO. EThcD spectra of O-Glycopeptide FGQAATMEGIGAIGGTPPAFNR from human NONO is shown. The site of O-GlcNAc modification was identified as threonine 440. The c and z fragments detected are as indicated in the sequence. F Human NONO protein structure. O-GlcNAcylation was indicated at threonine 440. (RRM; RNA recognition motif, NOPS; NONA/Paraspeckle). G IP assay to compare the O-GlcNAcylation levels of wild-type NONO with T440 to A mutant. 10 ug of either FLAG-tagged NONO WT or mutant were transfected into HEK293 cells and lysed after 24 h. NONO O-GlcNAcylation level was normalized to the immunoprecipitated NONO protein level (n = 3 per condition). H Sequence information indicates that NONO threonine 440 is a highly conserved sequence in mammals. Data are presented as mean ± SD; ***P < 0.001, Unpaired two-tailed t-test.

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