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. Author manuscript; available in PMC: 2020 Feb 1.
Published in final edited form as: Biochem Pharmacol. 2018 Dec 19;160:134–145. doi: 10.1016/j.bcp.2018.12.014

Fig. 3. m6A methylation determined missense protein expression in cells carrying p53 R273H mutation.

Fig. 3.

Fig. 3.

A, Protein expression of p53 and p53 target genes in cells exposed to Dox. Cells were treated with NPC (20 nM), PDMP (5 μM) or vehicle for 5 days, with exposed to Dox (100 nM) during the last 48 h of treatments. Equal amounts of detergent-soluble proteins extracted (50 μg/lane) were resolved using 4–20% gradient SDS-PAGE, and then immunoblotted with corresponding antibodies. Top panel, representative Western blots. METTL3, methyltransferase like 3; pp53, phosphorylated p53 (Ser15). Protein levels (bottom panel) are represented as mean ± SD of their optical densities normalized against GAPDH from three settings of blots. *, p<0.001 compared to SW48-Dox cells; #, p<0.001 compared to TP53-Dox cells treated with vehicle. B, Protein Expression of p53 and p53 target genes in cells exposed to vehicle. Cells were treated with NPC (20 nM), PDMP (5 μM) or vehicle for 5 days, with exposed to vehicle during the last 48 h of treatments. C, Immunostaining for METTL3 and pp53. Scale bar represents 25 μM in photomicrographs (100× magnification). Red, METTL3-Qdot 605; green, pp53-Alexa Fluor 488. D, Western blotting of p53 and pp53 after immunoprecipitation (IP). Cells were treated with PDMP (5 μM, 6 days) or vehicle, with exposed to Dox (100 nM) during the last 48 h of treatments. Equal amounts of detergent-soluble proteins (500 μg) were precipitated with indicated antibody and then immunoblotted after non-denaturing 12% PAGE resolution. Relative levels of p53 or pp53 are represented as means of their optical densities normalized against SW48-Dox cells with vehicle. *, p<0.001 compared to SW48-Dox with vehicle; **, p<0.001 compared to TP53-Dox with vehicle. E, MS/MS proteomics analysis of p53. Equal amounts of proteins were precipitated with pp53 antibody, resolved and assessed via mass spectroscopic (MS/MS) analysis.