Fig. 4
m6A methylation alters protein expression of TYK2 and DTX4
(A) Schematic of the DTX4 cDNA seq with the two specific point mutation scheme for altering A to G residues (red) that disrupt the m6A motifs. (B) RIP-rtPCR analysis of relative m6A level in DTX4 mRNAs in latent and reactivated LCL1 cells transfected with either mutated myc-DTX4 or wildtype myc-DTX4 control (C) Polysome profiling of the mutated myc tagged DTX4 transfected LCL cells followed by rtPCR of each set for finding percent of DTX4 mRNA. (D) Effect of the mutations on myc-DTX4 protein expression. (E) RIP-rtPCR analysis of relative YTHDF2 bound TYK2 mRNAs in latent and reactivated LCL1 cells (F) Protein expression of YTHDF2 and TYK2 in YTHDF2 knockdown latent and reactivated LCL1 cells. (G) RIP-rtPCR analysis of relative m6A level in TYK2 mRNAs in actinomycinD treated latent and reactivated LCL1 cells transfected with either sh-YTHDF2 or empty vector control H, I. mRNA expression of IFN-α and β by rtPCR in YTHDF2 knock down LCL1 cells during latency and reactivation. J. Effect of YTHDF2 knock down viral copy numbers per milliliter during latency and reactivation of LCL1 cells. Experiments were independently repeated three times, and results are presented as mean+/-s.d. from the three experiments. “***” represents p-value < 0.001; “**” represents p-value < 0.01; “*“represents p-value < 0.05 and “ns” represents no significance