Fig 1: BGLF2 inhibits multiple miRNAs.(A) Ago2 was immunoprecipitated from lysates of AGS-BGLF2 cells treated (+Dox) or untreated (-Dox) with Dox using anti-Ago2 antibody or IgG negative control antibody. Recovered miR-103a-3p, miR-20a-5p, and miR-17-5p miRNAs were quantified by RT-qPCR, normalized to spike in UniSp6 synthetic RNA control and calculated as fold enrichment in Ago2-IP over IgG control IP. Values were also normalized to recovery of Ago2 protein in the IP. Average values for +Dox samples from 3 independent experiments are shown relative to -Dox samples, which were set to 1. (B) miR-103a-3p, miR-20a-5p, miR-17-5p miRNAs from cell lysates in A (used for immunoprecipitations) were quantified by RT-qPCR, normalized to U6 RNA and plotted as in A. (C) Ago2-IP were performed using AGS-EBV-Z cells as in Fig 8D. miR-103a-3p, miR-20a-5p, and miR-17-5p miRNAs were quantified in input lysates and Ago2 IP samples and normalized to spike in UniSp6 synthetic RNA. miR-103a-3p, miR-20a-5p, and miR-17-5p values in Ago2-IP samples were also normalized to Ago2 protein recovered in each IP. Average values for 3 independent experiments are shown as percentage of input miR-103a-3p, miR-20a-5p, and miR-17-5p miRNA. (D) AGS-BGLF2 cells were treated with Dox for 48 hours or left untreated, then lysed in 9M urea and Western blotted for GPRC5A, FLAG, and actin. (E) Ago2 immunoprecipitations were performed as in C and transcripts for GPRC5A were quantified by RT-qPCR and normalized to Ago2 protein recovery. Average values for 4 independent experiments are shown as percentage of GPRC5A transcripts in the input. (F) The number of p-bodies per cells in AGS-EBV-Z cell pools containing a non-targeting (WT) or BGLF2-targeted (BGLF2 KO) CRISPR/Cas9, which were reactivated with Dox (+Dox) or left untreated (-Dox). * = 0.01