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. 2022 Nov 17;11:e69916. doi: 10.7554/eLife.69916

Figure 5. Evaluation of BG4 binding to G-quadruplex structure in mitochondrial genome.

(A) Schematic showing the experimental strategy used for mito-IP using anti-BG4. Briefly, cells were crosslinked and then mitochondria were isolated and sonicated to obtain the small fragments of mitochondrial DNA. Purified BG4 antibody was used along with protein A/G agarose beads to pull down the BG4 bound regions. (B) BG4 bound mtDNA was purified after reverse crosslinking and used for real-time PCR using primers derived from different regions of the mitochondrial genome, which include 5 G-quadruplex forming regions and 10 random regions. Input DNA served as template control. No antibody control was also used. Bars in blue (first 5) are for G-quadruplex forming regions, while green (last 10) are for random regions. Y-axis depicts threshold Ct value obtained following real time PCR for each primer. Error bar represents mean ± SEM. (C) Agarose gel profile showing the amplification of Input DNA (left panel) and BG4 pull down DNA (right panel). ‘M’ denotes 100 bp ladder. Refer also Figure 5—figure supplement 1.

Figure 5—source data 1. BG4 ChIP to show the binding of BG4 to mitochondrial G-quadruplex forming regions.

Figure 5.

Figure 5—figure supplement 1. Evaluation of existence of G-quadruplex in mitochondrial DNA.

Figure 5—figure supplement 1.

Related to Figure 5. Schematic showing the position of primers used for mito IP studies. GR1-GR5 represents primers that can amplify G-quadruplex forming motifs and CR1-CR10 represents the random control regions.