Research Paper Volume 13, Issue 9 pp 13300—13317

Figure 2. SnoRD126 combines with hnRNPK. (A) Confirmation of snoRD126-binding proteins by RNA pull-down followed by immunoblotting. Lanes 1, 10% of the cell extracts used in the pull-down assay. Lane 4, biotin-labelled snoRD126 probe. Lane 5, biotin-labelled and biotin-unlabelled mixed snoRD126 probe used as a competitor. Lane 6, without RNA probe. (B) Schematic diagrams of the wild-type (WT) snoRD126 and the mutants with double nucleic acid base mutations in its four conserved motifs. (C) RNA pull-down followed by immunoblotting using biotin-labelled WT (lane 3) and mutants of snoRD126 (lane 4-9). Lanes 1, 10% of the cell extracts used in the pull-down assay. Lane 11, without RNA probe. (D, E) Confirmation of snoRD126 presence in a hnRNPK ribonucleoprotein complex by RIP assay followed by a qRT-PCR assay using (D) HepG2 cells transfected with pcDNA3.1⁺ plasmid expressing an N-terminal 3xFlag-tagged hnRNPK (pcDNA3.1⁺-3xFlag-hnRNPK); (E) Huh7 cells stably expressing snoRD126, SNORD126 C’2 and SNORD126 D mutants. The data represent mean ± SD (n = 3). **P < 0.01. NS, not significant.