We first performed Proximity Ligation Assay (PLA) on Nalm6 lymphoblasts. Those cells express both RUNX1 and ETV6 transcripts and proteins  (Fig. 2a). Specificity of the assay was shown by lack of non-specific staining in the negative control, displayed by the use of mouse-RUNX1 antibody alone (Fig. 2b). On the contrary, incubation with two different-species-antibodies directed against RUNX1 showed on average 21.0 (±12.2, n = 425) fluorescent dots per cell validating the presence of RUNX1 proteins in Nalm6 cells. As expected, RUNX1 proteins were localized in the nuclei. Detection of RUNX1 and CBFB proximity showed on average 13.1 (±6.6, n = 299) dots per cell. This colocalization between RUNX1 and CBFB is in concordance with the literature [20, 21]. We next wanted to assess the proximity of the proteins RUNX1 and ETV6. PLA with anti-mouse RUNX1 antibodies and anti-rabbit ETV6 antibodies did not show any relevant interaction (about 1.2 ± 1.5 dot per nucleus, n = 366) in Nalm6 cells. This result is consistent with the literature and expression results; we did not expect to detect interactions between endogenous ETV6 and RUNX1 proteins in Nalm6 cells. According to this result, the cut-off assigned in this study was a mean of 4.2 PLA dots per nucleus (two standard deviations over the background signal ), representing the fluorescent background or the probability of the 2 proteins to be in a close proximity by chance.Fig. 2Validation of PLA on pre-B lymphoblasts. a Quantitative real-time RT-PCR and western blot analysis showed respectively mRNA and protein expression of RUNX1, ETV6 or ETV6-RUNX1 in Nalm6 and REH cells. All RT-PCR (left panel) were performed in triplicate and gene expression was normalized to ABL1 expression (error bars are S.D.) while western blot analyses (right panel) are representative images from the whole-cell lysates. b Technical controls demonstrate the specificity of PLA signals in Nalm6 cells and the proximity between two proteins (RUNX1 and CBFB). Nuclei were stained with DAPI. RUNX1 ab110035 antibodies were incubated alone (−) or with RUNX1 ab23980, CBFB ab133600 or ETV6 sc11382 antibodies. Each picture (upper panel) is representative of a typical cell staining observed in 5 fields randomly chosen. The quantification of the number of PLA dot per nucleus is presented with the mean values ± S.D