Our optimized PLA protocol effectively detects overexpressed or endogenous fusion proteins in pre-B lymphoblasts. The Nalm6 cell line overexpressing ETV6-RUNX1 (Nalm6+ETV6-RUNX1) was validated using RT-qPCR (a) and western blot (b). a Results are presented in terms of a fold change after normalizing ETV6-RUNX1 mRNA levels with GAPDH mRNA. Each value represents the mean of ± S.D. of three independent transduced cells. b Representative images of western blot showing expression of RUNX1 or HSC70 proteins in both cell lines are represented. c Pictures and quantification of PLA signals on Nalm6 cells overexpressing ETV6-RUNX1 protein (Nalm6+ETV6-RUNX1 cells) (a) or on REH cells that expressed endogenous ETV6-RUNX1 protein (b). 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 chosen at random. The quantification of the number of PLA dots per nucleus is presented with the mean values ± S.D