To evaluate the sensitivity of the assay, we performed PLA on Nalm6 cells displaying a gradient of expression of RUNX1. We generated Nalm6 cell lines depleted for RUNX1 transcript and protein (Nalm6shRUNX1) or overexpressing RUNX1 (Nalm6+RUNX1) (Fig. 3a and 3b). Quantification of RUNX1 protein level by PLA positively correlated with quantification of RUNX1 protein level demonstrated by western blot (Fig. 3b and 3c). This result demonstrates that PLA may be sensitive to protein level.Fig. 3PLA is sensitive to total protein level. a Nalm6 wild type, depleted for RUNX1 protein (Nalm6shRUNX1) or overexpressing RUNX1 (Nalm6+RUNX1) cell lines were validated using RT-qPCR. Results are presented in terms of a fold change after normalizing 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 (left panel) and densitometry analysis (right panel) showing the quantification of RUNX1 protein level normalized to HSC70 c Quantification of the PLA signal (dots plots) on Nalm6 cells displaying a gradient of expression of RUNX1 is represented. Nalm6, Nalm6shRUNX1 or Nalm6+RUNX1 cells were incubated with a pair of RUNX1 antibodies (the mean values ± S.D. are presented)