Figure 3: PRMT5 interacts with LKB1 in cellulo.

(a) Expression of PRMT5, MEP50 and LKB1 was evaluated in a wide range of human breast tumor cell lines by Western blot using the corresponding antibodies. GAPDH expression was used as a loading control. (b) The PRMT5-LKB1 interaction was assessed by co-immunoprecipitation in MCF-7 cell extracts using an anti-PRMT5 antibody. A rabbit irrelevant antibody was used as a negative control. The presence of PRMT5 and LKB1 was evaluated by Western blot analysis. (c) Detection of LKB1 interaction with PRMT5 and MEP50 was performed by Proximity Ligation Assay (PLA). MCF-7 cells were transfected with Scramble siRNA or siRNA targeting LKB1, PRMT5 or MEP50 for 48 hr. After fixation, PLA experiments were performed to evaluate the interactions between LKB1/PRMT5 and LKB1/MEP50 using LKB1-, PRMT5- and MEP50-specific antibodies. The detected dimers are represented by red dots. The nuclei were counterstained with mounting medium containing DAPI (blue) (Obj: X60). (d) Quantification of the number of dots per cell was performed by computer-assisted analysis as reported in the Materials and Methods section. The mean +/− s.e.m. of one experiment representative of three experiments is shown. The P-value was determined using the Student’s t-test. *** indicates a P < 0.001. (e) The efficacy of protein inhibition was verified by Western blot analysis using the corresponding antibodies.