Figure 3

Vimentin influences the IRBIT-IP3R1 interaction by sequestering IRBIT in perinuclear inclusions. A. HeLa cells were transfected with RFP or tested forms of RFP-vimentin. 24 hrs later, cells were lysed and immunopreciptitation was performed using 10A6 anti-IP3R1 antibody. For immunoblotting, KM1112 anti-IP3R1, anti-IRBIT and anti-RFP antibodies were used. B. Quantification of IRBIT-IP3R1 interaction. The densities of immunoprecipitated IRBIT were normalized to the densities of corresponding immunoprecipitated IP3R1. *p = 0.0002, **p = 0.009, ***p = 0.0001, ^#p = 0.0017, ^##p = 0.029, ^###p = 0.0009. Bars represent relative mean values ± s.d. from three independent experiments, with levels of IRBIT-IP3R1 interaction under control conditions normalized to a value of 1. C. Representative confocal images show distribution of immobile fraction of IRBIT in presence of RFP or tested RFP-vimentin forms. Neuro2a cells were transfected with RFP or RFP-vimentins, 24 hrs later permeabilized with saponin and immunostained for endogenous IRBIT (green). Fluorescence of RFP (red) and DAPI (blue) are also shown. Note that RFP was washed out from cells during saponin permeabilization. Scale bar, 5 μm.