Fig. 4

BCRP3 is mainly distributed to the cytoplasm and binds VPS34 complex. A Subcellular localization of BCRP3 in HeLa cells detected by cell fractionation followed by qRT-PCR analysis. GAPDH and Neat1 were used as cytoplasmic and nuclear RNA controls, respectively. B RNA FISH analysis of BCRP3 (red) in HeLa cells. Nuclei were stained with DAPI (blue). Bar, 10 μm. C In vitro RNA protein binding assay. GFP-ATG14 transiently expressed in 293T cells was immunoprecipitated with GFP-Trap beads and the immunocomplexes were incubated with in vitro transcribed BCRP3. The bound RNAs were extracted and analyzed by qRT-PCR, whereas the presence of VPS34 complex components in the immunocomplexes was analyzed by Western blot (left). Data are means ± SD from three independent experiments. P values are determined by unpaired t-test, ***P < 0.001. D-H RNA immunoprecipitation assay using indicated antibodies followed by qRT-PCR analysis. Data were normalized to the IgG control and expressed as means ± SD, n = 3. P values are determined by unpaired t-test, *P < 0.05, **P < 0.01, ***P < 0.001