GPR81 nuclear transportation is critical for cancer growth and progression in lung and other solid cancers

Figure 3 GPR81 is translocated to cell nucleus with importin beta. A: Anti-importin beta was used to immunoprecipitate proteins from lung cancer cell with lactate or vehicle overnight treatment and the precipitated material was used in Western blot analysis. Densitometry values summarizing western blot data are shown in the right panel. Ig served as a loading control. Cell H661 was used; B: GPR81 expression levels were quantified in lung cancer cell exposed to 5 mmol/L lactate, with or without ivermectin (10 μmol/L) vs vehicle control in western blot (middle panel). Densitometry values summarizing western blot data are shown in the right panel. GAPDH served as a loading control. A549, H661, H858, H645 were used; C-E: GPR81 expression level in cell fractions of GPR81 NLS mutant transduced lung cancer cells, GPR81 wild-type transduced and empty vector transduced cells cultured under lactate or vehicle conditions were measured with western blot; D and E: Loading markers were GAPDH for cytoplasm, cadherin for cell membrane and lamin for nucleus. Densitometry values summarizing western blot data are shown in the right panel. GAPDH served as a loading control. n = 4 Lung cancer cells (A549, H661, H858, H645); F: Immunoflurescent stain was performed on human lung cancer A549 cells (transduced with Empty vector, GPR81 wild-type and GPR81 NLSmutant as marked) with GPR81 antibody. Scale bar = 20 μmol/L.