Fig. 6.

UBXN6 promotes TFEB nuclear translocation-mediated lysosomal activation in macrophages during LPS stimulation. A–D Phosphorylated and total protein levels associated with the AMPK and mTOR signaling pathways in BMDMs stimulated with LPS (100 ng/mL) for the indicated times; ACTB was used as a loading control (A). Relative quantifications are shown for phospho-AMPK normalized to total AMPK (B), phospho-mTOR normalized to total mTOR (C), and phospho-S6K1 normalized to total S6K1 (D). E, F Confocal microscopy images of immunostained TFEB (green) and DAPI (blue, for nuclei) (E) and the percentage of TFEB nuclear translocation (F) obtained from BMDMs treated with LPS (100 ng/mL) for the indicated times. The white arrows indicate TFEB in the nucleus. G Representative images of BMDMs immunostained with LAMP1 (orange) and DAPI (blue, for nuclei) after stimulation with LPS (100 ng/mL) for 2 h. H Mean fluorescence intensities of LAMP1 in BMDMs stimulated with LPS (100 ng/mL) for the indicated periods determined by FIJI software. I, J Western blot analysis of LAMP1 proteins (I) and their relative levels normalized to those of ACTB (J) in BMDMs primed with LPS (100 ng/mL) for the indicated periods. Two-tailed Student’s t tests (B–D, F, H, and J) were used to determine statistical significance. LPS, lipopolysaccharide; n.s., not significant; MFI, mean fluorescence intensity. The data represent the means ± SD (B–D, F, H, and J) from at least three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001