FIG 6.

PIKfyve modulates lysosomal protein expression independently of transcription. (A) Immunoblot analysis of F4/80⁺ spleen macrophages for lysosomal and autophagy-related proteins: M6PR, LAMP1, p62, procathepsin D, cathepsin D, LC3-I, and LC3-II. Vinculin was used as a loading control. (B) qRT-PCR analysis of cathepsin D, LAMP1, and LC3 relative to GAPDH in F4/80⁺ spleen macrophages (n = 3 mice). (C) Immunoblot analysis of F4/80⁺ spleen macrophages probed with TFEB antibody. TFEB protein bands 1, 2, and 3 were cut for mass spectrometry analysis. (D) Proteomic analysis of TFEB protein bands 1, 2, and 3 in panel C. In red are the tryptic peptides identified in each band by mass spectrometry analysis. The sequence with UniProt number Q3UKG7 was used for the amino acid sequence of murine TFEB. (E) Immunoblot analysis of the F4/80⁺ spleen macrophages for PIKfyve, phospho-S6, S6, phospho-4EBP1, and 4EBP1. Probing for vinculin was used as the loading control. Statistical analysis was performed using unpaired two-tailed Student’s t test (NS, P > 0.05). All error bars indicate SEM.