Fig 1: No correlation in calf muscle or retinal CTSB to functional protection. (A) Active animals across all three exercise intensity groups were divided into high- and low-muscular CTSB levels. Animals with a calf muscle CTSB level 10% higher than the max inactive+LIRD animal were classified as “high CTSB” and compared with those below the threshold “low CTSB” for dark-adapted b-wave amplitude. The brightest flash dark-adapted b-wave amplitudes showed no correlation with calf muscle CTSB (B) and retinal CTSB protein (C). Circles represent measurements for individual animals. Data in A depicted as mean ± SEM. Linear regression line shown in B and C. Amp, amplitude.
Fig 2: CTSB gene expression and protein levels altered in retina after exercise. CTSB protein in calf muscle was analyzed by exercise level (A) and when comparing all inactive (inactive+LIRD and naïve) mice to all exercised mice (B). CTSB protein levels were also examined in the brain (C) and serum (D). In the retina, CTSB gene expression (E) and protein levels (F) were measured. Data are depicted as mean ± SEM normalized to inactive+LIRD for A and C–E and to all inactive in B. Circles represent measurements for individual animals. Colored asterisks refer to the difference between the group they are closest to and the group of the referenced color. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 3: Lysosome and autophagy in mutant cells. (A) Western blotting analysis of proteins associated with lysosome [CTSB, CTSK, LAMP2, ATP6V0D2, ATP6V1B2, GRN (Progranulin), LGALS3 (Galectin 3)], autophagy (ATG13, LC3, P62, mMTOR, p-mTOR, ULK1, p-ULK1), lipid metabolism and microglial functions (APOE, CD36) from cell lysates of WT and mutant BV-2 cells. The histogram represents the LC3-II/LC3-I ratio, p-mTOR/mTOR ratio, and p-ULK1/ULK1 ratio obtained from densitometric analysis shown in Supplementary Figure 3. Source data are available online for this figure. (B) Transmission electron micrographs of WT (1) and Abcd1−/−Abcd2−/− (2, 3, 4) BV-2 cells illustrating the presence of autophagic figures in the mutant cells (bar = 500 nm). The arrows indicate diaminobenzidine-stained peroxisomes (1), a late autophagic compartment (2), an autophagosome engulfing mitochondria (3) and pexophagy (4). Although less numerous, similar observations were made in the Abcd1−/− and in the Acox1−/− cells but not in the Abcd2−/− cells.
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