Fig 1: Effects of the different timing of α-syn incubation on mitochondrial stress and phagocytosis. (A) Total area of ROS signal (control = 22 ± 4) and (B) area of CytC (control = 58 µm2 ± 4) in TH(+) after intoxication with α-syn were studied after different incubation durations. (C) Representative pictures of TH neurons, control (left panel) and α-syn (right panel) for CytoC. (D) AIF (control = 42 ± 5) in TH(+) as well as (E) Lamp2(+) vesicles (control = 46 ± 3) and (F) area of LC3b(+) vesicles (control = 29 µm2 ± 3) after application of α-syn were also studied. All values were expressed as mean ± SEM; *, p < 0.05 with Two-way ANOVA followed by Fisher’s LSD test.
Fig 2: Gene expression in INS(832/13) cells. mRNA expression in cells cultured in LG, NT or EBSS in the presence or absence of E16 or UV-inactivated E16 (E16UV): Atg3 (a), Atg5 (b), Atg7 (c), Atg9a (d), Atg10 (e), Atg12 (f), Lamp2 (g), Stx17 (h) and Uvrag (i). Data are presented as mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001
Fig 3: α-syn/CBE mice present with progressive mitochondrial stress and autolysosomal stress in the substantia nigra. Total area of CytC (control = 1150 µm2 ± 10) (A), as well as area of LC3b (control = 7 µm2 ± 9) (B) and Lamp2 (control = 2027 µm2 ± 13) (C) in TH(+) after stereotaxic injection of α-syn were studied. (D) Representative pictures of TH(+) control (left panel) and α-syn (right panel) for Lamp2 (green). All values are expressed as mean ± SEM; *, p < 0.05 with one-way ANOVA followed by Fisher’s LSD test.
Fig 4: Protein expression levels of (A) LC3-II, (B) beclin-1, (C) LAMP-2 and (D) Rab-7 autophagy-associated markers using western blotting inrenal tissues (n=8/group) from rats with ischemia/perfusion injury following treatment with niclosamide (25 mg/kg), 3-MA (15 mg/kg) or rapamycin (10 mg/kg). Values are presented as the mean ± standard deviation. *P<0.05 vs. sham. LC3-II, microtubule-associated protein 1A/1B light chain 3B; LAMP-2, lysosome-associated membrane protein 2; 3-MA, 3-methyladenine; I/R, ischemia/perfusion injury.
Fig 5: BRD4 inhibition upregulated SIRT1 and inhibition of SIRT1 reversed the effects of BRD4 inhibition on autophagic flux. (A) mRNA level of Sirt1 at 4 h after CCK stimulation in isolated pancreatic acinar cells (n = 3). (B) Immunoblot analysis of SIRT1 level at 4 h after CCK stimulation (n = 5). (C) Immunoblot analysis for LC3B and p62 expression in isolated pancreatic acinar cells pretreated with 500 nmol·L-1 JQ1 or 10 μmol·L-1 EX527 followed by stimulation with 200 nmol·L-1 CCK for 4 h (n = 4). (D) Immunoblot analysis for ATG14, STX17, and LAMP2 expression (n = 5). (E) Immunoblot analysis for cathepsin B (n = 3) and cathepsin L expression (n = 5). (F) The activities of cathepsin B (n = 3) and cathepsin L (n = 3). Data represent the mean values ± SEM. Statistical analysis was performed by Student’s un-paired, two-tailed t-test between two groups, *P < 0.05, compared to the control; #P < 0.05, compared to CCK-stimulated group; +P < 0.05, compared to JQ1-treated group.
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