Phospho-PINK1 (Ser228) Antibody - #AF7081

Fig. 5 PINK1-PARK2 pathway contributed to EPC proliferation. a Representative western blots showed that shRNA targeting Pink1 (shPink1) effectively silenced PINK1 protein expression and shRNA targeting Park2 (shPark2) effectively silenced PARK2 protein expression after 24 h infection, respectively. Representative western blots and quantitative analysis revealed that either silencing Pink1 (b, d) or Park2 (c, e) significantly reduced MAP1LC3B expression. CCK-8 assay showed that either silencing Pink1 (f) or Park2 (g) before PTV treatment significantly reduced proliferative activity compared with PTV alone group. h EPCs of silencing Pink1 were seeded on E-plates for 24 h, 0.5 μM PTV was added to E-plates in PTV and shPink1 + PTV groups after 24 h incubation (black arrow). The normalized cell index indicated that EPC proliferative activity decreased in shPink1 + PTV group compared with PTV alone group. i EPCs of silencing Park2 were seeded on E-plates for 24 h, 0.5 μM PTV was added to E-plates in PTV and shPark2 + PTV groups after 24 h incubation (black arrow). The normalized cell index indicated that EPC proliferative activity decreased in shPark2 + PTV group compared with PTV alone group. j Western blots of the expression of PINK1, PARK2 and their phosphorylated form in WT and Pink1−/− mice. EPCs with or without 0.5 μM PTV treatment were seeded on E-plates for 24 h, 0.5 μM in PTV and Pink1−/− + PTV groups after 24 h incubation. The normalized cell index (k) and CCK-8 (l) indicated that EPC proliferative activity fell down in WT and Pink1−/− mice with or without PTV treatment. (EPCs were isolated from ApoE−/− mice fed with high-fat diet for 8 weeks, cells were isolated from 3 mice for 1 experiment and 3 independent experiments were performed, mean ± SD, *P < 0.05, **P < 0.01).

Fig 5 PRV infection activates mitophagy via the Pink1-Parkin pathway in PK-15 cells. (A, B) NIX, BNIP 3, FUNDC1, PINK1, p-PINK1(Ser228), Parkin, p-Parkin (Ser65), and gB protein levels were determined by Western blot upon G-PRV infection (MOI = 1) at 12, 18, and 24 hpi. (C) Parkin protein levels were measured by Western blot in mitochondrial lysates upon G-PRV infection (MOI = 1) at 12, 18, and 24 hpi or 10 µM CCCP treatment for 6 and 12 h. (D) Translocation of Parkin (green) to mitochondria (TOM20, red) was visualized by confocal microscopy upon W-PRV infection (MOI = 1) at 12 hpi or 10 µM CCCP treatment for 12 h. Co-localization analysis was performed using Image J. Scale bars: 10 µm. (E) Location of p-Ub (Ser65) (green) on mitochondria (TOM20, red) was visualized by confocal microscopy upon W-PRV infection (MOI = 1) at 18 hpi or 10 µM CCCP treatment for 12 h. Co-localization analysis was performed using Image J. Scale bars: 10 µm. (F) Ubiquitination of MFN2 was measured by Western blot upon G-PRV infection (MOI = 1) at 18 hpi or 10 µM CCCP treatment for 12 h. Normal rabbit IgG or mouse IgG was used as a negative control for immunoprecipitation. (G–I) Colocalization of mitochondria (TOM20, red) with autophagy receptors (p62, NDP52, Optineurin, green, respectively) was visualized by confocal microscopy upon W-PRV infection (MOI = 1) at 18 hpi. Co-localization analysis was performed using Image J. Scale bars: 10 µm. (J) p-PINK1(Ser228), p-Parkin (Ser65), and gB protein levels were determined by Western blot upon infection with G-PRV or treatment with UV-PRV (equivalent of MOI = 1) at 12, 18, and 24 hpi. (K) p-PINK1(Ser228), p-Parkin (Ser65), and gB protein levels were determined by Western blot upon G-PRV (MOI = 1) infection at 12, 18, and 24 hpi in the absence or presence of PAA (300 µM) treatment.

FIG. 6. Niclosamide activates PINK1-parkin-ubiquitin pathway to induce mitophagy and autophagy. (A, B) Western blot analysis after treatment with or without 100 nM 4-OHT, 100 nM MG-132, and niclosamide (0.63e20 m M) for 3 days. (A) Western blots were probed with anti-STAT3, anti-phospho-STAT3 (Tyr705), anti-TDP-43, and anti-GAPDH. (B) Western blots were probed with anti-PINK1, anti-phospho-PINK1 (Ser228), anti-parkin, anti-phospho-parkin (Ser65), anti-phospho-ubiquitin (Ser65), anti-LC3, anti-SQSTM1/p62 and anti-GAPDH. (C) Measurement of TDP-43 localization changes in the cytoplasm and nucleus after treatment with 100 nM MG-132 and STAT3 inhibitors (niclosamide, stattic, C188e9, S3I-201, or nifuroxazide) for 3 days. Blank, without MG-132; control,100 nM MG-132; STAT3 inhibitors,100 nM MG-132 with 0.63e20 m M compounds. N ¼ 3, mean ? SD. * P < 0.01, ** P < 0.001 vs control, Tukey’s multiple comparison test. (D) Western blot analysis after treatment with 100 nM 4-OHT, with or without 100 nM MG-132, and 10 m M STAT3 inhibitors (niclosamide, stattic, C188-9, S3I-201, nifuroxazide) for 3 days. Western blots were probed with anti-STAT3, anti-phospho-STAT3 (Tyr705), anti-phospho-ubiquitin (Ser65), anti-LC3, and anti-GAPDH. (AeD) Sililar results were obtained in N ¼ 3 biologically independent experiments.