Journal: The Journal of Biological Chemistry

Article Title: Y225A induces long-range conformational changes in human prion protein that are protective in Drosophila

doi: 10.1016/j.jbc.2023.104881

Figure Lengend Snippet: N174S and Y225A suppress the toxicity of human PrP-WT in the eye. Micrographs of eyes from frozen flies ( A – D ) or fixed for the scanning electron microscope ( E – H ) from control flies expressing mCD8-GFP or human PrP in the eyes ( GMR-Gal4 ). Control flies ( UAS-mCD8-GFP-attP2 ) show highly organized eyes with hexagonal ommatidia ( A and E , inset ). Flies expressing PrP-WT ( UAS-human PrP-V129-attP2 ) show small and disorganized eyes ( B ) with fused ommatidia and irregularly spaced bristles ( F , inset ). Flies expressing N174S ( UAS-human PrP-N174S - attP2 ) show mildly disorganized eyes ( C and G , arrowheads ). Flies expressing Y225A ( UAS-human PrP-Y225A - attP2 ) show almost normal eyes ( D and H , inset ). Scores: 0 (no change) to 3 (largest change) in size (S), organization (O), and pigmentation (P). I , Western blot from fly extracts expressing GFP or PrP in the fly eye probed with anti-β-Tubulin and 3F4 anti-PrP antibodies in two biological replicates. Quantification of total PrP shows no significant differences (pairwise t test): V129 versus N174S, p = 0.76; V129 versus Y225A, p = 0.538; Y225A versus N174S, p = 0.79. J and K , deglycosylation assays. Western blot of PrP treated with (+) or without (−) PNGase F probed with 3F4 ( J ) or 9A2 ( K ) anti-PrP antibodies. The first two lanes show human brain homogenate from homozygous PrP-V129 (VV). PNGase F, peptide N-glycosidase F; PrP, Prion protein.

Article Snippet: Membranes were blocked in TBS-T containing 5% nonfat milk and probed against the primary antibodies: anti-PrP 8H4 (1:10,000, Millipore-Sigma), anti-PrP 3F4 (1:10,000, Millipore-Sigma, Lot# 3150381), anti-β-Tubulin (1:50,000, Thermo Fisher Scientific, Lot# QC216972).

Techniques: Microscopy, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Y225A induces long-range conformational changes in human prion protein that are protective in Drosophila

doi: 10.1016/j.jbc.2023.104881

Figure Lengend Snippet: 2D blot analysis of PrP from human brain and Drosophila . 2D blotting of PrP from human brain and Drosophila homogenates with or without PNGase F treatment. A and B , human brain PrP-129VV; C and D , Drosophila expressing PrP-WT ( GMR-Gal4/UAS-human PrP-V129-attP2 ). E and F , PrP- Drosophila expressing N174S ( GMR-Gal4/UAS-human PrP-N174S - attP2 ); g and h: Drosophila expressing PrP-Y225A ( GMR-Gal4/UAS-human PrP-V129-Y225A - attP2 ). A , C , E , and G , homogenates not treated with PNGase F. B , D , F , and H , homogenates treated with PNGase F. Membranes were probed with the 3F4 anti-PrP antibody. PNGase F, peptide N-glycosidase F; PrP, Prion protein.

Article Snippet: Membranes were blocked in TBS-T containing 5% nonfat milk and probed against the primary antibodies: anti-PrP 8H4 (1:10,000, Millipore-Sigma), anti-PrP 3F4 (1:10,000, Millipore-Sigma, Lot# 3150381), anti-β-Tubulin (1:50,000, Thermo Fisher Scientific, Lot# QC216972).

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Y225A induces long-range conformational changes in human prion protein that are protective in Drosophila

doi: 10.1016/j.jbc.2023.104881

Figure Lengend Snippet: N174S and Y225A lower the toxicity of human PrP in mushroom body cell bodies. A – H , genotypes are the same as in Figure 6 . Some brains were immunostained with 3F4 anti-PrP antibody to examine PrP distribution ( insets ). Control flies expressing LacZ display compact, intense cell clusters at days 1 ( A ) and 35 ( E ). Flies expressing PrP-WT exhibit larger clusters at day 1 ( B ) that continue to expand by day 35 ( F ). Note the swollen cell bodies and the accumulation of intracellular PrP ( B and F , insets). Flies expressing N174S display enlarged clusters at day 1 ( C ) that shrink by day 35 ( G ). Some cell bodies appear swollen and accumulate intracellular PrP ( C and G , insets ). Flies expressing Y225A display small clusters at day 1 ( D ) that do not shrink by day 35 ( H ). The cell bodies are small and do not progressively accumulate intracellular PrP ( D and H , insets ). I , scatter plot for the area of cell body clusters for 1- and 35-day-old flies. Significant differences by pairwise t-tests and Holms post hoc analysis are illustrated. See Table 4 for p -values. J , NaPTA insolubility assay. Flies were aged for 20 days and heads homogenized. The homogenates were incubated in sarkosyl/NaPTA. An aliquot was used as control (T-total), and the rest was centrifuged to separate the soluble (S) and insoluble (P-pellet) fractions. The experiment was repeated in five biological replicates and quantified. PrP-WT and N174S accumulated similar amounts of insoluble PrP, whereas Y225A accumulated about half. NaPTA, Na+ phosphotungstic acid; PrP, Prion protein.

Article Snippet: Membranes were blocked in TBS-T containing 5% nonfat milk and probed against the primary antibodies: anti-PrP 8H4 (1:10,000, Millipore-Sigma), anti-PrP 3F4 (1:10,000, Millipore-Sigma, Lot# 3150381), anti-β-Tubulin (1:50,000, Thermo Fisher Scientific, Lot# QC216972).

Techniques: Expressing, Incubation

Journal: Clinical and Translational Medicine

Article Title: PINK1 modulates Prdx2 to reduce lipotoxicity‐induced apoptosis and attenuate cardiac dysfunction in heart failure mice with a preserved ejection fraction

doi: 10.1002/ctm2.70166

Figure Lengend Snippet: PTEN‐induced kinase 1 (PINK1) deficiency reduces peroxiredoxin‐2 (Prdx2) and exacerbates JNK and p38 phosphorylation leading to myocardial apoptosis. (A) Western blotting for the expression of PINK1, Prdx2, phospho‐p38, p38, phospho‐JNK, JNK, Bcl2, Bax, Cleaved‐Caspase3 and β‐actin in heart tissues. (B) Quantitative analyses of protein expression of PINK1, Prdx2, phospho‐p38, p38, phospho‐JNK, JNK, Bcl2, Bax, Cleaved‐Caspase3 and β‐actin. (C) immunohistochemical of PINK1 in heart tissues of WT and PINK1‐KO mice. Scale bar, 50 µm. (D) immunohistochemical of Prdx2 in heart tissues of WT and PINK1‐KO mice. Scale bar, 50 µm. (E) The apoptosis rate of cardiomyocytes by TUNEL assay Scale bar, 100 µm. For all statistical plots, the data are presented as the means ± SEs. * p < 0.05; ** p < 0.01; *** p < 0.001 using Student t test.

Article Snippet: The antibodies used in this study included anti‐PINK1 (Santa Cruz, sc‐517353, rabbit) and the following from Proteintech: anti‐Prdx2 (10545‐2‐AP, rabbit), anti‐P‐JNK (80024‐1‐RR, rabbit), anti‐P‐p38 (28796‐1‐AP, rabbit), anti‐JNK (24164‐1‐AP, rabbit), anti‐p38 (14064‐1‐AP, rabbit), anti‐Bcl2 (26593‐1‐AP, rabbit), anti‐Bax (50599‐2‐Ig, rabbit), anti‐C‐Caspase3 (68773‐1‐Ig, mouse), anti‐GFP (50430‐2‐AP, rabbit), anti‐ANP (27426‐1‐AP, rabbit) and anti‐β‐actin (66009‐1‐Ig, mouse).

Techniques: Western Blot, Expressing, Immunohistochemical staining, TUNEL Assay

Journal: Clinical and Translational Medicine

Article Title: PINK1 modulates Prdx2 to reduce lipotoxicity‐induced apoptosis and attenuate cardiac dysfunction in heart failure mice with a preserved ejection fraction

doi: 10.1002/ctm2.70166

Figure Lengend Snippet: PTEN‐induced kinase 1 (PINK1) overexpression attenuates myocardial apoptosis by restoring peroxiredoxin‐2 (Prdx2). (A) Western blotting for the expression of PINK1, Prdx2, phospho‐p38, p38, phospho‐JNK, JNK, Bcl2, Bax, Cleaved‐Caspase3 and β‐actin in heart tissues. (B) Quantitative analyses of protein expression of PINK1, Prdx2, phospho‐p38, p38, phospho‐JNK, JNK, Bcl2, Bax, Cleaved‐Caspase3 and β‐actin. (C) immunohistochemical of PINK1 in heart tissues of WT and PINK1‐KO mice. Scale bar, 50 µm. (D) immunohistochemical of Prdx2 in heart tissues of WT and PINK1‐KO mice. Scale bar, 50 µm. (E) The apoptosis rate of cardiomyocytes by TUNEL assay Scale bar, 100 µm. For all statistical plots, the data are presented as the means ± SEs. * p < 0.05; ** p < 0.01; *** p < 0.001 using Student t test.

Article Snippet: The antibodies used in this study included anti‐PINK1 (Santa Cruz, sc‐517353, rabbit) and the following from Proteintech: anti‐Prdx2 (10545‐2‐AP, rabbit), anti‐P‐JNK (80024‐1‐RR, rabbit), anti‐P‐p38 (28796‐1‐AP, rabbit), anti‐JNK (24164‐1‐AP, rabbit), anti‐p38 (14064‐1‐AP, rabbit), anti‐Bcl2 (26593‐1‐AP, rabbit), anti‐Bax (50599‐2‐Ig, rabbit), anti‐C‐Caspase3 (68773‐1‐Ig, mouse), anti‐GFP (50430‐2‐AP, rabbit), anti‐ANP (27426‐1‐AP, rabbit) and anti‐β‐actin (66009‐1‐Ig, mouse).

Techniques: Over Expression, Western Blot, Expressing, Immunohistochemical staining, TUNEL Assay

Journal: Clinical and Translational Medicine

Article Title: PINK1 modulates Prdx2 to reduce lipotoxicity‐induced apoptosis and attenuate cardiac dysfunction in heart failure mice with a preserved ejection fraction

doi: 10.1002/ctm2.70166

Figure Lengend Snippet: PTEN‐induced kinase 1 (PINK1) interacts with peroxiredoxin‐2 (Prdx2) and both are impaired in lipotoxicity. (A) Immunoprecipitation of PINK1 with Prdx2 in NRCM. (B) two alpha structure of PINK1: N‐terminal region (amino acids 1133) and C‐terminal region (amino acids 138500). (C) Detection of immunoprecipitation protein bands in a gel by silver staining. (D) Immunoblot of immunoprecipitation for the GFP and Prdx2. (E) Immunofluorescence co‐localisation of PINK1 with Prdx2 in NRCM. Scale bar, 25 µm. (F) Detection of LDs by Fluorescent Microscopy in palmitic acid gradient concentration. Scale bar, 25 µm. (G) Quantitative analyses of LDs. (H) Western blotting for the expression of PINK1, Prdx2, ANP and β‐actin in palmitic acid gradient concentration. (I) Cell viability of NRCM in palmitic acid gradient concentration. For all statistical plots, the data are presented as the means ± SEs. * p < 0.05; ** p < 0.01; *** p < 0.001 using Student t test.

Article Snippet: The antibodies used in this study included anti‐PINK1 (Santa Cruz, sc‐517353, rabbit) and the following from Proteintech: anti‐Prdx2 (10545‐2‐AP, rabbit), anti‐P‐JNK (80024‐1‐RR, rabbit), anti‐P‐p38 (28796‐1‐AP, rabbit), anti‐JNK (24164‐1‐AP, rabbit), anti‐p38 (14064‐1‐AP, rabbit), anti‐Bcl2 (26593‐1‐AP, rabbit), anti‐Bax (50599‐2‐Ig, rabbit), anti‐C‐Caspase3 (68773‐1‐Ig, mouse), anti‐GFP (50430‐2‐AP, rabbit), anti‐ANP (27426‐1‐AP, rabbit) and anti‐β‐actin (66009‐1‐Ig, mouse).

Techniques: Immunoprecipitation, Silver Staining, Western Blot, Immunofluorescence, Microscopy, Concentration Assay, Expressing

Journal: Clinical and Translational Medicine

Article Title: PINK1 modulates Prdx2 to reduce lipotoxicity‐induced apoptosis and attenuate cardiac dysfunction in heart failure mice with a preserved ejection fraction

doi: 10.1002/ctm2.70166

Figure Lengend Snippet: siPINK1 aggravates neonatal rat cardiomyocyte (NRCM) lipotoxicity through peroxiredoxin‐2 (Prdx2) reduction. (A) Western blotting for the expression of PINK1, Prdx2, phospho‐p38, p38, phospho‐JNK, JNK, Bcl2, Bax, Cleaved‐Caspase3 and β‐actin in myocardial lipotoxicity. (B) immunofluorescence of PINK1 in myocardial lipotoxicity. (C) Quantitative analyses of PINK1 positive area. (D) immunofluorescence of Prdx2 in myocardial lipotoxicity. (E) DCHF staining to reflect myocardial ROS production. Scale bar, 50 µm. For all statistical plots, the data are presented as the means ± SEs. * p < 0.05; ** p < 0.01; *** p < 0.001 using Student t test.

Article Snippet: The antibodies used in this study included anti‐PINK1 (Santa Cruz, sc‐517353, rabbit) and the following from Proteintech: anti‐Prdx2 (10545‐2‐AP, rabbit), anti‐P‐JNK (80024‐1‐RR, rabbit), anti‐P‐p38 (28796‐1‐AP, rabbit), anti‐JNK (24164‐1‐AP, rabbit), anti‐p38 (14064‐1‐AP, rabbit), anti‐Bcl2 (26593‐1‐AP, rabbit), anti‐Bax (50599‐2‐Ig, rabbit), anti‐C‐Caspase3 (68773‐1‐Ig, mouse), anti‐GFP (50430‐2‐AP, rabbit), anti‐ANP (27426‐1‐AP, rabbit) and anti‐β‐actin (66009‐1‐Ig, mouse).

Techniques: Western Blot, Expressing, Immunofluorescence, Staining

Journal: Clinical and Translational Medicine

Article Title: PINK1 modulates Prdx2 to reduce lipotoxicity‐induced apoptosis and attenuate cardiac dysfunction in heart failure mice with a preserved ejection fraction

doi: 10.1002/ctm2.70166

Figure Lengend Snippet: adPINK1 attenuates neonatal rat cardiomyocyte (NRCM) lipotoxicity by increasing peroxiredoxin‐2 (Prdx2). (A) Western blotting for the expression of PINK1, Prdx2, phospho‐p38, p38, phospho‐JNK, JNK, Bcl2, Bax, Cleaved‐Caspase3 and β‐actin in myocardial lipotoxicity. (B) immunofluorescence of PINK1 in myocardial lipotoxicity. (C) Quantitative analyses of PINK1 positive area. (D) immunofluorescence of Prdx2 in myocardial lipotoxicity. (E) DCHF staining to reflect myocardial ROS production. Scale bar, 50 µm. For all statistical plots, the data are presented as the means ± SEs. * p < 0.05; ** p < 0.01; *** p < 0.001 using Student t test.

Article Snippet: The antibodies used in this study included anti‐PINK1 (Santa Cruz, sc‐517353, rabbit) and the following from Proteintech: anti‐Prdx2 (10545‐2‐AP, rabbit), anti‐P‐JNK (80024‐1‐RR, rabbit), anti‐P‐p38 (28796‐1‐AP, rabbit), anti‐JNK (24164‐1‐AP, rabbit), anti‐p38 (14064‐1‐AP, rabbit), anti‐Bcl2 (26593‐1‐AP, rabbit), anti‐Bax (50599‐2‐Ig, rabbit), anti‐C‐Caspase3 (68773‐1‐Ig, mouse), anti‐GFP (50430‐2‐AP, rabbit), anti‐ANP (27426‐1‐AP, rabbit) and anti‐β‐actin (66009‐1‐Ig, mouse).

Techniques: Western Blot, Expressing, Immunofluorescence, Staining

Journal: Clinical and Translational Medicine

Article Title: PINK1 modulates Prdx2 to reduce lipotoxicity‐induced apoptosis and attenuate cardiac dysfunction in heart failure mice with a preserved ejection fraction

doi: 10.1002/ctm2.70166

Figure Lengend Snippet: siPrdx2 attenuates the alleviating effect of adPINK1 on neonatal rat cardiomyocyte (NRCM) lipotoxicity. (A) Western blotting for the expression of PINK1, Prdx2, phospho‐p38, p38, phospho‐JNK, JNK, Bcl2, Bax, Cleaved‐Caspase3 and β‐actin in myocardial lipotoxicity. (B) Quantitative analyses of protein expression of PINK1, Prdx2, phospho‐p38, p38, phospho‐JNK, JNK, Bcl2, Bax, Cleaved‐Caspase3 and β‐actin. (C) immunofluorescence of PINK1 in myocardial lipotoxicity. (D) Quantitative analyses of PINK1 positive area. (E) immunofluorescence of Prdx2 in myocardial lipotoxicity. (F) Quantitative analyses of Prdx2 positive area. (G) DCHF staining to reflect myocardial ROS production. Scale bar, 50 µm. (H) Quantitative analyses of DCFH staining. (I) The apoptosis rate of cardiomyocytes by TUNEL assay. Scale bar, 100 µm. (J) Quantitative analyses of TUNEL positive area. For all statistical plots, the data are presented as the means ± SEs. * p < 0.05; ** p < 0.01; *** p < 0.001 using Student t test.

Article Snippet: The antibodies used in this study included anti‐PINK1 (Santa Cruz, sc‐517353, rabbit) and the following from Proteintech: anti‐Prdx2 (10545‐2‐AP, rabbit), anti‐P‐JNK (80024‐1‐RR, rabbit), anti‐P‐p38 (28796‐1‐AP, rabbit), anti‐JNK (24164‐1‐AP, rabbit), anti‐p38 (14064‐1‐AP, rabbit), anti‐Bcl2 (26593‐1‐AP, rabbit), anti‐Bax (50599‐2‐Ig, rabbit), anti‐C‐Caspase3 (68773‐1‐Ig, mouse), anti‐GFP (50430‐2‐AP, rabbit), anti‐ANP (27426‐1‐AP, rabbit) and anti‐β‐actin (66009‐1‐Ig, mouse).

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, TUNEL Assay