Journal: Physiological Reports

Article Title: Sex‐specific mitochondrial dynamics and mitophagy response to muscle damage

doi: 10.14814/phy2.15230

Figure Lengend Snippet: Gene and protein expression analysis for (a and b) PINK1 and (c and d) Parkin. For gene expression analysis [men (M): n = 8; women (W): n = 8], data were normalized to Men ECC + REST at BL. A time × sex × condition interaction was observed for PINK1 . For protein expression analysis [M: n = 6; W: n = 6], data were normalized to Men ECC + REST at BL. A sex × condition × time interaction was observed for Parkin. A time × sex interaction was observed for Parkin. A condition × sex interaction was observed for Parkin. The box and whisker plots display an example of BL, 12 h, and 24 h loading gene and protein expressions for PINK1 and Parkin (see Figure 2b for the corresponding GAPDH). MW, molecular weight (kDa). * p < 0.05 versus ECC + REST. φ P < 0.05 versus BL. $ p < 0.05 versus ECC+REST. γ P< 0.05 versus BL. # p < 0.05 versus BL

Article Snippet: Samples were then transferred electrophoretically to a PVDF membrane at 70 V at 4°C for 2.5 h. Next, 5% non‐fat milk powder was used to block the membranes for 1 h, and after that, the membrane was incubated with primary antibodies against OPA1 (1:1000; Cell Signaling, 80471S; 80 kDa), DRP1 (1:1000; Novus, NB110‐55288; 80 kDa), PINK1 (1:2000; Novus, NBP1‐49678; 55 kDa), Parkin (1:1000; Cell Signaling, 2132S; 55 kDa), and GAPDH (1:5000; Santa Cruz, sc‐365062; 37 Membranes were stripped) with 3% non‐fat milk at 4°C overnight.

Techniques: Expressing, Whisker Assay, Molecular Weight

Journal: Physiological Reports

Article Title: Sex‐specific mitochondrial dynamics and mitophagy response to muscle damage

doi: 10.14814/phy2.15230

Figure Lengend Snippet: Bivariate correlational analysis for gene expression of (a) DRP1 , (b) PINK1 and the protein expression of (c) OPA1, (d) PINK1, and (e) Parkin with testosterone area under the curve. The average of 12 and 24 h were used for gene and protein expression in correlational analyses

Article Snippet: Samples were then transferred electrophoretically to a PVDF membrane at 70 V at 4°C for 2.5 h. Next, 5% non‐fat milk powder was used to block the membranes for 1 h, and after that, the membrane was incubated with primary antibodies against OPA1 (1:1000; Cell Signaling, 80471S; 80 kDa), DRP1 (1:1000; Novus, NB110‐55288; 80 kDa), PINK1 (1:2000; Novus, NBP1‐49678; 55 kDa), Parkin (1:1000; Cell Signaling, 2132S; 55 kDa), and GAPDH (1:5000; Santa Cruz, sc‐365062; 37 Membranes were stripped) with 3% non‐fat milk at 4°C overnight.

Techniques: Expressing

Journal: Physiological Reports

Article Title: Sex‐specific mitochondrial dynamics and mitophagy response to muscle damage

doi: 10.14814/phy2.15230

Figure Lengend Snippet: Bivariate correlational analysis for gene expression of (a) MFN1 , (b) DRP1 , and protein expression of (c) Parkin with the cortisol area under the curve (AUC) and gene expression of (d) PINK1 with growth hormone AUC. The average of 12 and 24 h were used for gene and protein expression in correlational analyses

Article Snippet: Samples were then transferred electrophoretically to a PVDF membrane at 70 V at 4°C for 2.5 h. Next, 5% non‐fat milk powder was used to block the membranes for 1 h, and after that, the membrane was incubated with primary antibodies against OPA1 (1:1000; Cell Signaling, 80471S; 80 kDa), DRP1 (1:1000; Novus, NB110‐55288; 80 kDa), PINK1 (1:2000; Novus, NBP1‐49678; 55 kDa), Parkin (1:1000; Cell Signaling, 2132S; 55 kDa), and GAPDH (1:5000; Santa Cruz, sc‐365062; 37 Membranes were stripped) with 3% non‐fat milk at 4°C overnight.

Techniques: Expressing

Journal: British Journal of Pharmacology

Article Title: Afzelin ameliorates D‐galactosamine and lipopolysaccharide‐induced fulminant hepatic failure by modulating mitochondrial quality control and dynamics

doi: 10.1111/bph.13669

Figure Lengend Snippet: Effect of afzelin on mitophagy in GalN/LPS‐treated mice. Mice received an i.p. injection of vehicle or afzelin (200 mg·kg−1) 1 h before GalN (800 mg·kg−1)/LPS (40 μg·kg−1) treatment. Western blot analysis was performed to measure the protein levels of (A) mitochondrial PINK1 and (B) parkin as well as (C) the hepatic ratio of LC3‐II/LC3‐I and (D) p62 expression level in the afzelin‐treated group. The expression of each protein was adjusted to GAPDH or β‐actin as the loading control. The ratio of the LC3‐II and LC3‐I bands was evaluated by densitometric analysis. All values are expressed as mean ± SEM of 10 mice per group. * Significantly different (P < 0.05) from the control group. + Significantly different (P < 0.05) from the GalN/LPS group.

Article Snippet: The primary antibodies used were as follows: mitofusin 2 (Mfn2), nuclear respiratory factor 1 (Nrf1), sequestosome1/p62 (p62), PGC1α, SIRT1, mitochondrial transcription factor A (TFAM) (Abcam, Cambridge, MA, USA), phosphorylated AMPK (phospho‐AMPK), Rev‐Erb‐α (Cell Signalling Technology, Beverly, MA, USA), microtubule‐associated protein 1 light chain 3 (LC3) (Novus Biologicals, Littleton, CO, USA), dynamin‐related protein 1 (Drp1), GAPDH, Parkin, PTEN‐induced putative kinase 1 (PINK1) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and β‐actin (Sigma‐Aldrich).

Techniques: Injection, Western Blot, Expressing

Journal: British Journal of Pharmacology

Article Title: Afzelin ameliorates D‐galactosamine and lipopolysaccharide‐induced fulminant hepatic failure by modulating mitochondrial quality control and dynamics

doi: 10.1111/bph.13669

Figure Lengend Snippet:

Article Snippet: The primary antibodies used were as follows: mitofusin 2 (Mfn2), nuclear respiratory factor 1 (Nrf1), sequestosome1/p62 (p62), PGC1α, SIRT1, mitochondrial transcription factor A (TFAM) (Abcam, Cambridge, MA, USA), phosphorylated AMPK (phospho‐AMPK), Rev‐Erb‐α (Cell Signalling Technology, Beverly, MA, USA), microtubule‐associated protein 1 light chain 3 (LC3) (Novus Biologicals, Littleton, CO, USA), dynamin‐related protein 1 (Drp1), GAPDH, Parkin, PTEN‐induced putative kinase 1 (PINK1) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and β‐actin (Sigma‐Aldrich).

Techniques:

Journal: bioRxiv

Article Title: Lipid Dysregulation Unveil the Intricate Interplay of Lysosomal and Mitochondrial Changes in Frontotemporal Dementia with GRN Haploinsufficiency

doi: 10.1101/2024.01.22.576606

Figure Lengend Snippet: A. Protein Levels of LC3B-II and P62 Assessed by Western Blotting in Primary Human Fibroblasts. Primary human fibroblasts were cultured with or without a 6-hour STV pre-assay period and treated with CLQ at 30 µM for 5 hours. Western blotting was performed to assess the protein levels of LC3B-II and P62, and quantification are shown as bar plot graphs. B. Representative Images of LC3B Puncta (green) and DAPI (blue) Staining in Primary Human Fibroblasts. Primary human fibroblasts were treated with or without a 5-hour pre-assay period with CLQ at 30 µM. LC3B puncta (green) staining and DAPI (blue) staining were used to visualize autophagosomes and nuclei, respectively. Scale bar = 100 µm. C. Representative TEM images of primary human fibroblasts, showing autophagosomes (purple). Insets: Autophagosome. Scale bar is indicated in the figure. D. Protein Levels of LC3B-II and PINK1 Analyzed by Western Blotting in Cytoplasmic and Mitochondrial Fractions of Primary Human Fibroblasts. Primary human fibroblasts were treated with or without a 5-hour pre-assay period with CLQ at 30 µM. Western blotting was performed to analyze the protein levels of LC3B-II and PINK1 in both cytoplasmic and mitochondrial fractions. E. Representative Images of LC3B (green) and TOM20 (red) Staining in Primary Human Fibroblasts. Bar plot shows the quantification of the relative area of LC3B colocalized with TOM20. Scale bar = 10 µm. Healthy control fibroblasts(CTL), FTD-GRN patient fibroblast (GRN). Starving (STV), Chloroquine (CLQ), non treated (NT). Data are presented as means ± SEMs (n = 3). Statistical significance represented by *p < 0.05 and **p < 0.01, determined using Student’s t test.

Article Snippet: Primary Antibodies: PGRN (1μg/mL, AF2420, R&D systems), LAMP-1(1:10 000, ab25630, Abcam), LAMP-2 (1:1 000, #49067, Cell Signaling), LC3Bxp (1:1 000, #3868, Cell Signaling), SQSTM1/P62 (1:1 000, #5114, Cell Signaling), β-Tubulin (1:5 000, MA5-16308, Thermo Fisher) PINK1 (1:1 000, BC100-494, Novus Bio) TOM20 (1:1 000, 11802-1-AP, Proteintech), GAPDH (1:50 000, 60004-1-Ig, Proteintech), Lamin A+C (1:10 000, ab108595, Abcam), TDP-43 (1:1 000, ab104223, Abcam).

Techniques: Western Blot, Cell Culture, Staining

Journal: Autophagy

Article Title: The pro-oxidant adaptor p66SHC promotes B cell mitophagy by disrupting mitochondrial integrity and recruiting LC3-II

doi: 10.1080/15548627.2018.1505153

Figure Lengend Snippet: Commercial antibodies.

Article Snippet: Antibody Host Species Catalog number Source WB dilution IF dilution IP dilution Anti-ACTB mouse MAB1501 Merck Millipore 1:10,000 - - Anti-LC3B rabbit 3868 Cell Signaling Technology 1:500 1:100 1:500 Anti-MT-CO2 mouse Ab110258 Abcam 1:200 - - Anti-COX4I1/COXIV mouse Ab33985 Abcam 1:20,000 - - Anti-p-PRKAA/AMPKα rabbit 2535 Cell Signaling Technology 1:1000 1:100 - Anti- PRKAA/AMPKα rabbit 5832 Cell Signaling Technology 1:1000 - - Anti-p-MTOR rabbit 2448 Cell Signaling Technology 1:200 - - Anti-MTOR rabbit 2972 Cell Signaling Technology 1:250 - - Anti-Ubiquitin mouse 3936 Cell Signaling Technology 1:300 1:100 - Anti-PINK1 rabbit sc-33,796 Santa Cruz Biotechnology 1:500 - - Anti-GFP mouse A11120 Invitrogen 1:100 1:100 1:500 Anti-GFP rabbit A11122 Invitrogen 1:500 - 1:500 Anti-GOLGA2/GM130 mouse 610,822 BD Pharmingen 1:500 - - Anti-LAMP1 mouse 328,602 BioLegend 1:500 1:400 - Anti-PHB mouse CP34 Calbiochem 1:1000 - - Anti-PRKN rabbit sc-30,130 Santa Cruz Biotechnology - 1:10 - Anti-TOMM20 mouse sc-17,764 Santa Cruz Biotechnology 1:500 1:500 - Anti-TOMM20 rabbit sc-11,415 Santa Cruz Biotechnology 1:500 1:500 - Anti-BECN1 rabbit 3495 Cell Signaling Technology 1:500 - - Anti-PIK3C3/VPS34 rabbit D9A5 Cell Signaling Technology 1:1000 - - Anti-SHC rabbit 06–203 Merck Millipore 1:500 - 1:500 Anti-OPA1 mouse 612,606 BD Pharmingen 1:1000 - - Anti-MFN1 mouse ABC41 Merck Millipore 1:1000 - - Anti-DNM1L/DRP1 rabbit 611,112 BD Pharmingen 1:500 - - Anti-TIMM23 rabbit ab116329 Abcam 1:5000 - - Anti-HIF1A mouse 610,958 BD Pharmingen 1:2000 - - APC-anti-IgG1 mouse 560,089 BD Pharmingen - 1:50 - PE-anti-SDC1/CD138 mouse 142,503 BioLegend - 1:50 - Anti-RFP rabbit 600–401-379 Rockland Immunochemicals - 1:100 - Open in a separate window WB, western blot; IF, immunofluoscence; IP, immunoprecipitation Commercial antibodies.

Techniques: