Journal: EMBO Reports

Article Title: Lipid and protein content profiling of isolated native autophagic vesicles

doi: 10.15252/embr.202153065

Figure Lengend Snippet:

Article Snippet: Donkey polyclonal anti‐mouse‐Cy5 (IF 1:200) , Jackson ImmunoResearch , 715‐175‐150.

Techniques: Protease Inhibitor, Software, Microscopy, Laser-Scanning Microscopy, Mass Spectrometry, Transmission Assay

Journal: The EMBO Journal

Article Title: p21 regulates expression of ECM components and promotes pulmonary fibrosis via CDK4 and Rb

doi: 10.1038/s44318-024-00246-7

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Cy5 AffiniPure Goat Anti-Rabbit IgG (H + L) antibody , , Jackson ImmunoResearch , , , Cat# 111-175-144; RRID: AB_2338013.

Techniques: Sequencing, Electron Microscopy, Formulation, SYBR Green Assay, Red Blood Cell Lysis, Plasmid Preparation, Software, Real-time Polymerase Chain Reaction, Diagnostic Assay, Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: Cellular Prion Protein Regulates Its Own ?-Cleavage through ADAM8 in Skeletal Muscle *

doi: 10.1074/jbc.M112.360891

Figure Lengend Snippet: ADAM8 protein level correlates with PrP C1/full-length ratio in skeletal muscle tissue of Dox-induced Tg(HQK) mice. Tg(HQK) mice were treated with 6 g of Dox/kg food for 0–60 days as indicated and 3–6 animals were taken at each time point. Skeletal muscles (quadriceps) from the hind legs of these animals were homogenized and subjected to SDS-PAGE and immunoblot analysis in three Western blots. The homogenates were either treated (for PrP probing) or not treated (for ADAM8 probing) with PNGase F before SDS-PAGE. A, representative immunoblot probed with anti-PrP antibody 8H4 followed by probing with an anti-actin antibody after stripping; samples for day 3, 5, and 6 are not shown. B, representative immunoblot probed with an antibody against ADAM8 followed by probing with an anti-actin antibody after stripping; samples for day 3, 5, and 6 are not shown. C and D, diagrams of PrP C1/full-length ratios (C) and ADAM8 protein level (D) over increasing duration of Dox treatment. The ADAM8 protein level for each sample was normalized against the actin level for the sample in each blot and expressed as the ratio against the normalized ADAM8 protein level in the untreated wild type FVB mouse on the same blot. The error bars denote standard deviations calculated from the duplicate blots. E, plot of PrP C1/full-length ratio against ADAM8 protein level over the time course of Dox treatment.

Article Snippet: Then 20 μg of total proteins for each sample were separated by SDS-PAGE on a 10–20% Criterion Tris-Triton Precast gel (Bio-Rad), transferred onto PVDF membranes under 360 mA for 90 min, incubated with the 8H4 antibody (for residues 176–186 of human PrP or residues 175–185 of mouse PrP) ( 63 ) (1:5000 diluted in 0.5% normal goat serum, 1× TBS and 0.05% Tween) at 4 °C with gentle shaking overnight, followed by incubation with sheep anti-mouse IgG (Amersham Biosciences, Buckinghamshire, UK).

Techniques: SDS Page, Western Blot, Stripping Membranes

Journal: The Journal of Biological Chemistry

Article Title: Cellular Prion Protein Regulates Its Own ?-Cleavage through ADAM8 in Skeletal Muscle *

doi: 10.1074/jbc.M112.360891

Figure Lengend Snippet: ADAM8 protein level correlates with PrP C1/full-length ratio in skeletal muscle tissue of Tg mice constitutively expressing human PrP at different levels. Skeletal muscles (quadriceps) from the hind legs of wild type FVB mice (Wt) and four different Tg mice lines (Tg43, Tg4, Tg17, and Tg21) constitutively expressing human PrP at different levels were collected. Muscle homogenates were either treated (for PrP probing) or not treated (for ADAM8 probing) with PNGase F, subjected to SDS-PAGE, and probed with an anti-PrP antibody (8H4) or an antibody against ADAM8. A, representative immunoblot probed with anti-PrP antibody 8H4 followed by probing with an anti-actin antibody after stripping. B, representative immunoblot probed with an antibody against ADAM8 followed by probing with an anti-actin antibody after stripping. C and D, diagrams of ADAM8 protein level (C) and PrP C1/full-length ratios (D) in the skeletal muscles of the four Tg mouse lines. The ADAM8 protein level for each sample was normalized against the actin level and expressed as the ratio against the normalized ADAM8 protein level in the untreated sex and age-matched wild type FVB mouse on the same blot. The error bars denote standard errors calculated from the three animals for each Tg line. The bars with asterisk(s) indicate statistical significance when compared with the Tg43 mouse samples; *, p < 0.05; **, p < 0.01. E, plot of PrP C1/full-length ratios against ADAM8 protein levels in the four Tg lines. Linear regression analysis revealed an R2 value of 0.93, highly indicative of a linear correlation between ADAM8 protein level and the PrP C1/full-length ratio.

Article Snippet: Then 20 μg of total proteins for each sample were separated by SDS-PAGE on a 10–20% Criterion Tris-Triton Precast gel (Bio-Rad), transferred onto PVDF membranes under 360 mA for 90 min, incubated with the 8H4 antibody (for residues 176–186 of human PrP or residues 175–185 of mouse PrP) ( 63 ) (1:5000 diluted in 0.5% normal goat serum, 1× TBS and 0.05% Tween) at 4 °C with gentle shaking overnight, followed by incubation with sheep anti-mouse IgG (Amersham Biosciences, Buckinghamshire, UK).

Techniques: Expressing, SDS Page, Western Blot, Stripping Membranes

Journal: The Journal of Biological Chemistry

Article Title: Cellular Prion Protein Regulates Its Own ?-Cleavage through ADAM8 in Skeletal Muscle *

doi: 10.1074/jbc.M112.360891

Figure Lengend Snippet: ADAM8 protein level correlates with PrP C1/full-length ratio in skeletal muscle tissue of mice constitutively expressing mouse PrP at different levels. Skeletal muscles (quadriceps) from the hind legs of wild type C57BL6 mice (BL6) and the Tga20 line constitutively expressing mouse PrP at high levels were collected. Muscle homogenates were either treated (for PrP probing) or not treated (for ADAM8 probing) with PNGase F, and subjected to SDS-PAGE and immunoblotting with an anti-PrP antibody (8H4) or an antibody against ADAM8. A, immunoblot probed with anti-PrP antibody 8H4 followed by probing with an anti-actin antibody after stripping. B, immunoblot probed with an antibody against ADAM8 followed by probing with an anti-actin antibody after stripping. C, plot of PrP C1/full-length ratios against relative ADAM8 protein levels. The error bars denote standard errors calculated from the three animals for each mouse line. The bars with an asterisk indicate statistical significance (p < 0.05) when compared with the wild type C57BL6 (BL6) samples.

Article Snippet: Then 20 μg of total proteins for each sample were separated by SDS-PAGE on a 10–20% Criterion Tris-Triton Precast gel (Bio-Rad), transferred onto PVDF membranes under 360 mA for 90 min, incubated with the 8H4 antibody (for residues 176–186 of human PrP or residues 175–185 of mouse PrP) ( 63 ) (1:5000 diluted in 0.5% normal goat serum, 1× TBS and 0.05% Tween) at 4 °C with gentle shaking overnight, followed by incubation with sheep anti-mouse IgG (Amersham Biosciences, Buckinghamshire, UK).

Techniques: Expressing, SDS Page, Western Blot, Stripping Membranes

Journal: The Journal of Biological Chemistry

Article Title: Cellular Prion Protein Regulates Its Own ?-Cleavage through ADAM8 in Skeletal Muscle *

doi: 10.1074/jbc.M112.360891

Figure Lengend Snippet: The PrP C1/full-length fragment ratio is significantly reduced in skeletal muscles of ADAM8 knock-out mice. Skeletal muscles (quadriceps) from the hind legs of three each of wild type C57BL/6 mice (Wt) and ADAM8−/− mice were collected. Muscle homogenates were either treated (for PrP probing) or not treated (for ADAM8 probing) with PNGase F, subjected to SDS-PAGE, and probed with an anti-PrP antibody (8H4) or an antibody against ADAM8. A, immunoblots probed with an antibody against ADAM8 or 8H4 followed by probing with an anti-actin antibody after stripping. B, diagram of PrP C1/full-length ratio in the skeletal muscles of Wt and ADAM8−/− mice. The error bars denote standard errors calculated from the three animals for each mouse line. The bars with asterisk(s) indicate statistical significance when compared with the Wt mouse samples. **, p < 0.005.

Article Snippet: Then 20 μg of total proteins for each sample were separated by SDS-PAGE on a 10–20% Criterion Tris-Triton Precast gel (Bio-Rad), transferred onto PVDF membranes under 360 mA for 90 min, incubated with the 8H4 antibody (for residues 176–186 of human PrP or residues 175–185 of mouse PrP) ( 63 ) (1:5000 diluted in 0.5% normal goat serum, 1× TBS and 0.05% Tween) at 4 °C with gentle shaking overnight, followed by incubation with sheep anti-mouse IgG (Amersham Biosciences, Buckinghamshire, UK).

Techniques: Knock-Out, SDS Page, Western Blot, Stripping Membranes

Journal: The Journal of Biological Chemistry

Article Title: Cellular Prion Protein Regulates Its Own ?-Cleavage through ADAM8 in Skeletal Muscle *

doi: 10.1074/jbc.M112.360891

Figure Lengend Snippet: Overexpression of mouse PrP leads to up-regulation of ADAM8 in C2C12 cells. C2C12 cells were transfected with a vector expressing mouse PrP and selected with hygromycin to establish seven stable cell clones (numbered 1–7). The original C2C12 cells (CO) and blank vector-transfected C2C12 cells (VC) serve as controls. Lysates from these clones were directly subjected to Western blotting and the experiments repeated three times. A, representative immunoblot probed with 8H4 followed by probing with an anti-actin antibody after stripping. B, representative immunoblot probed with an antibody against ADAM8 followed by probing with an anti-actin antibody after stripping. C, diagram of total PrP levels and ADAM8 levels in the C2C12 clones. The total PrP and ADAM8 protein levels for each sample were normalized against the actin level and expressed as the ratio against the normalized protein levels in the #1 clone. The error bars denote standard deviations calculated from the three blots. The bars with asterisk(s) indicate statistical significance when compared with the #1 clone; *, p < 0.05; **, p < 0.01.

Article Snippet: Then 20 μg of total proteins for each sample were separated by SDS-PAGE on a 10–20% Criterion Tris-Triton Precast gel (Bio-Rad), transferred onto PVDF membranes under 360 mA for 90 min, incubated with the 8H4 antibody (for residues 176–186 of human PrP or residues 175–185 of mouse PrP) ( 63 ) (1:5000 diluted in 0.5% normal goat serum, 1× TBS and 0.05% Tween) at 4 °C with gentle shaking overnight, followed by incubation with sheep anti-mouse IgG (Amersham Biosciences, Buckinghamshire, UK).

Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Stable Transfection, Clone Assay, Western Blot, Stripping Membranes

Journal: The Journal of Biological Chemistry

Article Title: Cellular Prion Protein Regulates Its Own ?-Cleavage through ADAM8 in Skeletal Muscle *

doi: 10.1074/jbc.M112.360891

Figure Lengend Snippet: The PrP C1/full-length ratio is proportional to ADAM8 protein level in C2C12 myoblast cells. Two stable C2C12 clones expressing mouse PrP (MoPrP-6 and MoPrP-7) were transfected with miR RNAi sequence targeting the ORF region of ADAM8 and several stable cell lines were obtained. Lysates from these stable cell lines were either treated (for PrP probing) or not treated (for ADAM8 probing) with PNGase F, then subjected to electrophoresis and immunoblotting with an anti-PrP antibody (8H4) or an antibody against ADAM8. The immunoblotting was repeated three times. A, representative immunoblots probed with anti-PrP antibody 8H4 or with an antibody against ADAM8 followed by probing with an anti-actin antibody after stripping; the relative ADAM8 protein level and PrP C1/full-length ratio for each cell line were quantified and diagramed underneath the Western blot. B, plot of PrP C1/full-length ratios against ADAM8 protein levels in ADAM8-knockdown C2C12 cell lines. The ADAM8 protein level for each C2C12 cell line was normalized against the actin level and expressed as the ratio against the normalized ADAM8 protein level in the original MoPrP-6 control cell line (CO) on the same blot. Data from the average of three duplicate blots are plotted. CO, the original MoPrP6/MoPrP7 cell line; VC, blank vector-transfected MoPrP6/MoPrP7 cell line; KD, knockdown.

Article Snippet: Then 20 μg of total proteins for each sample were separated by SDS-PAGE on a 10–20% Criterion Tris-Triton Precast gel (Bio-Rad), transferred onto PVDF membranes under 360 mA for 90 min, incubated with the 8H4 antibody (for residues 176–186 of human PrP or residues 175–185 of mouse PrP) ( 63 ) (1:5000 diluted in 0.5% normal goat serum, 1× TBS and 0.05% Tween) at 4 °C with gentle shaking overnight, followed by incubation with sheep anti-mouse IgG (Amersham Biosciences, Buckinghamshire, UK).

Techniques: Clone Assay, Expressing, Transfection, Sequencing, Stable Transfection, Electrophoresis, Western Blot, Stripping Membranes, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Cellular Prion Protein Regulates Its Own ?-Cleavage through ADAM8 in Skeletal Muscle *

doi: 10.1074/jbc.M112.360891

Figure Lengend Snippet: ADAM8 directly cleaves PrP to generate the C1 fragment. Recombinant human PrP (rhPrP) was cleaved at 37 °C for 14 h with a recombinant human ADAM8 that had been treated or untreated with thermolysin for activation, followed by SDS-PAGE and subsequent immunoblot analysis (A and B) or direct staining with Coomassie Blue (C), or by direct mass spectrometry analysis (D). A, Western blot probed with antibodies 8H4 (against human PrP176–186). B, Western blot probed with 3F4 (against human PrP 106–112). C, SDS-PAGE gel directly stained with Coomassie Blue. D, mass spectrometry analysis. An in vitro reaction sample with thermolysin-activated ADAM8 and rhPrP was analyzed. Monoisotopic distributions of protonated (M+11H)11+ N1 fragment ion (top) and protonated (M+13H)13+ C1 fragment ion (bottom) are shown; Mm: molecular mass. In panels A-B, skeletal muscle tissue homogenate from a Tg(HQK) mouse treated with Dox for 60 days was loaded as a control. In panels A–C, the arrows point to the C1 band. The rhPrP sample contained an extension of Gly-Ser at the N terminus as a leftover from the His tag; accordingly, the resulting N1 fragment also contained the extra Gly-Ser residues at the N terminus.

Article Snippet: Then 20 μg of total proteins for each sample were separated by SDS-PAGE on a 10–20% Criterion Tris-Triton Precast gel (Bio-Rad), transferred onto PVDF membranes under 360 mA for 90 min, incubated with the 8H4 antibody (for residues 176–186 of human PrP or residues 175–185 of mouse PrP) ( 63 ) (1:5000 diluted in 0.5% normal goat serum, 1× TBS and 0.05% Tween) at 4 °C with gentle shaking overnight, followed by incubation with sheep anti-mouse IgG (Amersham Biosciences, Buckinghamshire, UK).

Techniques: Recombinant, Activation Assay, SDS Page, Western Blot, Staining, Mass Spectrometry, In Vitro

Journal: EMBO Reports

Article Title: Lipid and protein content profiling of isolated native autophagic vesicles

doi: 10.15252/embr.202153065

Figure Lengend Snippet:

Article Snippet: Donkey polyclonal anti‐rabbit‐Cy5 (FlowCyt 3 μg/12 μg; IF 1:200) , Jackson ImmunoResearch , 711‐175‐152.

Techniques: Protease Inhibitor, Software, Microscopy, Laser-Scanning Microscopy, Mass Spectrometry, Transmission Assay