Journal: PLoS ONE

Article Title: Pno1 Tissue-Specific Expression and Its Functions Related to the Immune Responses and Proteasome Activities

doi: 10.1371/journal.pone.0046093

Figure Lengend Snippet: A. Targeting strategy to generate Pno1 KO mice. The black square marked as 3′ probe represents the sequence used as probe in Southern blotting for genotyping. A 18.6-kb BamH1 fragment detected by this probe represents the WT allele, and a 7.5-kb BamH1 fragment, the KO allele. B. Genotyping of Pno1 mutant mice . Tail DNA was digested with BamHI, and analyzed by Southern blotting (left panel), with a 3′ probe whose sequence location is indicated in A. A 18.6-kb band representing the WT allele and a 7.5-kb band representing the recombinant allele are indicated by arrows. Ear lobe DNA without digestion was analyzed by PCR for routine genotyping (right panel). A 548-bp band representing the WT allele and a 224-bp band representing the recombinant allele are indicated by arrows. C. Reduced Pno1 mRNA expression in Pno1 +/− tissues . mRNA from the Li, Th and Spl of WT and heterozygous Pno1 +/− (HET) mice were analyzed by reverse transcription-real time PCR (RT-qPCR) for Pno1 mRNA levels. The results are expressed as ratios of Pno1 versus β-actin signals with means ± SD indicated. D. Reduced Pno1 mRNA up-regulation in Pno1 +/− T cells upon activation . T cells from WT and HET Spl were stimulated with solid phase anti-CD3 mAb and anti-CD28 mAb (0.5 µg/ml and 4 µg/ml respectively for coating) for 1 to 24 h, and their Pno1 mRNA levels were quantified by RT-qPCR. The results are expressed as ratios of Pno1 versus β-actin signals with means ± SD indicated.

Article Snippet: The proteasome β5 subunit and β-actin were detected by blotting with rabbit anti-human β5 antibody (Ab, Millipore, Billerica, MA, USA), followed by horse radish peroxidase-conjugated goat anti-rabbit IgG (GE Healthcare, Little Chalfont, Bucks, United Kindom.).

Techniques: Sequencing, Southern Blot, Mutagenesis, Recombinant, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Activation Assay

Journal: PLoS ONE

Article Title: Pno1 Tissue-Specific Expression and Its Functions Related to the Immune Responses and Proteasome Activities

doi: 10.1371/journal.pone.0046093

Figure Lengend Snippet: e1.5 embryos were harvested, and cultured in M16 medium and photographed daily until e4.5. Phase contrast micrographs are shown. At the end of culture on e4.5, the embryos were genotyped with qPCR.

Article Snippet: The proteasome β5 subunit and β-actin were detected by blotting with rabbit anti-human β5 antibody (Ab, Millipore, Billerica, MA, USA), followed by horse radish peroxidase-conjugated goat anti-rabbit IgG (GE Healthcare, Little Chalfont, Bucks, United Kindom.).

Techniques: Cell Culture

Journal: PLoS ONE

Article Title: Pno1 Tissue-Specific Expression and Its Functions Related to the Immune Responses and Proteasome Activities

doi: 10.1371/journal.pone.0046093

Figure Lengend Snippet: A. T cell subpopulations in the spleen and LN. CD4 and CD8 T-cell populations in WT and HET spleens, LN and thymuses were analyzed by 2-color flow cytometry. Percentages are indicated. B. B cell population in lymphoid organs. B cell populations in the spleen and LN were analyzed according to B220 and CD19 expression by 2-color flow cytometry. C. T cell proliferation. WT and HET spleen T cells were stimulated with solid phase anti-CD3 mAb (0.5 µg/ml for coating). The cells were pulsed with 3 H-thymidine 16 h before harvesting. 3 H-thymidine uptake by the cells was measured at 24, 48 and 72 h. Samples were tested in triplicate, and means ± SD of CPM are shown. D. C69 and CD25 expression on activated WT and HET T cells. WT and HET T cells were stimulated overnight by solid phase anti-CD3 mAb plus anti-CD28 mAb (0.5 µg/ml and 4 µg/ml respectively for coating). CD69 and CD25 expression on CD4 (left panel) and CD8 (right panel) T cells was measured by 2-color flow cytometry. All experiments in this figure were repeated at least 3 times and representative data are reported.

Article Snippet: The proteasome β5 subunit and β-actin were detected by blotting with rabbit anti-human β5 antibody (Ab, Millipore, Billerica, MA, USA), followed by horse radish peroxidase-conjugated goat anti-rabbit IgG (GE Healthcare, Little Chalfont, Bucks, United Kindom.).

Techniques: Flow Cytometry, Expressing

Journal: PLoS ONE

Article Title: Pno1 Tissue-Specific Expression and Its Functions Related to the Immune Responses and Proteasome Activities

doi: 10.1371/journal.pone.0046093

Figure Lengend Snippet: A. Normal marginal zone B cell and follicular B cell populations in the spleen of HET mice. Marginal Zone B cell (B220 + CD21 hi CD23 low/− ) and follicular B cell (B220 + CD21 int/hi CD23 hi ) populations in WT and HET spleens were analyzed by 3-color flow cytometry. Percentages are indicated. B. B1 B cell and B2 B cell subpopulation in peritoneal exudates of HET mice. Peritoneal exudate B1a (B220 + CD23 − CD5 + IgM hi ), B1b (B220 + CD23 − CD5 − IgM hi ) and B2 (B220 + CD23 + IgM int/hi ) B cells of WT and HET mice were analyzed by by 4-color flow cytometery. Percentages are indicated. C. Proliferation of B cells from HET mice. WT and HET spleen B cells were stimulated with different stimuli as indicated (anti-IgM: 5 µg/ml; IL-4∶10 ng/ml; anti-CD40 mAb: 2 µg/ml; LPS: 2 µg/ml). The cells were pulsed with 3 H-thymidine 6 h before harvesting. 3 H-thymidine uptake by the cells was measured at 24 h and 48h after the initiation of the culture. Samples were in triplicate, and means ± SD of CPM are shown. D. B cell activation markers CD80 and CD86 expression on activated WT and HET B cells . WT and HET spleen B cells were stimulated as described in C . CD80 and CD86 expression on B220 + B cells was measured by 3-color flow cytometry 24 h after the initiation of the culture. E. Plasma cells in the draining lymph nodes of immunized WT and HET mice . WT and HET mice were immunized with chick type II collagen with adjuvants at the tail base and sacrificed 21 days after the immunization. Isotype-switched plasmablast/plasma cells (IgD − IgM − CD138 + B220 lo/− ) from the draining lymph nodes of WT and HET were analyzed by 4-color flow cytometry. F. Serum collagen-specific antibody production in WT and HET mice . Sera from mice (WT n = 7, HET n = 4) as described in E were collected on day 21 after the immunization. Chick collagen-specific IgG Abs were measured by ELISA. The data are expressed as arbitrary titres. The titres between WT and HET groups were not statiscially significant (p = 0.8724, Student’s t test). Experiments A-E were repeated at least 3 times and representative data are shown.

Article Snippet: The proteasome β5 subunit and β-actin were detected by blotting with rabbit anti-human β5 antibody (Ab, Millipore, Billerica, MA, USA), followed by horse radish peroxidase-conjugated goat anti-rabbit IgG (GE Healthcare, Little Chalfont, Bucks, United Kindom.).

Techniques: Flow Cytometry, Activation Assay, Expressing, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Pno1 Tissue-Specific Expression and Its Functions Related to the Immune Responses and Proteasome Activities

doi: 10.1371/journal.pone.0046093

Figure Lengend Snippet: A. Proteasome activities in spleen and thymus. Lysates from the Sp and Th of WT and HET mice were assayed for chymotrypsin-like and trypsin-like proteasome protease activities. Samples were tested in triplicate, and means ± SD are shown. B. HET and WT organs have similar proteasome levels. Lysate proteins from the lung, spleen, thymus, liver and kidney were analyzed for proteasome content according to proteasome β5 subunit levels based on immunoblotting (upper panel). β-actin levels were used to show even loading of lysate proteins (lower panel).

Article Snippet: The proteasome β5 subunit and β-actin were detected by blotting with rabbit anti-human β5 antibody (Ab, Millipore, Billerica, MA, USA), followed by horse radish peroxidase-conjugated goat anti-rabbit IgG (GE Healthcare, Little Chalfont, Bucks, United Kindom.).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Pno1 Tissue-Specific Expression and Its Functions Related to the Immune Responses and Proteasome Activities

doi: 10.1371/journal.pone.0046093

Figure Lengend Snippet: A. T cell sub-populations in the spleen and LN. CD4 and CD8 T cell populations in the WT and Tg spleen LN and thymus were analyzed by 2-color flow cytometry. Percentages are indicated. B. B cell population in lymphoid organs. The B cell population in the spleen and LN was analyzed according to B220 and CD19 expression by 2-color flow cytometry. C. T cell proliferation. WT and Tg spleen T cells were stimulated with solid phase anti-CD3 mAb (0.5 µg/ml for coating). The cells were pulsed with 3 H-thymidine 16 h before harvesting. 3 H-thymidine uptake by cells was measured at 24, 48 and 72 h. The samples were tested in triplicate, and means ± SD of CPM are shown. D. C69 and CD25 expression on activated WT and HET T cells.WT and Tg T cells were stimulated overnight by solid phase anti-CD3 plus anti-CD28 mAbs (0.5 µg/ml and 4 µg/ml respectively for coating). CD69 and CD25 expression on CD4 (left panel) and CD8 (right panel) T cells was measured by 2-color flow cytometry. All experiments in this figure were repeated at least 3 times and representative data are shown.

Article Snippet: The proteasome β5 subunit and β-actin were detected by blotting with rabbit anti-human β5 antibody (Ab, Millipore, Billerica, MA, USA), followed by horse radish peroxidase-conjugated goat anti-rabbit IgG (GE Healthcare, Little Chalfont, Bucks, United Kindom.).

Techniques: Flow Cytometry, Expressing

Journal: PLoS ONE

Article Title: Pno1 Tissue-Specific Expression and Its Functions Related to the Immune Responses and Proteasome Activities

doi: 10.1371/journal.pone.0046093

Figure Lengend Snippet: A. Normal marginal zone B cell and follicular B cell populations in the spleen of Tg mice. Marginal Zone B cell (B220 + CD21 hi CD23 low/− ) and follicular B cell (B220 + CD21 int/hi CD23 hi ) populations in WT and Tg spleens were analyzed by 3-color flow cytometry. Percentages are indicated. B. B1 B cell and B2 B cell subpopulation in peritoneal exudates of Tg mice. Peritoneal exudate B1a (B220 + CD23 − CD5 + IgM hi ), B1b (B220 + CD23 − CD5 − IgM hi ) and B2 (B220 + CD23 + IgM int/hi ) B cells of WT and Tg mice were analyzed by by 4-color flow cytometery. Percentages are indicated. C. Proliferation of B cells from Tg mice . WT and Tg spleen B cells were stimulated with different stimuli as indicated (anti-IgM: 5 µg/ml; IL-4∶10 ng/ml; anti-CD40 mAb: 2 µg/ml; LPS: 2 µg/ml). The cells were pulsed with 3 H-thymidine 6 h before harvesting. 3 H-thymidine uptake by the cells was measured at 24 h and 48 h after the initiation of the culture. Samples were in triplicate, and means ± SD of CPM are shown. D. B cell activation markers CD80 and CD86 expression on activated WT and Tg B cells . WT and Tg spleen B cells were stimulated as described in C . CD80 and CD86 expression on B220 + B cells was measured by 3-color flow cytometry 24 h after the initiation of the culture. E. Plasma cells in the draining lymph nodes of immunized WT and Tg mice . WT and Tg mice were immunized with chick type II collagen with adjuvants at the tail base and sacrificed 21 days after the immunization. Isotype-switched plasmablast/plasma cells (IgD − IgM − CD138 + B220 lo/− ) from the draining lymph nodes of WT and Tg were analyzed by 4-color flow cytometry. F. Serum collagen-specific antibody production in WT and Tg mice . Sera from mice (WT n = 7, Tg n = 4) as described in E were collected on day 21 after the immunization. Chick collagen-specific IgG Abs were measured by ELISA. The data are expressed as arbitrary titres. The titres between WT and Tg groups were not statiscially significant (p = 0.1927, Student’s t test). Experiments A-E were repeated at least 3 times and representative data are shown.

Article Snippet: The proteasome β5 subunit and β-actin were detected by blotting with rabbit anti-human β5 antibody (Ab, Millipore, Billerica, MA, USA), followed by horse radish peroxidase-conjugated goat anti-rabbit IgG (GE Healthcare, Little Chalfont, Bucks, United Kindom.).

Techniques: Flow Cytometry, Activation Assay, Expressing, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Pno1 Tissue-Specific Expression and Its Functions Related to the Immune Responses and Proteasome Activities

doi: 10.1371/journal.pone.0046093

Figure Lengend Snippet: A. Pno1 mRNA over-expression in L cells stably transfected with the Pno1-expressing construct pCEP-Pno1-HA . Pno1 mRNA levels were quantified by RT-qPCR. Samples were tested in triplicate and means ± SD of Pno1 versus β-actin signal ratios are shown. L cells stably transfected with empty vectors were used as controls. B. L cells over-expressing Pno1 presented similar proteasome activity as vector-transfected cells . The chymotrypsin-like activity of L cells stably transfected with pCEP-Pno1-HA was quantified, and L cells transfected with empty vectors were used as controls. The assay was conducted in triplicate, and means ± SD are shown. C. Proteasome activities in glycerol density gradient fractions . Lysates from L cells stably transfected with Pno1-expressing constructs or empty vectors were fractionated with glycerol gradients, and chymotrypsin-like proteasome activity in fractions 1 to 27 was quantified. D-G. Location of Pno1, β5, S6 and L7 in glycerol density gradients . Proteins in fractions 1 to 27 of the glycerol gradient of L cells overexpressing Pno1 were analyzed by immunoblotting. The β5 proteasome subunit (D), Pno1 (E), S6 subunit of the small ribosome complex (F) and L7 subunit of the large ribosome complex (G) were detected by their respective Abs (Pno1 was detected by anti-HA Ab).

Article Snippet: The proteasome β5 subunit and β-actin were detected by blotting with rabbit anti-human β5 antibody (Ab, Millipore, Billerica, MA, USA), followed by horse radish peroxidase-conjugated goat anti-rabbit IgG (GE Healthcare, Little Chalfont, Bucks, United Kindom.).

Techniques: Over Expression, Stable Transfection, Transfection, Expressing, Construct, Quantitative RT-PCR, Activity Assay, Plasmid Preparation, Western Blot

Journal: bioRxiv

Article Title: Fibrin fragment E potentiates TGF-β-induced myofibroblast activation and recruitment

doi: 10.1101/829945

Figure Lengend Snippet: Interactions between soluble recombinant αVβ 3 (5-50 nM) and immobilized FnE (A) or FnDD (B) were determined by SPR. Immobilization was at 952 RU and 1251 RU respectively. (C) SPR sensorgrams of the interaction between FnE and integrin αVβ 3 fitted to a bivalent analyte model (i), a heterogeneous ligand model (ii) or to the 1:1 two state interaction model (iii). (D) Representative western blot and (E) quantification of α-SMA protein levels of HFL1 fibroblasts in the presence of the cyclic-RGD peptide EMD 66203 in combination with TGF-β (5 ng/ml) and/or FnE (2 nM). α-SMA was normalized against β-actin and expressed as compared to vehicle (DMSO) treated controls. (F) Chemotaxis and (G) net migration of HFL1 cells in presence of 5 µM cyclic-RGD peptide. Dashed grey lines indicate mean values for cells without addition of cyclic-RGD. (H) Integrin β3 and β5 protein levels in HFL1 cells after transfection with siRNA selectively targeting these integrins, as indicated. (I) α-SMA ( ACTA2 ) mRNA levels of siRNA transfected cells stimulated with TGF-β (5 ng/ml) with or without addition of FnE (2 nM) for 48 h. (J) Chemotaxis and (K) net migration of HFL1 cells treated with siRNA to suppress integrin β3 or β5 in response to a PDGF-BB (0-20 ng/ml) or FnE (0-2 nM) gradients. * p<0.05. Error bars represent standard error of the mean ( n ≥3).

Article Snippet: Primary antibodies used were mouse anti-α-SMA (clone 1A4, Sigma-Aldrich) 1:10 000, rabbit anti-β-actin (ab8227, Abcam) 1:5000, mouse anti-PARP1 (BD-bioscience) 1:1500, mouse anti-integrin β3 (Cell signaling) 1:500, rabbit anti-integrin β5 (Abnova) 1:250, all added in blocking buffer with 0.1% Tween-20.

Techniques: Recombinant, Western Blot, Chemotaxis Assay, Migration, Transfection

Journal: Cell reports

Article Title: The nociceptive activity of peripheral sensory neurons is modulated by the neuronal membrane proteasome

doi: 10.1016/j.celrep.2024.114058

Figure Lengend Snippet: (A) Representative micrographs of immuno-EM labeling on primary DRG cultures. White arrowheads: gold particles localized on the cytoplasm. Black arrowheads: gold particles localized on the membrane. Bar graphs to the right show the proportion of gold particles localized on the cytoplasm (Cyto) and membrane (Mem). Violin plots show the distribution of gold particles within 240 nm of the neuronal membranes. Data are presented as mean ± SEM ( n = 14–20 micrographs analyzed from two independent animals). (B) Representative images of antibody feeding experiments in DRG cultures against the β5 proteasome subunit followed by cytoplasmic NF-H staining. White box: magnified section shown on bottom of composite image. (C) Representative 3D projection images of antibody feeding experiments in DRG cultures against the β5 proteasome subunit followed by cytoplasmic NF-H staining. (D) Representative 3D projection images of antibody feeding experiments in DRG cultures against the P2X3 and β5 proteasome subunit co-labeling. Bar graph shows quantification of surface P2X3 and β5 co-labeling under the conditions that at least one neuron in the field of view was positive for both NMP and P2X3. Data are presented as mean ± SEM ( n = 13 images analyzed). See also .

Article Snippet: Rabbit anti-β5 proteasome subunit , Enzo , BML-PW8895; RRID: AB_10540901.

Techniques: Labeling, Membrane, Staining

Journal: Cell reports

Article Title: The nociceptive activity of peripheral sensory neurons is modulated by the neuronal membrane proteasome

doi: 10.1016/j.celrep.2024.114058

Figure Lengend Snippet:

Article Snippet: Rabbit anti-β5 proteasome subunit , Enzo , BML-PW8895; RRID: AB_10540901.

Techniques: Recombinant, Saline, Protease Inhibitor, Synthesized, Bradford Protein Assay, Software, Imaging

Journal: Molecular cell

Article Title: Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α

doi: 10.1016/j.molcel.2023.05.008

Figure Lengend Snippet: (A) Fluorescence confocal images showing A488-irisin binding in HEK293T cells. HEK293T cells were either transiently transfected with control vector or full-length αV and β5 plasmids. 2 nM Hsp90α was used for 1 hr pretreatment, and 2 nM A488-irisin-His was subsequently used for 5 min treatment. Scale bar: 20 μm. (B) Anti-phosphorylated FAK (Y397) and anti-FAK western blots showing the levels of integrin signaling upon irisin and/or Hsp90α treatments. HEK293T cells were transfected and treated in the same way as (A), except for the addition of the shown amounts of unlabeled irisin-His (0.1 nM or 1 nM) and Hsp90α (1 nM). Anti-αV and anti-β5 antibodies were used to probe the levels of the ectopically expressed αV and β5. (C) Immunofluorescence confocal images showing cell surface Hsp90α in SK-Mel2 cells. Live cells were treated with either control IgG or anti-Hsp90α at 4°C. Scale bar: 50 μm. (D) Quantification of the percentage of SK-Mel2 cells expressing cell surface Hsp90α in (C) (significant if p-value < 0.05 by unpaired t-test). (E) Fluorescence confocal images showing A647-irisin binding in SK-Mel2 cells. Live cells were pretreated with either control IgG or anti-Hsp90α at 4°C for 1 hr followed by 2 nM A647-irisin-His treatment at room temperature for 5 min. Scale bar: 50 μm. (F) Quantification of the percentage of A647-positive cells in (E) (significant if p-value < 0.05 by unpaired t-test). (G) Co-immunoprecipitation assay of endogenous cell surface αV and β5 using SK-Mel2 cells. Endogenous cell surface Hsp90α was captured by anti-Hsp90α in live cells at 4°C. (H) Crystal violet assay showing does-dependent inhibition of the cell viability of SK-Mel2 upon irisin treatment. Grey bar: control treatment with PBS. Concentrations of irisin-His used for the treatments were indicated (one-way ANOVA). (I) Crystal violet assay showing the inhibition of irisin-mediated effect in SK-Mel2 cells by anti-Hsp90α or control antibody. Grey bar: control treatment with PBS. 50 ng/mL of irisin-His was used (one-way ANOVA). (J) Western blot of mouse inguinal fat tissue lysates using the indicated antibodies to probe integrin signaling. Mice were given anti-Hsp90α antibody or control IgG (500 μg/kg) subcutaneously 24 hrs before a bolus injection of recombinant irisin (5 mg/kg) directly into the inguinal fat pads. The mice were sacrificed and inguinal fat tissues were harvested 20 min after irisin injection.

Article Snippet: Rabbit Monoclonal Anti- Integrin β5 , Abcam , Cat. # ab184312.

Techniques: Fluorescence, Binding Assay, Transfection, Control, Plasmid Preparation, Western Blot, Immunofluorescence, Expressing, IF-P, Co-Immunoprecipitation Assay, Crystal Violet Assay, Inhibition, Injection, Recombinant

Journal: Molecular cell

Article Title: Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α

doi: 10.1016/j.molcel.2023.05.008

Figure Lengend Snippet: (A) Flow charts of the steps used in three different methods for analyzing αVβ5-Apo and αVβ5/Hsp90α cryo-EM samples. (B) 2D classes (generated by method 1) of αVβ5 particles in each of the three conformational states and the numbers (quantified by all three methods) of particles in each state. (C) Quantification of the percentage of distinguished particles (“likely open” particles were not included) in each of the three conformational states. (D) Fluorescence anisotropy assay for A488-irisin binding by αVβ5, the αVβ5/Hsp90α complex in the presence of 1 mM MgCl2 and 1 mM CaCl2, or αVβ5 in the presence of 1 mM MnCl2. 50 nM A488-irisin-His was used in the assay. (E) Cartoon diagram showing a two-step process of the irisin action through αVβ5. Irisin alone has low affinity for the closed-state αVβ5. Hsp90α, Mn2+ ion, or other possible factors, “opens” αVβ5, allowing for high-affinity irisin binding and effective signaling transduction through its integrin receptor. (F) and (G) TALON pull-downs performed using 1 μM bead-bound clasped and tagged αVβ5. These were mixed with 2 μM untagged Hsp90α without bound nucleotide (Hsp90α-Apo) or Hsp90α charged with the indicated nucleotides (F), or Hsp90α nonhydrolyzing mutant (G95D) (G), and bound samples were analyzed by Coomassie staining and anti-Hsp90α western blot.

Article Snippet: Rabbit Monoclonal Anti- Integrin β5 , Abcam , Cat. # ab184312.

Techniques: Cryo-EM Sample Prep, Generated, Fluorescence, Binding Assay, Transduction, Mutagenesis, Staining, Western Blot

Journal: Molecular cell

Article Title: Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α

doi: 10.1016/j.molcel.2023.05.008

Figure Lengend Snippet: Figure 5C

Article Snippet: Rabbit Monoclonal Anti- Integrin β5 , Abcam , Cat. # ab184312.

Techniques: Control, Virus, Recombinant, Expressing, Protease Inhibitor, Lysis, Transfection, Electron Microscopy, Immunodepletion, Clone Assay, Protein Purification, Endotoxin Assay, Bradford Assay, Staining, Mass Spectrometry, Mutagenesis, Software, Isolation