Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: PLY-EVs induce dendritic cell maturation and inflammatory cytokine release upon internalization (A) Confocal microscopy images showing the internalization of CFSE-labelled PLY (0.1) and naive EVs (green) by THP-1-monocyte-derived DCs at 24 h post-treatment. Scale bars, 25 μm. (B) Flow cytometry histograms ( N = 3) to quantify the DC uptake of CFSE-labeled PLY(0.5)EVs and naive EVs. (C) Dose-dependent uptake of PLY (0.1, 0.5) EVs by DCs. (D) Phase-contrast microscopy images of immature day 5 DCs coincubated with PLY (0.1, 0.5) EVs and naive EVs for 24 h. Arrows indicate matured DCs (magnified in inset). Scale bars, 50 μm. Images are representative of three independent experiments. (E–G) Flow cytometry histograms ( N = 3) to quantify the expression levels of (E) CD80, (F) CD86, and (G) CD83 on THP-1-monocyte-derived DCs treated with PLY(0.5) and naive EVs. (H and I) Flow cytometry histograms ( N = 2) showing the expression levels of DC maturation marker CD83 at 96 h post-incubation of primary human monocytes with (H) PLY(0.5) and naive EVs and (I) naive EVs pre-treated with recombinant PLY protein (naive EVs+rPLY). (J and K) Cytokine ELISA showing the levels of secreted TNF-α from (J) DCs treated with PLY (0.1) EVs or naive EVs alone ( N = 3) for 24 h and (K) DCs pre-treated with PLY (0.1,0.5) or naive EVs for 24 h followed by subsequent infection with S. pneumoniae , T4R strain ( N = 2). Recombinant PLY (0.5 μg/mL) was used as positive control. All data are represented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. n.s., not significant. See also Figures S6–S9 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Confocal Microscopy, Derivative Assay, Flow Cytometry, Labeling, Microscopy, Expressing, Marker, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Infection, Positive Control

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: Adoptive transfer of EVs from infected mice drives inflammation and pathology in a PLY-dependent manner (A) C57BL/6 mice were intranasally administered with 4 × 10 6 CFU of serotype 4 strain, T4 or the isogenic PLY mutant strain, T4Δply. At day 4 post-infection, EVs isolated from BALF were labeled and administered to healthy recipient mice at 35 μg/mice. The EV retention in murine respiratory tract was imaged by IVIS imaging and immune infiltration into lungs, and cytokine levels in BALF was measured. (B) Bacterial load in murine BALF ( N = 5 mice/group) upon infection with T4 and T4Δply strains was measured by CFU dilution assay. ∗∗ in (B) indicates p < 0.01 by Mann-Whitney test. (C) Quantification of relative total EV protein content from mice ( N = 3 mice/group) infected with T4 and T4Δply strains by BCA protein assay. PBS-treated mice served as control. ∗ and ∗∗ in (C) indicates p < 0.05 and p < 0.005, respectively, by unpaired t test. (D) IVIS imaging of mice intranasally administered with Nile-red-labeled EVs isolated from mice infected with T4 (EVs-T4) or T4Δply (EVs-T4Δply). EVs from PBS-treated mice (naive EVs) served as control. ROI intensity values indicate the total flux (photons/sec) recorded from the given region showing higher intensity of EVs from T4-infected mice in the respiratory tract. The color scale (photons/sec/cm 2 ) indicates the relative intensities of individual signals. (E and F) Flow cytometry analysis of inflammatory macrophages (F4/80 + ) and neutrophils (Ly6G + ) in BALF of mice ( N = 6 mice/group) administered with EVs from infected or untreated mice at 18 h. (G) TNF-α levels in the BALF of mice ( N = 5 mice/group) treated with EVs isolated from infected or untreated mice were measured post-sacrifice at 18 h by ELISA. ∗∗ and ∗∗∗ in (G) indicates p < 0.01 and p < 0.001, respectively, by unpaired t test. (H) Hematoxylin and eosin (H&E) staining of mouse lungs ( N = 6 mice/group) at 18 h post-administration of EVs from infected or PBS-treated mice. Mice treated with EVs from T4-infected mice showed tissue microlesions (MLEs) and immune cell infiltration in the alveolar interstitium indicative of PLY-induced tissue damage (magnified in the inset). BR, bronchiole; MLE, microlesions. Scale bars, 200 μm. Blind histopathological scoring was performed based on presence or absence of cellularity in alveolar interstitium and lesions. A score of “0” was given when no lesions were found, and a score of “1” was given to tissue showing increasing cellularity and lesions. Mouse BALF flow cytometry and histology data are representative of three independent experiments. All data are represented as mean ± SEM. See also Figure S12 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Adoptive Transfer Assay, Infection, Mutagenesis, Isolation, Labeling, Imaging, Dilution Assay, MANN-WHITNEY, Bicinchoninic Acid Protein Assay, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet:

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Virus, Mutagenesis, Isolation, Recombinant, Modification, Saline, Labeling, Staining, Electron Microscopy, Lysis, Western Blot, Buffer Exchange, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Software, Membrane

Journal: The Journal of Biological Chemistry

Article Title: Death Receptor 6 Induces Apoptosis Not through Type I or Type II Pathways, but via a Unique Mitochondria-dependent Pathway by Interacting with Bax Protein *

doi: 10.1074/jbc.M112.362038

Figure Lengend Snippet: Time course of DR6-induced apoptosis. After induction of DR6 expression by the addition of tetracycline (Tet), cells were harvested at various time points as indicated and divided into two portions. Half of the harvested cells were directly lysed and separated by SDS-PAGE, followed by Western blot analysis. The other half of the cells were used for preparation of cytosolic extracts and mitochondria-containing membrane fraction as described under “Experimental Procedures.” A, top panel shows induction of DR6 expression. Second panel shows PARP cleavage. Third, fourth, fifth, and sixth panels show activation of caspase-3, caspase-9, caspase-8, and caspase-7, respectively. The activation of caspases was determined by the formation of active forms of these caspases. This membrane was also reprobed with anti-actin antibody to indicate relative loading of samples (bottom panel). B, top panels show release of cytochrome c from mitochondria (right column) to the cytosol (left column). Second panels show translocation of Bax from cytosol (left panel) to mitochondria (right panel). Third panel show tBid formation and translocation to mitochondria. Fourth panel shows the reprobe of the second panels with anti-COX I antibody confirming the mitochondria remained intact during preparation.

Article Snippet: The plasmid pcDNA4/TO/LacZ-Myc-His, which expresses LacZ protein with a Myc tag after the addition of tetracycline, and the conventional plasmid pcDNA3.1/LacZ-Myc-His, which expresses Myc-tagged LacZ protein, were provided in the vector packages by the vendor (Invitrogen).

Techniques: Expressing, SDS Page, Western Blot, Membrane, Activation Assay, Translocation Assay

Journal: The Journal of Biological Chemistry

Article Title: Death Receptor 6 Induces Apoptosis Not through Type I or Type II Pathways, but via a Unique Mitochondria-dependent Pathway by Interacting with Bax Protein *

doi: 10.1074/jbc.M112.362038

Figure Lengend Snippet: Overexpression of antiapoptotic protein Bcl-2 and Bcl-xL inhibited DR6-induced apoptosis. Cell lysis and fractionation were performed as described previously. A and B, after 24-h transfection, a significant amount of Bcl-2 and Bcl-xL was detected (lanes 3 and 4, top panel). Because the recombinant Bcl-2 and Bcl-xL were expressed as a FLAG-tagged protein, the recombinant Bcl-2 and Bcl-xL were detected with a slower migration rate than endogenous Bcl-2 and Bcl-xL (compare lanes 3 and 4 with lanes 1 and 2). This membrane was also reprobed with anti-actin antibody to indicate relative loading of samples (fourth panel). Second and third panels show tetracycline (Tet)-induced DR6 expression, respectively; fifth and sixth panels, cytochrome c release from mitochondria; seventh and eighth panels, Bax translocation from cytosol to mitochondria. C, top panel shows Western blot for PARP; second to sixth panels, Western blots for caspases; seventh panel, Western blot for actin to indicate relative loading of lysate samples; eighth and ninth panels, Western blots for Bid; tenth and eleventh panels were stained for mitochondrial protein COX I.

Article Snippet: The plasmid pcDNA4/TO/LacZ-Myc-His, which expresses LacZ protein with a Myc tag after the addition of tetracycline, and the conventional plasmid pcDNA3.1/LacZ-Myc-His, which expresses Myc-tagged LacZ protein, were provided in the vector packages by the vendor (Invitrogen).

Techniques: Over Expression, Lysis, Fractionation, Transfection, Recombinant, Migration, Membrane, Expressing, Translocation Assay, Western Blot, Staining

Journal: The Journal of Neuroscience

Article Title: N- glycosylation of Acid-sensing Ion Channel 1a Regulates its Trafficking and Acidosis-induced Spine Remodeling

doi: 10.1523/JNEUROSCI.5021-11.2012

Figure Lengend Snippet: (A) Representative Western blot showing the sensitivity of WT ASIC1a, N366Q, N393Q or N366Q/N393Q (QQ) mutants to Endo H and PNGase F treatment. CHO cells were transfected with different constructs as indicated and surface proteins were biotinylated. Cell lysates were treated with PNGase F or Endo H as described in Methods. Surface proteins were then isolated and analyzed by Western blot. PNGase F treated samples all ran at the same position, indicating that the difference in migration patterns between untreated samples were due to differential glycosylation. Note that N366Q increased the % of Endo H resistant populations. Also note the small shift in migration between untreated and PNGase F or Endo H treated N393Q, which shows that the majority of N393Q was glycosylated but was sensitive to Endo H (the lower blot has contrast enhanced to better show this result of N393Q). Bottom plot shows the quantification. Asterisks indicate significant differences from WT (p < 0.001, ANOVA). (B) Western blots and quantification (from 7 different repeats) showing the effect of Asn366 and Asn393 mutations on surface expression of ASIC1a. Asterisks indicate significant differences from WT (p < 0.05, Wilcoxon signed rank test). (C & D) The effect of Tunicamycin on ASIC1a glycosylation (C) and surface trafficking (D). ASIC1a-transfected CHO cells were treated with 0.5 μg/ml Tunicamycin for 24 hrs. Untreated or PNGase F treated lysates (C) or surface and total fractions (D) were analyzed by Western blot. Note that Tunicamycin had no effect on surface:total ratio of QQ, which has both glycosylation sites mutated. Asterisk indicate significant differences (p < 0.01, paired t-test)

Article Snippet: Other reagents used: Endo H and PNGase F (New England Biolabs, Ipswich, MA); NHS-sulfo-LC-biotin and NeutrAvidin Beads (Pierce); Tunicamycin (ThermoFisher Scientific); culture media and serum (HyClone and Invitrogen); Fugene HD (Roche) and lipofectamine 2000 (Invitrogen).

Techniques: Western Blot, Transfection, Construct, Isolation, Migration, Glycoproteomics, Expressing

Journal: The Journal of Neuroscience

Article Title: N- glycosylation of Acid-sensing Ion Channel 1a Regulates its Trafficking and Acidosis-induced Spine Remodeling

doi: 10.1523/JNEUROSCI.5021-11.2012

Figure Lengend Snippet: Hippocampal slices were co-transfected with HA-tagged wild-type ASIC1a, N366Q, N393Q or QQ mutants together with a membrane targeted Lck-GFP (Benediktsson et al., 2005). Localization of transfected ASIC1a was visualized by immunofluorescence using an anti-HA antibody. (A) Representative confocal images showing the overall distribution of ASIC1a in transfected neurons. Left panel shows ASIC1a localization, middle panel shows GFP fluorescence, right panel shows the merged image (HA immunofluorescence in red and GFP fluorescence in green). Note that WT and N366Q were present in most dendritic branches, with N366Q showing stronger dendritic staining in distal dendrites. Also note that the majority of Tunicamycin-treated WT and almost all N393Q and QQ were restricted to the cell body. (B) Line profile showing the fluorescence intensity of ASIC1a (red line) and Lck-GFP (green line) along the two lines illustrated in panel A. (C) Quantification of the effect of Tunicamycin on relative ASIC1a levels at mid-apical dendrites. The ratio of dendritic to cell body ASIC1a fluorescence was normalized to that of Lck-GFP, which controls for passive diffusion of membrane proteins. Numbers in parenthesis indicate the number of neurons quantified from 2 different experiments. (D) Quantification of the effect of glycosylation mutants on relative ASIC1a levels at mid-apical dendrites. Numbers in parenthesis indicate the number of neurons quantified from 6 (for WT), 4 (for N366Q) and 3 (for N393Q and QQ) different experiments. Asterisks indicate significant differences from WT. (E) High magnification view of a segment of apical dendrite. Note the presence of wild-type and N366Q ASIC1a in most dendritic spines. (F) Line profile of the relative fluorescence intensity along the line illustrated in (E). Note the difference in relative ASIC1a:Lck-GFP fluorescence intensity in spine versus shaft. (G) Quantification of Lck-GFP and ASIC1a spine:shaft ratio. Numbers on the bars indicate the total number of spines quantified from 9 wild-type and 12 N366Q transfected neurons from three separate experiments. Asterisks indicate significant differences from Lck-GFP (p < 0.0001, student’s t-test).

Article Snippet: Other reagents used: Endo H and PNGase F (New England Biolabs, Ipswich, MA); NHS-sulfo-LC-biotin and NeutrAvidin Beads (Pierce); Tunicamycin (ThermoFisher Scientific); culture media and serum (HyClone and Invitrogen); Fugene HD (Roche) and lipofectamine 2000 (Invitrogen).

Techniques: Transfection, Membrane, Immunofluorescence, Fluorescence, Staining, Diffusion-based Assay, Glycoproteomics

Journal: The Journal of Neuroscience

Article Title: N- glycosylation of Acid-sensing Ion Channel 1a Regulates its Trafficking and Acidosis-induced Spine Remodeling

doi: 10.1523/JNEUROSCI.5021-11.2012

Figure Lengend Snippet: CHO cells were transfected with ASIC1a and treated with DMSO (control) or 0.5 μg/ml Tunicamycin for 24 hrs. (A–D) Representative traces (A) and quantification showing the effect of Tunicamycin on the amplitude (B), density (C), and rate of desensitization (D) of pH6-activated current. Asterisks indicate significant (p < 0.001, paired t-test) differences. Numbers on the bars indicate total number of cells recorded.

Article Snippet: Other reagents used: Endo H and PNGase F (New England Biolabs, Ipswich, MA); NHS-sulfo-LC-biotin and NeutrAvidin Beads (Pierce); Tunicamycin (ThermoFisher Scientific); culture media and serum (HyClone and Invitrogen); Fugene HD (Roche) and lipofectamine 2000 (Invitrogen).

Techniques: Transfection, Control

Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: Identification of CD74 as a putative receptor for IBDV. (A) Affinity purification with mAb against the IBDV major capsid protein VP2 in DT40 cells, incubated with VP2 (or the empty vector as a negative control), followed by mass spectrometry analysis. Many host proteins were associated with VP2, including chicken CD74. (B and C) Interaction between VP2 and the CD74 extracellular domain detected via a coimmunoprecipitation (co-IP) assay. (B) Western blot (WB) analysis using an antibody against the HA tag showing the bands corresponding to VP2 in the Flag co-IP assay. (C) Western blot analysis using an antibody against the HA tag showing the bands corresponding to the CD74 extracellular domain in the Flag co-IP assay. (D) To determine the distribution of CD74 in organs, the heart, liver, spleen, lung, kidney, thymus, and bursa of Fabricius of uninfected SPF chickens were collected to measure CD74 transcription levels. The most abundant CD74 expression was found in the bursa. (E) To confirm the involvement of chicken CD74 in the IBDV infection process, SPF chickens were challenged by vvIBDV or PBS. Bursas were collected for RT-qPCR analysis of CD74 transcription levels. CD74 mRNAs were significantly induced by vvIBDV at 12 h p.i. (*, P < 0.05; **, P < 0.01). The arithmetic means and standard deviations for at least three independent experiments performed in duplicate are shown.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Affinity Purification, Incubation, Plasmid Preparation, Negative Control, Mass Spectrometry, Co-Immunoprecipitation Assay, Western Blot, Expressing, Infection, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: CD74 knockdown suppresses IBDV replication. The expression of the CD74 Ii-2 isoform was downregulated by siRNA interference or knockdown by shRNA in DT40 cells. The vvIBDV Gx strain at an MOI of 1 was added to the CD74 siRNA interference groups or the CD74 KD cell line. Infected cells were washed with PBS at 4 h p.i., and DT40 complete medium was then added. Cells and supernatants were collected at 24, 48, and 72 h p.i. for Western blotting and qPCR and at 72 h p.i. for ELD50 analysis. (A) CD74 expression levels in DT40 cells determined by Western blotting, showing that CD74 downregulation was effective. siSc., scrambled control siRNA. (B) Western blot assays showing IBDV VP2 expression declining at 24 to 48 h p.i. (C) qPCR analysis showing IBDV copy numbers dropping off at 48 h p.i. (6.34-fold decrease compared with the siRNA negative control; *, P < 0.05). (D) ELD50 assay showing the IBDV titer being downregulated at 48 h p.i. (14.0-fold decrease compared with the siRNA negative control; *, P < 0.05). (E) CD74 mRNA level in DT40 cells determined by qPCR, indicating that CD74 knockdown was effective. (F) IBDV VP2 protein expression was downregulated significantly at 24 to 72 h p.i. in the CD74 KD groups. (G) The IBDV copy number was downregulated at 24 to 72 h p.i. (103- to 104-fold decrease compared with the wild-type [WT] control; P < 0.05). (F) ELD50 assay showing the IBDV titer being downregulated at 72 h p.i. (4.42 × 104-fold decrease compared with wild-type cells; P < 0.05). The arithmetic means and standard deviations for at least three independent experiments performed in duplicate are shown.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Knockdown, Expressing, shRNA, Infection, Western Blot, Control, Negative Control

Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: CD74 overexpression promotes IBDV replication. CD74 Ii-2 isoform overexpression promotes IBDV infectivity. The expression of the CD74 Ii-2 isoform was upregulated by plasmid transfection in DT40 cells. Next, vvIBDV at an MOI of 1 was used to infected the cells as described in the text. Cells and supernatants were collected at 24, 48, and 72 h p.i. for Western blotting and at 48 h p.i. for qPCR analysis. (A) CD74 Ii-2 isoform overexpression remarkably promotes IBDV replication. The IBDV copy number was increased in the overexpression groups at 72 h p.i. compared with the nonoverexpression group (2.90-fold increase; *, P < 0.05). (B) IBDV VP2 protein expression is upregulated at 48 to 72 h p.i. in the overexpression group.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Over Expression, Infection, Expressing, Plasmid Preparation, Transfection, Western Blot

Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: CD74 isoform Ii-2 confers to vvIBDV the ability to attach to a vvIBDV-nonpermissive cell line. (A) 293T cells (nonpermissive to vvIBDV) were transfected with a full-length chicken CD74 Ii-2 plasmid with an HA tag (or the empty vector as a negative control). Twenty-four hours after transfection, cells were incubated with vvIBDV at an MOI of 5 at 37°C for 36 h to investigate whether CD74 confers susceptibility to IBDV infection. (Top) Cells were processed for confocal analysis, using IBDV VP2 mAb and an HA tag antibody as the primary antibodies. No virus (green fluorescence) was detected in the empty vector control. (Bottom) In the CD74 Ii-2 overexpression group, CD74 (red fluorescence) accumulated on the cell membrane, colocalizing with the IBDV particles (green fluorescence). No virus was observed to enter CD74-overexpressing nonpermissive cells. CD74 could confer attachment ability but not susceptibility to IBDV infection. (B) 293T cells overexpressed HA-tagged chicken CD74 Ii-2 or the empty vector for 24 h and were then incubated with 200 μg SVPs at 4°C for 1 h. After washing with PBS 5 times, cells were processed for confocal analysis as described above. SVPs (green fluorescence) were bound to the membranes of CD74-overexpressing cells (red fluorescence) and colocalized with CD74, but no binding was observed for the empty vector group. (C and D) 293T and HeLa cells (both of which are nonpermissive to vvIBDV) were transfected with a eukaryotic expression plasmid of HA-tagged chicken CD74 Ii-2 (or the empty vector as a negative control) and maintained for 24 h under normal culture conditions. The cells were then incubated with vvIBDV at an MOI of 50 at 4°C for 1 h for the binding assay and washed with PBS 5 times to remove the unbound viruses. Cells were processed for confocal analysis using the same antibodies and procedure as the ones described above. No virus (green fluorescence) was found to bind or infect 293T (C) or HeLa (D) cells transfected with the empty vector. In contrast, IBDV (green fluorescence) colocalized with overexpressed chicken CD74 (red fluorescence) on the membrane of nonpermissive 293T (C) and HeLa (D) cells. (E and F) qPCR analysis indicating that CD74 overexpression promotes IBDV binding to 293T or HeLa cells compared with the empty vector transfection group (9.55-fold increase in 293T cells [E] and 5.07-fold increase in HeLa cells [F]; both P < 0.05). The arithmetic means and standard deviations for at least three independent experiments performed in duplicate are shown.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Transfection, Plasmid Preparation, Negative Control, Incubation, Infection, Virus, Fluorescence, Control, Over Expression, Membrane, Binding Assay, Expressing

Journal: Nature Communications

Article Title: Loss of TRIM29 mitigates viral myocarditis by attenuating PERK-driven ER stress response in male mice

doi: 10.1038/s41467-024-44745-x

Figure Lengend Snippet: a Immunoblot (IB) analysis of endogenous proteins TRIM29 and PERK precipitated with anti-PERK or immunoglobulin G (IgG) from whole-cell lysates of mouse primary neonatal cardiomyocytes from Trim29 fl/fl and WTMyHC-Cre mice with or without (Mock) CVB3 and EMCV infection for 6 h at an MOI of 5. Schematic diagram showing full-length PERK (Full, b ) or TRIM29 (Full, c ) and serial truncations of PERK ( b ) or TRIM29 ( c ) with deletion (Δ) of various domains (left margin); numbers at ends indicate amino acid positions (top). Luminal, ER luminal domain; TM, transmembrane domain; Kinase, protein kinase domain; BBOX, the B-box zinc-finger domain; CC, the coil-coil domain; OmpH, the outer membrane protein H domain. Immunoblot analysis of purified HA-tagged full-length PERK and serial truncations of PERK with deletion of various domains alone ( d ) or purified HA-tagged full-length TRIM29 and serial truncations of TRIM29 with deletion of various domains alone ( e ) with anti-HA antibody (top blot) or after incubation with Myc-tagged TRIM29 ( d ) or Myc-tagged PERK ( e ) and immunoprecipitation with anti-Myc antibody (second blot), and immunoblotting analysis of purified Myc-tagged TRIM29 ( d ) or Myc-tagged PERK ( e ) with anti-Myc antibody (third blot) or after incubation with Myc-tagged TRIM29 ( d ) or Myc-tagged PERK ( e ) and immunoprecipitation with anti-Myc antibody (bottom blot). f Immunoblot analysis of endogenous phosphorylated PERK (pPERK), HA-tagged PERK and Myc-tagged TRIM29 from whole-cell lysates of HEK293T cells co-transfected with HA-PERK and vector or Myc-TRIM29 for 24 h. g Immunoblot analysis of endogenous proteins TRIM29 and phosphorylated PERK (pPERK) precipitated with anti-pPERK or immunoglobulin G (IgG) from whole-cell lysates of mouse primary neonatal cardiomyocytes from Trim29 fl/fl mice followed by CVB3 infection at an MOI of 5 and treatment with the PERK inhibitor GSK2656157 or DMSO for 6 h. The position of protein markers (shown in kDa) is indicated on the right. Data are representative of three independent experiments. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used for immunoblot analysis: anti-TRIM29 (1:1000; A301-210A; Bethyl), anti-TRIM29 (1:1000; sc-33151; Santa Cruz), anti-ANP (1:1000; sc-515701; Santa Cruz), anti-FITC (1:2000; 71–1900; Thermo Fisher Scientific), antibody to phosphorylated PERK (1:1000; MA5-15033; Thermo Fisher Scientific), anti-PERK (1:1000; 3192 S; Cell Signaling Technology), anti-IRE1α (1:1000; 3194 S; Cell Signaling Technology), anti-ATF6 (1:1000; 65880 S; Cell Signaling Technology), anti-CHOP (1:1000; 2895 S; Cell Signaling Technology), anti-cleaved caspase-3 (1:1000; 9664 S; Cell Signaling Technology), anti-BCL2 (1:1000; 3498 S; Cell Signaling Technology), anti-BAX (1:1000; 2772 S; Cell Signaling Technology), anti-STING (1:1000; 13647 S; Cell Signaling Technology), anti-IRF3 (1:1000; 4302 S; Cell Signaling Technology), anti-TBK1 (1:1000; 3504 S; Cell Signaling Technology), anti-SUMO1 (1:1000; 4930 S; Cell Signaling Technology), anti-SUMO2/3 (1:1000; 4971 S; Cell Signaling Technology), antibody to phosphorylated IRF3 at Ser396 (1:1000; 4947 S; Cell Signaling Technology), antibody to phosphorylated TBK1 at Ser172 (1:1000; 5483 S; Cell Signaling Technology), antibody to phosphorylated IRE1α (1:1000; ab48187, Abcam), anti-His (1:5000; 652504; BioLegend), anti-Flag (1:5000; A8592; Sigma), anti-GAPDH (1:20,000; G9295; Sigma), anti-HA (1:5000; H6533; Sigma) and anti-Myc (1:5000; 16–213; Sigma).

Techniques: Western Blot, Infection, Membrane, Purification, Incubation, Immunoprecipitation, Transfection, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: Cellular Levels and Binding of c-di-GMP Control Subcellular Localization and Activity of the Vibrio cholerae Transcriptional Regulator VpsT

doi: 10.1371/journal.ppat.1002719

Figure Lengend Snippet: (A) Representative epifluorescence micrographs of wild-type V. cholerae expressing GFP-VpsT, CdgA-GFP or CdgH-GFP fusion proteins. Marker is 2 µm. (B) Subcellular fractionation of V. cholerae strains containing vpsT , cdgA or cdgH tagged with an HA epitope in their native chromosomal loci. Western immunoblot was performed on cellular fractions representing whole cell (WC), cytoplasmic (C) and total membrane (M) fractions. HA-tagged proteins were detected using a polyclonal anti-HA antibody. gfp was constitutively expressed from a chromosomal locus. GFP was detected using monoclonal anti-GFP antibody and is used as a cytoplasmic fraction control. OmpU was detected using a polyclonal anti-OmpU antibody and is used as a total membrane fraction control. One representative experiment of three biological replicates is shown. (C) Bacterial two-hybrid analysis of VpsT, CdgA and CdgH. Reconstitution of CyaA, indicative of protein-protein interaction, was detected by β-galactosidase activity on LB plates containing ampicillin (100 µg/ml), kanamycin (50 µg/ml), IPTG (500 µM) and X-gal (40 µg/ml). Plates were incubated at 30°C for 48 h.

Article Snippet: Mouse monoclonal antibody against GFP (Santa Cruz Biotechnology) and rabbit polyclonal antibody against the HA epitope (Santa Cruz Biotechnology) were used according to the manufacturer's instructions.

Techniques: Expressing, Marker, Fractionation, Western Blot, Membrane, Control, Activity Assay, Incubation

Journal: The Journal of Neuroscience

Article Title: GluN2B-Containing NMDA Receptors Regulate AMPA Receptor Traffic through Anchoring of the Synaptic Proteasome

doi: 10.1523/jneurosci.3567-14.2015

Figure Lengend Snippet: Figure 3. GluN1 and GluN2A total and surface levels are decreased in GluN2B / cortical neurons. Membrane extracts from cortical cultures from GluN2B /, heterozygous GluN2B /, and GluN2B / mice at 7 DIV (A) or 15 DIV (B) were used to evaluatetotalGluN1andGluN2AproteinlevelsbyWesternblot.C,D,TotalGluN1andGluN2Aproteinlevelswerenormalizedwith transferrinreceptor(TransfR)levelsandpresentedaspercentageoftheproteinlevelsdetectedinwild-typeneurons7DIV.Dataare presentedasmean SEMfromthreetofourindependentexperiments.*p0.05,**p0.01,***p0.0001,one-wayANOVA followed by Bonferroni’s multiple-comparisons test. Relative mRNA levels for GluN1 (E) and GluN2A (F) in 15 DIV mouse cortical culturesweredeterminedbyquantitativereal-timePCR.Dataarepresentedasmean SEMfromfiveindependentexperiments forGluN1mRNAlevelsandfourindependentexperimentsforGluN2AmRNAlevels.p0.05,pairedttest.G,Totalextractsandcell surface biotinylated-purified proteins isolated from high-density cortical culture 15 DIV from GluN2B / and / mice were assayed by Western blot using an antibody against GluN1. H, Transferrin receptor protein levels were used as a loading control. GluN1 levels are normalized to GluN1 levels in wild-type neurons. Data are presented as mean SEM from three independent experiments. **p 0.01, paired t test.

Article Snippet: Primary: anti-GluN1 (1:500 WB, #MAB363), anti-GluN2A (1:1000, #AB1555P), anti-GluA1 (1:1000 WB or 1:100 ICC, #AB1504), anti-GluA2 (1:1000 WB or 1:500 ICC, #MAB397), anti-phospho-GluA1 Ser845 (1:1000, #AB5849), anti-phospho-GluA2 Ser880 (1:1000, #07- 294), phospho-stargazin (Ser239/240) (1:500, #ab3713), phosphoCaMKII / subunit (Thr286/287) (1:1000, #06 – 881) were from Millipore; anti-GluN1 (1:1500 ICC, #MAb 54.1) was from Invitrogen; N-terminal of GluA1 (1:50) was a kind gift from Dr. Andrew Irving; anti-PSD-95 (D27E11) (1:2000 WB or 1:100 ICC, #3450) and antiphospho-protein kinase C (PKC) substrates (1:1000, #2261S) were from Cell Signaling Technology; anti-SynGAP (1:1000, #PA1-046) and antiPSD-95 (1:500, #MA 1.046 C7E3-1B8) were from Affinity Bioreagents; anti-Synaptophysin (1:10000, #101 011) was from Synaptic Systems; anti-CaMKII (K-19) (1:500, #sc-100366) was from Tebu-Bio, Santa Cruz Biotechnology); CaMKII (6G9) (1:2000, #C265) was from SigmaAldrich; anti-phospho-GluA1 Ser 831 (1:5000, #2041) was from Tocris Bioscience; anti-phospho-GluA1 T840 was a kind gift from R. Huganir (JH2811); anti-Transferrin receptor antibody (1:1000, #13-6800) was from Invitrogen; anti-vesicular glutamate transporter 1 (VGLUT1; 1:5000, #AB5905) was from Millipore; anti-MAP2 (1:5000, #ab5392) was from Abcam; and anti- -actin (1:5000, #1378996) was from Roche Molecular Biochemicals.

Techniques: Membrane, Purification, Isolation, Western Blot, Control