Journal: Journal of Neuroinflammation

Article Title: β-arrestin1 regulates astrocytic reactivity via Drp1-dependent mitochondrial fission: implications in postoperative delirium

doi: 10.1186/s12974-023-02794-x

Figure Lengend Snippet: Orthopedic surgery induces glial activation and pro-inflammatory phenotypes in the hippocampus. A Mice were established orthopedic surgery-induced POD model. Levels of pro-inflammatory cytokines in the hippocampus were analyzed by ELISA. B Levels of anti-inflammatory and neurotrophic cytokines in the hippocampus were analyzed by ELISA. C mRNA levels of pro-inflammatory and anti-inflammatory genes as well as neurotrophic genes in the hippocampus were analyzed by RT-PCR. D Immunohistochemical staining of Iba-1 in the hippocampus of CON and POD mice. E Immunohistochemical staining of GFAP in the hippocampus of CON and POD mice. F Analysis of Iba-1-positive area and cell numbers in the hippocampus. G Analysis of GFAP-positive area and cell numbers in the hippocampus. H Expression of GFAP and Iba-1 in the hippocampus of CON and POD mice. I Densitometric analysis of GFAP and Iba-1. J Heatmap of neurotoxic astrocytes transcripts in the hippocampus of CON and POD mice. K Heatmap of neuroprotective astrocytes transcripts in the hippocampus of CON and POD mice. L Expression of C3, Serping1 and Psmb8 in the hippocampus of CON and POD mice. M Densitometric analysis of C3, Serping1 and Psmb8. N Double immunofluorescent staining of astrocytic pan-active marker GFAP and neurotoxic astrocytic marker C3 in hippocampus of CON and POD mice. O Relative co-localized signals of the GFAP-positive and C3-positive immunofluorescent particles between CON and POD group. Data are analyzed by unpaired Student’s t-test. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs . the CON group. Values are presented as means ± SEM from at least three independent experiments

Article Snippet: After blocking with 10% nonfat dry milk in Tris-buffered saline (20 mM Tris–HCl, 500 mM NaCl, pH 7.4) with Tween 20 (Aladdin, #T104863), the membranes were then probed with the following primary antibodies overnight at 4 °C: mouse anti-GFAP antibody (1:1000, Cell Signaling Technology, #3670), rabbit anti-Iba-1 antibody (1:1000, Wako, #019-19741), rabbit anti-β-arrestin1 (1:1000, Cell Signaling Technologies, #12697), rabbit anti-β-arrestin2 (1:200, Cell Signaling Technologies, #3857), rabbit anti-C3 antibody (1:1000, abcam, #ab11887), rabbit anti-Serping1 antibody (1:1000, proteintech, #12259-1-AP), rabbit anti-Psmb8 antibody (1:1000, proteintech, #14859-1-AP), mouse anti-Fis1 antibody (1:1000, Santa Cruz, #sc-376447), mouse anti-Drp1 antibody (1:1000, Santa Cruz, #sc-101270), rabbit anti-Drp1 antibody (1:1000, proteintech, #12957-1-AP), rabbit anti-COX IV antibody (1:1000, Cell Signaling Technology, #4850), mouse anti-β-actin antibody (1:3000, sigma, #a1978).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Immunohistochemical staining, Staining, Expressing, Marker

Journal: Journal of Neuroinflammation

Article Title: β-arrestin1 regulates astrocytic reactivity via Drp1-dependent mitochondrial fission: implications in postoperative delirium

doi: 10.1186/s12974-023-02794-x

Figure Lengend Snippet: β-arrestin1 in astrocytes modulates cognitive impairments and astrocytic reactivity in the mouse model for POD. A β-arrestin1 protein levels in brain lysate of WT and β-arrestin1 −/− mice. B Experimental protocol and timeline of the POD mouse model. C Representative moving track plots (red curve) of mice in the second trial of Y-maze test. Blue box represents the novel arm. D Time (%) spent in the novel arm in the Y-maze test. E Bouts of novel arm entry in the Y-maze test. F Representative moving track plots (red curve) of mice in the probe trial of Morris water maze test. Black circle represents the invisible platform. G Latency (s) to reach the hidden platform in the probe test of Morris water maze test. H Crossing times in target quadrant in the probe test of Morris water maze test. I Representative immunofluorescent staining of GFAP in the hippocampus. J Analysis of GFAP-positive cell body area in the hippocampus. K Analysis of GFAP-positive cell numbers in the hippocampus. L Heatmap of the expression level of the neurotoxic astrocytes-specific transcripts in the hippocampus. M Expression of C3, Serping1 and Psmb8 in the hippocampus. N Densitometric analysis of C3, Serping1 and Psmb8. O Schematic diagram of the mice model with micro-injection of AAV-siβ-arrestin1 into the hippocampus. P Immunofluorescent co-localization of GFP and GFAP (red) after the AAV micro-injection. Q Representative immunofluorescent staining of GFAP (red) in the hippocampus. R Analysis of astrocytic reactivity by GFAP-positive cell body area and GFAP-positive cell numbers in the hippocampus. S Bouts of novel arm entry in the Y-maze test. T Time (%) spent in the novel arm in the Y-maze test. U Latency (s) to reach the hidden platform in the probe test of Morris water maze test. V Crossing times in target quadrant in the probe test of Morris water maze test. For C-M except for R, data were analyzed by two-way ANOVA followed by Tukey's multiple comparisons test. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. the WT-CON group or NC AAV CON group. # P < 0.05, ## P < 0.01 and ### P < 0.001 vs. the WT-POD mice or NC AAV POD. For R, data are analyzed by unpaired Student’s t-test. * P < 0.05 and *** P < 0.001 vs. the NC AAV POD group. n = 6 mice per group for immunofluorescent staining. n = 3 for western blotting. n = 7–10 mice for behavioral tests. Values are presented as means ± SEM

Article Snippet: After blocking with 10% nonfat dry milk in Tris-buffered saline (20 mM Tris–HCl, 500 mM NaCl, pH 7.4) with Tween 20 (Aladdin, #T104863), the membranes were then probed with the following primary antibodies overnight at 4 °C: mouse anti-GFAP antibody (1:1000, Cell Signaling Technology, #3670), rabbit anti-Iba-1 antibody (1:1000, Wako, #019-19741), rabbit anti-β-arrestin1 (1:1000, Cell Signaling Technologies, #12697), rabbit anti-β-arrestin2 (1:200, Cell Signaling Technologies, #3857), rabbit anti-C3 antibody (1:1000, abcam, #ab11887), rabbit anti-Serping1 antibody (1:1000, proteintech, #12259-1-AP), rabbit anti-Psmb8 antibody (1:1000, proteintech, #14859-1-AP), mouse anti-Fis1 antibody (1:1000, Santa Cruz, #sc-376447), mouse anti-Drp1 antibody (1:1000, Santa Cruz, #sc-101270), rabbit anti-Drp1 antibody (1:1000, proteintech, #12957-1-AP), rabbit anti-COX IV antibody (1:1000, Cell Signaling Technology, #4850), mouse anti-β-actin antibody (1:3000, sigma, #a1978).

Techniques: Staining, Expressing, Microinjection, Western Blot

Journal: Journal of Neuroinflammation

Article Title: β-arrestin1 regulates astrocytic reactivity via Drp1-dependent mitochondrial fission: implications in postoperative delirium

doi: 10.1186/s12974-023-02794-x

Figure Lengend Snippet: β-arrestin1 deletion aggravates the neurotoxic reactivity of primary astrocytes. A Schematic of the experimental design. B Expression of C3, Serping1 and Psmb8 in the primary astrocytes. C Densitometric analysis of C3, Serping1 and Psmb8. D Heat map of A1 astrocytic genes in primary cell cultures. E Immunofluorescent staining of C3 (green) and GFAP (red) in primary astrocytes. F Immunofluorescent staining of Serping1 (red) and GFAP (green) in primary astrocytes. G Relative co-localized signals of the GFAP-positive and C3-positive immunofluorescent particles between groups. H Relative co-localized signals of the GFAP-positive and Serping1-positive immunofluorescent particles between groups. I Astrocytes were stained with MitoSOX and analyzed by flow cytometry. J JC-1 staining in astrocytes were analyzed by flow cytometry. K Quantification of the mitochondrial ROS in MitoSOX staining. L Quantification of the loss of mitochondrial membrane potential in JC-1 staining measured by flow cytometry. M Oxygen consumption rates were evaluated by Seahorse. N Quantification of oxygen consumption for ATP production, basal respiration and proton leak. O ATP levels in astrocytes. Data were analyzed by two-way ANOVA followed by Tukey's multiple comparisons test. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. the WT CON-MCM group. # P < 0.05, ## P < 0.01 and ### P < 0.001 vs. the WT LPS-MCM group. Values are presented as means ± SEM from at least three independent experiments

Article Snippet: After blocking with 10% nonfat dry milk in Tris-buffered saline (20 mM Tris–HCl, 500 mM NaCl, pH 7.4) with Tween 20 (Aladdin, #T104863), the membranes were then probed with the following primary antibodies overnight at 4 °C: mouse anti-GFAP antibody (1:1000, Cell Signaling Technology, #3670), rabbit anti-Iba-1 antibody (1:1000, Wako, #019-19741), rabbit anti-β-arrestin1 (1:1000, Cell Signaling Technologies, #12697), rabbit anti-β-arrestin2 (1:200, Cell Signaling Technologies, #3857), rabbit anti-C3 antibody (1:1000, abcam, #ab11887), rabbit anti-Serping1 antibody (1:1000, proteintech, #12259-1-AP), rabbit anti-Psmb8 antibody (1:1000, proteintech, #14859-1-AP), mouse anti-Fis1 antibody (1:1000, Santa Cruz, #sc-376447), mouse anti-Drp1 antibody (1:1000, Santa Cruz, #sc-101270), rabbit anti-Drp1 antibody (1:1000, proteintech, #12957-1-AP), rabbit anti-COX IV antibody (1:1000, Cell Signaling Technology, #4850), mouse anti-β-actin antibody (1:3000, sigma, #a1978).

Techniques: Expressing, Staining, Flow Cytometry, Membrane

Journal: Journal of Neuroinflammation

Article Title: β-arrestin1 regulates astrocytic reactivity via Drp1-dependent mitochondrial fission: implications in postoperative delirium

doi: 10.1186/s12974-023-02794-x

Figure Lengend Snippet: β-arrestin1-biased ligand Carvedilol recovers the neurotoxic astrocytes reactivity. A Heat map of the expression levels of the signature genes for neurotoxic astrocytes in primary cell cultures. B Expression of C3, Serping1 and Psmb8 in primary cell cultures. C Densitometric analysis of C3, Serping1 and Psmb8. D Immunofluorescent staining of GFAP (red) and C3 (green) in primary astrocytes. E Immunofluorescent staining of GFAP (green) and Serping1 (red) in primary astrocytes. F Relative co-localized signals of the GFAP-positive and C3-positive immunofluorescent particles between groups. G Relative co-localized signals of the GFAP-positive and Serping1-positive immunofluorescent particles between groups. H Astrocytes were stained with MitoSOX and analyzed by flow cytometry. I JC-1 staining in astrocytes was analyzed by flow cytometry. J Quantification of the mitochondrial ROS in MitoSOX staining. K Quantification of the loss of mitochondrial membrane potential in JC-1 staining measured by flow cytometry. L Oxygen consumption rates were evaluated by Seahorse. M Quantification of oxygen consumption for ATP production, basal respiration and proton leak. N ATP levels in astrocytes. Data were analyzed by one-way ANOVA followed by Dunnet’s post-hoc test. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. the CON group. # P < 0.05, ## P < 0.01 and ### P < 0.01 vs. the LPS-MCM group. Values are presented as means ± SEM from at least three independent experiments

Article Snippet: After blocking with 10% nonfat dry milk in Tris-buffered saline (20 mM Tris–HCl, 500 mM NaCl, pH 7.4) with Tween 20 (Aladdin, #T104863), the membranes were then probed with the following primary antibodies overnight at 4 °C: mouse anti-GFAP antibody (1:1000, Cell Signaling Technology, #3670), rabbit anti-Iba-1 antibody (1:1000, Wako, #019-19741), rabbit anti-β-arrestin1 (1:1000, Cell Signaling Technologies, #12697), rabbit anti-β-arrestin2 (1:200, Cell Signaling Technologies, #3857), rabbit anti-C3 antibody (1:1000, abcam, #ab11887), rabbit anti-Serping1 antibody (1:1000, proteintech, #12259-1-AP), rabbit anti-Psmb8 antibody (1:1000, proteintech, #14859-1-AP), mouse anti-Fis1 antibody (1:1000, Santa Cruz, #sc-376447), mouse anti-Drp1 antibody (1:1000, Santa Cruz, #sc-101270), rabbit anti-Drp1 antibody (1:1000, proteintech, #12957-1-AP), rabbit anti-COX IV antibody (1:1000, Cell Signaling Technology, #4850), mouse anti-β-actin antibody (1:3000, sigma, #a1978).

Techniques: Expressing, Staining, Flow Cytometry, Membrane

Journal: Journal of Neuroinflammation

Article Title: β-arrestin1 regulates astrocytic reactivity via Drp1-dependent mitochondrial fission: implications in postoperative delirium

doi: 10.1186/s12974-023-02794-x

Figure Lengend Snippet: β-arrestin1-biased ligand Carvedilol protects POD mice from pro-inflammatory phenotypes. A Experimental protocol and timeline of the mouse model. B Representative immunofluorescent staining of GFAP in the hippocampus. C Relative GFAP-positive cell body area in the hippocampus. D Relative GFAP-positive cell numbers in the hippocampus. E Heatmap of the expression level of the A1-specific transcripts in hippocampal samples. F Expression of C3, Serping1 and Psmb8 in the hippocampus. G Densitometric analysis of C3, Serping1 and Psmb8. H Representative moving track plots (red curve) of mice in the second trial of Y-maze test. Blue box represents the novel arm. I Time (%) spent in the novel arm in the Y-maze test. J Bouts of novel arm entry in the Y-maze test. K Representative moving track plots (red curve) of mice in the probe trial of Morris water maze test. Black circle represents the invisible platform. L Latency (s) to reach the hidden platform in the probe test of Morris water maze test. M Crossing times in target quadrant in the probe test of Morris water maze test. Data were analyzed by one-way ANOVA followed by Dunnet’s post-hoc test. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. the CON group. # P < 0.05, ## P < 0.01 and ### P < 0.01 vs. the POD group. n = 6 mice per group for immunofluorescent staining. n = 3 for western blotting. n = 10 mice for behavioral tests. Values are presented as means ± SEM

Article Snippet: After blocking with 10% nonfat dry milk in Tris-buffered saline (20 mM Tris–HCl, 500 mM NaCl, pH 7.4) with Tween 20 (Aladdin, #T104863), the membranes were then probed with the following primary antibodies overnight at 4 °C: mouse anti-GFAP antibody (1:1000, Cell Signaling Technology, #3670), rabbit anti-Iba-1 antibody (1:1000, Wako, #019-19741), rabbit anti-β-arrestin1 (1:1000, Cell Signaling Technologies, #12697), rabbit anti-β-arrestin2 (1:200, Cell Signaling Technologies, #3857), rabbit anti-C3 antibody (1:1000, abcam, #ab11887), rabbit anti-Serping1 antibody (1:1000, proteintech, #12259-1-AP), rabbit anti-Psmb8 antibody (1:1000, proteintech, #14859-1-AP), mouse anti-Fis1 antibody (1:1000, Santa Cruz, #sc-376447), mouse anti-Drp1 antibody (1:1000, Santa Cruz, #sc-101270), rabbit anti-Drp1 antibody (1:1000, proteintech, #12957-1-AP), rabbit anti-COX IV antibody (1:1000, Cell Signaling Technology, #4850), mouse anti-β-actin antibody (1:3000, sigma, #a1978).

Techniques: Staining, Expressing, Western Blot

Journal: Journal of Neuroinflammation

Article Title: β-arrestin1 regulates astrocytic reactivity via Drp1-dependent mitochondrial fission: implications in postoperative delirium

doi: 10.1186/s12974-023-02794-x

Figure Lengend Snippet:

Article Snippet: After blocking with 10% nonfat dry milk in Tris-buffered saline (20 mM Tris–HCl, 500 mM NaCl, pH 7.4) with Tween 20 (Aladdin, #T104863), the membranes were then probed with the following primary antibodies overnight at 4 °C: mouse anti-GFAP antibody (1:1000, Cell Signaling Technology, #3670), rabbit anti-Iba-1 antibody (1:1000, Wako, #019-19741), rabbit anti-β-arrestin1 (1:1000, Cell Signaling Technologies, #12697), rabbit anti-β-arrestin2 (1:200, Cell Signaling Technologies, #3857), rabbit anti-C3 antibody (1:1000, abcam, #ab11887), rabbit anti-Serping1 antibody (1:1000, proteintech, #12259-1-AP), rabbit anti-Psmb8 antibody (1:1000, proteintech, #14859-1-AP), mouse anti-Fis1 antibody (1:1000, Santa Cruz, #sc-376447), mouse anti-Drp1 antibody (1:1000, Santa Cruz, #sc-101270), rabbit anti-Drp1 antibody (1:1000, proteintech, #12957-1-AP), rabbit anti-COX IV antibody (1:1000, Cell Signaling Technology, #4850), mouse anti-β-actin antibody (1:3000, sigma, #a1978).

Techniques:

Journal: bioRxiv

Article Title: C1q binding to surface-bound IgG is stabilized by C1r 2 s 2 proteases

doi: 10.1101/2021.02.08.430229

Figure Lengend Snippet: (A) Schematic presentation of C1 consisting of the recognition molecule C1q in complex with a tetramer of proteases C1r and C1s (C1r 2 s 2 ). C1q consists of six polypeptide chains that come together in an N-terminal stalk. At their C-terminus, all six chains end in a globular domain (gC1q) that recognizes Fc domains of IgM and clustered IgGs. The C1r 2 s 2 tetramer associates to the collagen arms of C1q via Ca 2+ -dependent interactions . The proteases dissociate from C1q in the presence of EDTA, a calcium-chelator. (B) Binding of different forms of purified C1 to DNP-beads (1 µg/ml DNP) coated with 20 nM anti-DNP IgG1-4. ‘C1’ indicates the fully assembled C1 complex (= C1qr 2 s 2 ), ‘C1q’ is the recognition molecule C1q only, ‘C1-EDTA’ sample consists of C1q, C1r and C1s, but the proteases are not attached to C1q (because 10 mM EDTA disrupts the Ca 2+ -dependent association between proteases and C1q). (C) Binding of different forms of purified C1 to IgG3-labelled beads coated with 0.003 µg/ml (∼300-fold lower than in (B) ). The dotted lines show aspecific binding of the C1q molecules in absence of IgG3. (B-C) Bound C1q was detected by polyclonal anti-C1q antibodies and flow cytometry. Data represent mean ± SD of three independent experiments.

Article Snippet: To detect C1r, C1s, C1-INH or C1q, the membrane was incubated in PBS-T with 1% ELK for 45 minutes with either goat anti-human C1r (1:300, R&D Systems, AF1807), sheep anti-human C1s (1:300, R&D Systems, AF2060), rabbit anti-C1-INH (1 µg/mL, Sino Biologicals, 10995-R018) or rabbit anti-human C1q (1:300), respectively.

Techniques: Binding Assay, Purification, Flow Cytometry

Journal: bioRxiv

Article Title: C1q binding to surface-bound IgG is stabilized by C1r 2 s 2 proteases

doi: 10.1101/2021.02.08.430229

Figure Lengend Snippet: (A) Detection of C1q on IgG-coated DNP-beads that were first labelled with purified C1 and subsequently incubated with 10 mM EDTA or 200 nM C1-INH to remove C1r and C1s proteases. Data represent geometric mean ± SD of three independent experiments. Unpaired t test (buffer vs. EDTA; buffer vs. C1-INH); * P < 0.05, all other conditions not significant. (B) Schematic cartoon of our hypothesis that C1r 2 s 2 dissociation by C1-INH can result in C1q dislodgement depending on the stability of the C1q-IgG complex. Our data suggest that removal of the C1r and C1s proteases from surface-bound C1 by C1-INH can result in two situations: (I) C1q dissociates from the surface-bound IgGs in the case the remaining C1q-IgG complexes are unstable, e.g. for C1q-IgG2 complexes; or (II) C1q remains bound since it has formed a stable interaction with the surface-bound IgGs.

Article Snippet: To detect C1r, C1s, C1-INH or C1q, the membrane was incubated in PBS-T with 1% ELK for 45 minutes with either goat anti-human C1r (1:300, R&D Systems, AF1807), sheep anti-human C1s (1:300, R&D Systems, AF2060), rabbit anti-C1-INH (1 µg/mL, Sino Biologicals, 10995-R018) or rabbit anti-human C1q (1:300), respectively.

Techniques: Purification, Incubation

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Differential gene expression of serine protease inhibitors in bovine ovarian follicle: possible involvement in follicular growth and atresia

doi: 10.1186/1477-7827-9-72

Figure Lengend Snippet: Details of the primers used for quantitative real-time RT-PCR analysis

Article Snippet: The 7-μm-thick follicular sections were incubated at room temperature for 4 h with rabbit polyclonal anti-SERPINA5 antibody (H00005104, Abnova, Taipei, Taiwan) diluted 1:10, rabbit polyclonal anti-SERPINB6 antibody (GTX114637, GeneTex Inc, Irvine, CA, USA) diluted 1:100, rabbit polyclonal anti-SERPINF2 antibody (H00005345, Abnova) diluted 1:20 or rabbit polyclonal anti-SERPING1 antibody (GTX105316, GeneTex) diluted 1:300 in Discovery Ab diluents (Roche).

Techniques: Quantitative RT-PCR

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Differential gene expression of serine protease inhibitors in bovine ovarian follicle: possible involvement in follicular growth and atresia

doi: 10.1186/1477-7827-9-72

Figure Lengend Snippet: QPCR analysis of SERPINA5, SERPINB6, SERPINE1, SERPINE2, SERPINF2 and SERPING1 in healthy and atretic follicles . Total RNA from the follicular wall (i.e., granulosa plus theca interna) was extracted from three healthy follicles and three atretic follicles. The expression of mRNA was normalized to the expression of GAPDH measured in the same RNA preparation. The black and white bars indicate healthy and atretic follicles, respectively. Data are shown as the mean ± SEM. Different superscript letters denote significant differences ( P < 0.05).

Article Snippet: The 7-μm-thick follicular sections were incubated at room temperature for 4 h with rabbit polyclonal anti-SERPINA5 antibody (H00005104, Abnova, Taipei, Taiwan) diluted 1:10, rabbit polyclonal anti-SERPINB6 antibody (GTX114637, GeneTex Inc, Irvine, CA, USA) diluted 1:100, rabbit polyclonal anti-SERPINF2 antibody (H00005345, Abnova) diluted 1:20 or rabbit polyclonal anti-SERPING1 antibody (GTX105316, GeneTex) diluted 1:300 in Discovery Ab diluents (Roche).

Techniques: Expressing

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Differential gene expression of serine protease inhibitors in bovine ovarian follicle: possible involvement in follicular growth and atresia

doi: 10.1186/1477-7827-9-72

Figure Lengend Snippet: mRNA localization of SERPINA5, SERPINB6, SERPINF2 and SERPING1 in E 2 -active and E 2 -inactive follicles . SERPINA5, SERPINB6 and SERPINF2 mRNA was expressed more in healthy than in atretic follicles, while SERPING1 mRNA was expressed more in atretic than in healthy follicles in QPCR analysis. (A, C, E, G, I, K, M and O) Digoxigenin (DIG)-labeled anti-sense cRNA probes were used. (B, D, F, H, J, L, N and P) DIG-labeled sense cRNA probes were used. Sections (7 μm) of bovine follicles were hybridized with each probe. SERPINA5 (A, B, C and D), SERPINB6 (E, F, G and H) and SERPINF2 (I, J, K and L) mRNA were found in the GCs of E 2 -active follicles and a weak hybridization signal was detected in GCs of E 2 -inactive follicles. SERPING1 mRNA (M, N, O and P) was detected in both GCs and the TL of E 2 -inactive follicles and a weak hybridization signal was also detected in both GCs and the TL of E 2 -active follicles. Scale bars = 20 μm.

Article Snippet: The 7-μm-thick follicular sections were incubated at room temperature for 4 h with rabbit polyclonal anti-SERPINA5 antibody (H00005104, Abnova, Taipei, Taiwan) diluted 1:10, rabbit polyclonal anti-SERPINB6 antibody (GTX114637, GeneTex Inc, Irvine, CA, USA) diluted 1:100, rabbit polyclonal anti-SERPINF2 antibody (H00005345, Abnova) diluted 1:20 or rabbit polyclonal anti-SERPING1 antibody (GTX105316, GeneTex) diluted 1:300 in Discovery Ab diluents (Roche).

Techniques: Labeling, Hybridization

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Differential gene expression of serine protease inhibitors in bovine ovarian follicle: possible involvement in follicular growth and atresia

doi: 10.1186/1477-7827-9-72

Figure Lengend Snippet: Protein localization of SERPINA5, SERPINB6, SERPINF2 and SERPING1 in E 2 -active and E 2 -inactive follicles . Localization of SERPINA5 (A and B), SERPINB6 (C and D), SERPINF2 (E and F) and SERPING1 (G and H) protein was detected by immunohistochemistry. Sections (7 μm) of bovine E 2 -active (A, C, E and G) and E 2 -inactive follicles (B, D, F and H) were incubated with anti-SERPINA5, anti-SERPINB6, anti-SERPINF2 and anti-SERPING1 polyclonal antibodies. SERPINA5, SERPINB6 and SERPINF2 were detected in the GCs of E 2 -active and E 2 -inactive follicles. SERPING1 was detected in both GCs and the TL of E 2 -active and E 2 -inactive follicles. Negative control (I and J) was incubated without anti-SERPIN antibodies. Scale bars = 20 μm.

Article Snippet: The 7-μm-thick follicular sections were incubated at room temperature for 4 h with rabbit polyclonal anti-SERPINA5 antibody (H00005104, Abnova, Taipei, Taiwan) diluted 1:10, rabbit polyclonal anti-SERPINB6 antibody (GTX114637, GeneTex Inc, Irvine, CA, USA) diluted 1:100, rabbit polyclonal anti-SERPINF2 antibody (H00005345, Abnova) diluted 1:20 or rabbit polyclonal anti-SERPING1 antibody (GTX105316, GeneTex) diluted 1:300 in Discovery Ab diluents (Roche).

Techniques: Immunohistochemistry, Incubation, Negative Control

Journal: PLoS Pathogens

Article Title: Acquisition of C1 inhibitor by Bordetella pertussis virulence associated gene 8 results in C2 and C4 consumption away from the bacterial surface

doi: 10.1371/journal.ppat.1006531

Figure Lengend Snippet: (A) Vag8 binds to C1-inh in a dose dependent manner. No binding was observed to the serpins alpha-1-antichymotrypsin and alpha-2-antiplasmin or the complement components C1, C2, C3 and C4 by ELISA. (B) Vag8 forms a stable complex with C1-inh in fluid phase as shown by making use of the gel filtration chromatography method. (C) The successful construction of the B . pertussis B1917ΔVag8 mutant strain was confirmed by immunoblot. No Vag8 could be detected in the pellet or the bacterial supernatant or OMV’s of B1917ΔVag8. In addition, the B . pertussis wild type strain B1917 expressed both the full length Vag8 as well as the passenger domain in the supernatant. (D) Using ImageJ, the intensity of the Vag8 bands were semi quantified relative to known concentrations of recombinant Vag8. We show that 10 7 bacteria of the B1917 parental strain contain 10 μg/ml Vag8 and 10 9 bacteria secrete 5–10 μg/ml of full length and 1 μg/ml of passenger Vag8. Moreover, 10 μg/ml OMV contains 5 μg/ml of Vag8. (E) The B1917ΔVag8 mutant strain shows increased sensitivity to serum-mediated killing compared to the B1917 parent strain. Data shown in Fig 1A, 1D and 1E represent the mean ± SEM of three separate experiments while Fig 1B and 1C are representative of three separate experiments.

Article Snippet: All samples mixed with 2x SB or SB-DTT were boiled for 10 minutes, run on SDS-PAGE gels and transferred to PVDF membranes.Membranes were blocked with 4% skimmed milk in PBS-T and then incubated with a primary antibody either directed against C1-inh (0.3 μg/ml, rabbit-anti-human, Sino Biologicals, Beijing, China), C1r (1 μg/ml, goat-anti-human, R&D systems), C1s (2 μg/ml, goat-anti-human, Nordic Immunology), C1q (1 μg/ml, rabbit-anti-human, Dako), C4 (0.5 μg/ml, goat-anti-human, Complement Technology), C2 (3.3 μg/ml, Complement Technology) or C3 (2 μg/ml, goat-anti-human, Complement Technology).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Filtration, Chromatography, Mutagenesis, Western Blot, Recombinant

Journal: PLoS Pathogens

Article Title: Acquisition of C1 inhibitor by Bordetella pertussis virulence associated gene 8 results in C2 and C4 consumption away from the bacterial surface

doi: 10.1371/journal.ppat.1006531

Figure Lengend Snippet: (A) B . pertussis strain B1917 was opsonized with 1.25% NHS with or without Vag8 or Prn. Supernatant was analysed using anti C1-inh. The presence of Vag8 results in a decreased signal of the ~150 kDa band. (B) In a purified system consisting of C1q, C1s or C1r with C1-inh in the presence or absence of Vag8 or Prn, we show, using anti-C1-inh, that C1s and C1r, but not C1q, bind to C1-inh and that this binding is inhibited by the addition of Vag8 but not Prn. (C) C1r was detected using anti-C1r. The presence of C1r bound to C1 and C1-inh is inhibited in the presence of Vag8 but not Prn. Additionally, we show the presence of active C1r in the presence of Vag8. (D) Purified MASP-2 was incubated with C1-inh in the presence of Vag8 or Prn and analyzed using anti-C1-inh. We show inhibition of MASP-2 binding to C1-inh in the presence of Vag8, but not Prn. Immunoblots are representative of three separate experiments.

Article Snippet: All samples mixed with 2x SB or SB-DTT were boiled for 10 minutes, run on SDS-PAGE gels and transferred to PVDF membranes.Membranes were blocked with 4% skimmed milk in PBS-T and then incubated with a primary antibody either directed against C1-inh (0.3 μg/ml, rabbit-anti-human, Sino Biologicals, Beijing, China), C1r (1 μg/ml, goat-anti-human, R&D systems), C1s (2 μg/ml, goat-anti-human, Nordic Immunology), C1q (1 μg/ml, rabbit-anti-human, Dako), C4 (0.5 μg/ml, goat-anti-human, Complement Technology), C2 (3.3 μg/ml, Complement Technology) or C3 (2 μg/ml, goat-anti-human, Complement Technology).

Techniques: Purification, Binding Assay, Incubation, Inhibition, Western Blot

Journal: PLoS Pathogens

Article Title: Acquisition of C1 inhibitor by Bordetella pertussis virulence associated gene 8 results in C2 and C4 consumption away from the bacterial surface

doi: 10.1371/journal.ppat.1006531

Figure Lengend Snippet: (A) Purified C1s and C2 were incubated alone, or with C1-inh, in the presence of Vag8 or Prn and visualized using Instant Blue. Increased C2 cleavage is shown in the presence of Vag8. B . pertussis B1917 was opsonized with 1.25% NHS with or without Vag8 or Prn. Supernatant was analysed by immunoblot and shows cleavage of (B) C4, (C) C2, but not (D) C3 in the presence of Vag8 compared to control situation. Incubation of 1.25% NHS alone with or without Vag8 or Prn shows increased cleavage of (E) C4 and (F) C2 in the presence of Vag8. All figures are representative for three separate experiments.

Article Snippet: All samples mixed with 2x SB or SB-DTT were boiled for 10 minutes, run on SDS-PAGE gels and transferred to PVDF membranes.Membranes were blocked with 4% skimmed milk in PBS-T and then incubated with a primary antibody either directed against C1-inh (0.3 μg/ml, rabbit-anti-human, Sino Biologicals, Beijing, China), C1r (1 μg/ml, goat-anti-human, R&D systems), C1s (2 μg/ml, goat-anti-human, Nordic Immunology), C1q (1 μg/ml, rabbit-anti-human, Dako), C4 (0.5 μg/ml, goat-anti-human, Complement Technology), C2 (3.3 μg/ml, Complement Technology) or C3 (2 μg/ml, goat-anti-human, Complement Technology).

Techniques: Purification, Incubation, Western Blot

Journal: PLoS Pathogens

Article Title: Acquisition of C1 inhibitor by Bordetella pertussis virulence associated gene 8 results in C2 and C4 consumption away from the bacterial surface

doi: 10.1371/journal.ppat.1006531

Figure Lengend Snippet: (A) In the absence of Vag8, C1 and MBL/MASP complexes are formed on the bacterial surface which results in the activation of the CP and LP proteases. These proteases cleave C4 and C2 and give rise to the C3 convertase (C4bC2a). Any C1s, C1r, MASP-1 or MASP-2 not part of a bacterial surface bound complex remains inactive by association with C1-inh and hence free intact C4 and C2 is available for recruitment upon complement activation on bacteria. (B) In the presence of Vag8, either the secreted passenger or as part of an OMV, the interaction between C1s, C1r, MASP-1 or MASP-2 and C1-inh is interrupted as Vag8 hijacks this inhibitor. This results in the presence of active proteases in the bacterial environment which cleave and hence deplete the free C4 and C2. The lack of C4 and C2 to be cleaved and deposited on the bacterial membrane upon recognition of B pertussis by C1 and MBL complexes will lead to decreased complement deposition on the surface of B . pertussis and subsequent bacterial killing. Epithelial cells were adapted from Servier Medical Art, provided by Servier under a CC-BY 3.0 license (available at: http://www.servier.com/powerpoint-image-bank ).

Article Snippet: All samples mixed with 2x SB or SB-DTT were boiled for 10 minutes, run on SDS-PAGE gels and transferred to PVDF membranes.Membranes were blocked with 4% skimmed milk in PBS-T and then incubated with a primary antibody either directed against C1-inh (0.3 μg/ml, rabbit-anti-human, Sino Biologicals, Beijing, China), C1r (1 μg/ml, goat-anti-human, R&D systems), C1s (2 μg/ml, goat-anti-human, Nordic Immunology), C1q (1 μg/ml, rabbit-anti-human, Dako), C4 (0.5 μg/ml, goat-anti-human, Complement Technology), C2 (3.3 μg/ml, Complement Technology) or C3 (2 μg/ml, goat-anti-human, Complement Technology).

Techniques: Activation Assay

Journal: Journal of Alzheimer's Disease

Article Title: A New Serum Biomarker Set to Detect Mild Cognitive Impairment and Alzheimer’s Disease by Peptidome Technology

doi: 10.3233/JAD-191016

Figure Lengend Snippet: List of 4 identified peptides

Article Snippet: After incubation in 0.3% hydrogen peroxide/methanol followed by bovine serum albumin, the sections were stained overnight at 4°C with the following primary antibodies: rabbit anti-fibrinogen β chain (FBC) antibody (1:2500, Sigma, St. Louis, MO); rabbit anti-alpha-2-HS-glycoprotein (AHSG) antibody (1:50, Cloud-Clone Corp, Houston, TX, USA); rabbit anti-fibrinogen α chain (FAC) antibody (1:125, Sigma, St. Louis, MO); rabbit anti-plasma protease C1 Inhibitor (PPC1I) antibody (1:50, Proteintech Group, Chicago, IL).

Techniques: Clinical Proteomics

Journal: Journal of Alzheimer's Disease

Article Title: A New Serum Biomarker Set to Detect Mild Cognitive Impairment and Alzheimer’s Disease by Peptidome Technology

doi: 10.3233/JAD-191016

Figure Lengend Snippet: Diagnostic performance of single marker peptide

Article Snippet: After incubation in 0.3% hydrogen peroxide/methanol followed by bovine serum albumin, the sections were stained overnight at 4°C with the following primary antibodies: rabbit anti-fibrinogen β chain (FBC) antibody (1:2500, Sigma, St. Louis, MO); rabbit anti-alpha-2-HS-glycoprotein (AHSG) antibody (1:50, Cloud-Clone Corp, Houston, TX, USA); rabbit anti-fibrinogen α chain (FAC) antibody (1:125, Sigma, St. Louis, MO); rabbit anti-plasma protease C1 Inhibitor (PPC1I) antibody (1:50, Proteintech Group, Chicago, IL).

Techniques: Diagnostic Assay, Marker, Clinical Proteomics

Journal: Scientific Reports

Article Title: Serping1 associated with α-synuclein increase in colonic smooth muscles of MPTP-induced Parkinson’s disease mice

doi: 10.1038/s41598-024-51770-9

Figure Lengend Snippet: Increased serping1 and α-synuclein (α-syn) in colonic smooth muscle in the Parkinson’s disease model. Immunohistochemical analysis of serping1 ( a - f ) and α-syn ( g - l ) conducted in colon tissue in control (CTL) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) groups (n = 3/group). Serping1 increased in the MPTP group (c, f); increased serping1 indicated by arrows. α-Syn increased in the MPTP group (i, l; α-syn indicated by arrows).

Article Snippet: Mouse anti-TH (1:2000; cat. no. sc-25269, Santa Cruz Biotechnology), rabbit anti-α-syn (1:500; cat. no. NBP2-15,365, Novus Biologicals), rabbit anti-serping1 (1:2000; cat. no. PAA235Mu01, Cloud-Clone Corp.), mouse anti-bradykinin (1:500; cat. no. sc-293304, Santa Cruz Biotechnology), mouse anti-B1R (1:500; cat. no. sc-293196, Santa Cruz Biotechnology), and mouse anti-β-actin (1:5000; cat. no. sc-47778, Santa Cruz Biotechnology) were used as primary antibodies.

Techniques: Immunohistochemistry

Journal: Scientific Reports

Article Title: Serping1 associated with α-synuclein increase in colonic smooth muscles of MPTP-induced Parkinson’s disease mice

doi: 10.1038/s41598-024-51770-9

Figure Lengend Snippet: Increased serping1 and α-synuclein (α-syn) in colon in the Parkinson’s disease model. ( A ) Western blot analyses of serping1 and α-syn were investigated in colon in control (CTL) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) groups. Serping1 and α-syn increased in the MPTP group. ( B ) Bar graph of the immunoblot analysis results, showing significantly increased serping1 and α-syn (n = 3, * p < 0.05).

Article Snippet: Mouse anti-TH (1:2000; cat. no. sc-25269, Santa Cruz Biotechnology), rabbit anti-α-syn (1:500; cat. no. NBP2-15,365, Novus Biologicals), rabbit anti-serping1 (1:2000; cat. no. PAA235Mu01, Cloud-Clone Corp.), mouse anti-bradykinin (1:500; cat. no. sc-293304, Santa Cruz Biotechnology), mouse anti-B1R (1:500; cat. no. sc-293196, Santa Cruz Biotechnology), and mouse anti-β-actin (1:5000; cat. no. sc-47778, Santa Cruz Biotechnology) were used as primary antibodies.

Techniques: Western Blot

Journal: Scientific Reports

Article Title: Serping1 associated with α-synuclein increase in colonic smooth muscles of MPTP-induced Parkinson’s disease mice

doi: 10.1038/s41598-024-51770-9

Figure Lengend Snippet: Immunofluorescence microscopy analysis of serping1 and α-synuclein (α-syn) in colonic smooth muscle in the Parkinson’s disease model. Immunofluorescence analysis of serping1 and α-syn in control (CTL; a - f ) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; g - l ) groups (n = 3/group). Nuclei were stained with DAPI ( a , g ; 400X). α-Syn staining is shown in panels b and h , and serping1 staining is shown in panels c and i in CTL and MPTP groups. Merge of α-syn and serping1 staining ( d , j ; 400X) and merge with DAPI ( e , k ; merged serping1 and α-syn indicated with arrows). Panels f and l and magnifications of panels e and k , respectively. More clear and brighter merged serping1 and α-syn staining surrounding nuclei in the MPTP group than in the CTL group (f, l; merged serping1 and α-syn indicated with arrows).

Article Snippet: Mouse anti-TH (1:2000; cat. no. sc-25269, Santa Cruz Biotechnology), rabbit anti-α-syn (1:500; cat. no. NBP2-15,365, Novus Biologicals), rabbit anti-serping1 (1:2000; cat. no. PAA235Mu01, Cloud-Clone Corp.), mouse anti-bradykinin (1:500; cat. no. sc-293304, Santa Cruz Biotechnology), mouse anti-B1R (1:500; cat. no. sc-293196, Santa Cruz Biotechnology), and mouse anti-β-actin (1:5000; cat. no. sc-47778, Santa Cruz Biotechnology) were used as primary antibodies.

Techniques: Immunofluorescence, Microscopy, Staining

Journal: Scientific Reports

Article Title: Serping1 associated with α-synuclein increase in colonic smooth muscles of MPTP-induced Parkinson’s disease mice

doi: 10.1038/s41598-024-51770-9

Figure Lengend Snippet: Western blot analysis of changes in factors related to serping1 in colon in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson’s disease. ( A ) The factors related to the kinin system regulated by serping1, bradykinin and bradykinin B1 receptor (B1R), were investigated. B1R increased significantly in the MPTP group. ( B ) Bar graph of the Western blotting analysis results (n = 3, * p < 0.05).

Article Snippet: Mouse anti-TH (1:2000; cat. no. sc-25269, Santa Cruz Biotechnology), rabbit anti-α-syn (1:500; cat. no. NBP2-15,365, Novus Biologicals), rabbit anti-serping1 (1:2000; cat. no. PAA235Mu01, Cloud-Clone Corp.), mouse anti-bradykinin (1:500; cat. no. sc-293304, Santa Cruz Biotechnology), mouse anti-B1R (1:500; cat. no. sc-293196, Santa Cruz Biotechnology), and mouse anti-β-actin (1:5000; cat. no. sc-47778, Santa Cruz Biotechnology) were used as primary antibodies.

Techniques: Western Blot

Journal: Scientific Reports

Article Title: Serping1 associated with α-synuclein increase in colonic smooth muscles of MPTP-induced Parkinson’s disease mice

doi: 10.1038/s41598-024-51770-9

Figure Lengend Snippet: Changes in factors related to the kinin system and α-synuclein (α-syn) according to the extent of serping1 knockdown in C2C12 cells. ( A ) Immunoblot analysis of down-regulation of serping1 expression by serping1 siRNA and the levels of α-syn and the factors associated with the kinin system, bradykinin and bradykinin B1 receptor (B1R). The negative control (NC) group was transfected using non-targeting siRNA. We applied 10 and 100 nM of the siRNA targeting serping1 . The NC group was also treated with 100 nM of siRNA. ( B ) Bar graph of the immunoblotting results, showing that knockdown of serping1 expression correlated with reduction of the level of α-syn, bradykinin and B1R, significantly in C2C12 cells. (n = 3, * p < 0.05, ** p < 0.005, *** p < 0.0005).

Article Snippet: Mouse anti-TH (1:2000; cat. no. sc-25269, Santa Cruz Biotechnology), rabbit anti-α-syn (1:500; cat. no. NBP2-15,365, Novus Biologicals), rabbit anti-serping1 (1:2000; cat. no. PAA235Mu01, Cloud-Clone Corp.), mouse anti-bradykinin (1:500; cat. no. sc-293304, Santa Cruz Biotechnology), mouse anti-B1R (1:500; cat. no. sc-293196, Santa Cruz Biotechnology), and mouse anti-β-actin (1:5000; cat. no. sc-47778, Santa Cruz Biotechnology) were used as primary antibodies.

Techniques: Western Blot, Expressing, Negative Control, Transfection