Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Molecular characterization of a precision-cut rat liver slice model for the evaluation of antifibrotic compounds

doi: 10.1152/ajpgi.00281.2018

Figure Lengend Snippet: ELISA analysis of secreted biomarkers. SB525334, nintedanib, and sorafenib suppressed secretion of hyaluronic acid (HA), insulin-like growth factor binding protein 5 (IGFBP5), and WNT1-inducible signaling pathway protein 1 (WISP1) in the conditioned medium after 48 h of incubation. Data are means ± SE; n = 3 rats for both sham and bile duct ligation (BDL); n = 8–10 liver slices for each condition. **P < 0.01 and ***P < 0.001.

Article Snippet: Secreted biomarkers including procollagen I, HA, IGFBP5, and WISP1 in the conditioned medium were measured using procollagen I ( {"type":"entrez-nucleotide","attrs":{"text":"Ab210579","term_id":"70570352","term_text":"AB210579"}} Ab210579 ; Abcam, Cambridge, UK), hyaluronan quantikine (DHYAL0; R&D Systems, Minneapolis, MN), IGFBP5 ( {"type":"entrez-nucleotide","attrs":{"text":"Ab208345","term_id":"76667415","term_text":"AB208345"}} Ab208345 ; Abcam), and WISP1 quantikine (MWSP10; R&D Systems) ELISA kits following manufacturer's instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation, Ligation

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Molecular characterization of a precision-cut rat liver slice model for the evaluation of antifibrotic compounds

doi: 10.1152/ajpgi.00281.2018

Figure Lengend Snippet: The small molecule αV integrins inhibitor CWHM12 attenuated fibrotic phenotype in fibrotic precision-cut liver tissue slices (PCLSs) after 48 h of incubation. A: CWHM12 suppressed expression of a panel of fibrotic genes. B: CWHM12 decreased secretion of procollagen I, hyaluronic acid (HA), insulin-like growth factor binding protein 5 (IGFBP5), and WNT1-inducible signaling pathway protein 1 (WISP1) in the conditioned medium. Data are means ± SE; n = 3 rats for both sham and bile duct ligation (BDL); n = 6–8 liver slices for each condition. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Secreted biomarkers including procollagen I, HA, IGFBP5, and WISP1 in the conditioned medium were measured using procollagen I ( {"type":"entrez-nucleotide","attrs":{"text":"Ab210579","term_id":"70570352","term_text":"AB210579"}} Ab210579 ; Abcam, Cambridge, UK), hyaluronan quantikine (DHYAL0; R&D Systems, Minneapolis, MN), IGFBP5 ( {"type":"entrez-nucleotide","attrs":{"text":"Ab208345","term_id":"76667415","term_text":"AB208345"}} Ab208345 ; Abcam), and WISP1 quantikine (MWSP10; R&D Systems) ELISA kits following manufacturer's instructions.

Techniques: Incubation, Expressing, Binding Assay, Ligation

Journal: The Journal of Neuroscience

Article Title: Subretinal Human Umbilical Tissue-Derived Cell Transplantation Preserves Retinal Synaptic Connectivity and Attenuates Müller Glial Reactivity

doi: 10.1523/JNEUROSCI.1532-17.2018

Figure Lengend Snippet: TSP-receptor α2δ-1 is synaptically expressed in the retina, and its expression is reduced in RCS rats. A, Representative images of the retina stained for α2δ-1 from LE (healthy) and RCS (degenerative) retinas at P14 and (B) P30. C, Quantitative staining intensity analysis demonstrated that α2δ-1 expression was reduced in RCS rat as early as P14. D, α2δ-1 was enriched in both the OPL and IPL, and the expression gap became more distinct at P30. Representative images of the (E) OPL and (F and G) IPL with the synapses labeled for Bassoon (F, green), VGluT1 (G, green), α2δ-1 (red), and NR1 (blue) from LE retina on P21 demonstrated postsynaptic expression of α2δ-1. Representative images of the (H) OPL and (J) IPL synapses labeled for Bassoon (green) and α2δ-1 (red) from LE (healthy) and RCS (degenerative) retinas on P21. Quantification of α2δ-1-containing synapses formed in the (I) OPL and (K) IPL reveals that the number of α2δ-1 synapse is already reduced in RCS rat by P21. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. ***p < 0.0001.

Article Snippet: Primary antibodies (mouse anti-Bassoon 1:500 [RRID: AB_10618753 , ADI-VAM-PS003-F, Enzo], rabbit anti-mGluR6 1:150 [RRID:not applicable, RA13105, Neuromics], guinea pig anti-VGlut1 1:750 [AB5905, Millipore], rabbit anti-PSD95 1:500 [RRID: AB_87705 , 51-6900, Invitrogen], mouse anti-Gephyrin 1:250 [RRID: AB_1279448 , 147-021, Synaptic Systems], goat anti-TSP1 1:200 [RRID: AB_2201958 , AF3074, R&D Systems], goat-anti-TSP2 1:200 [RRID: AB_2202068 , AF1635, R&D Systems], mouse anti-glutamine synthetase 1:1000 [RRID: AB_397879 , 610517, BD Biosciences], rabbit anti-SOX9 1:4000 [RRID: AB_2239761 , AB5535, Millipore], goat anti-ChAT[RRID: AB_11214092 , AB144P, Millipore], rabbit anti-α2δ-1 [RRID: AB_258885 , C5105, Sigma-Aldrich]), rabbit anti-α2δ-1 [RRID: AB_2039785 , ACC-015, Alomone Labs], and rabbit anti-GFAP [RRID: AB_10013382 , Z033429-2, Dako] were diluted in 5% NGS or 5% BSA containing PBST.

Techniques: Expressing, Staining, Labeling

Journal: The Journal of Neuroscience

Article Title: Subretinal Human Umbilical Tissue-Derived Cell Transplantation Preserves Retinal Synaptic Connectivity and Attenuates Müller Glial Reactivity

doi: 10.1523/JNEUROSCI.1532-17.2018

Figure Lengend Snippet: Subretinal hUTC transplantation preserves OPL synapses in RCS rats. Representative images of the OPL with the PR ribbon synapses labeled for (A) Bassoon (green) and mGluR6 (red) and (B) Bassoon (green) and α2δ-1 (red) from LE (control), RCS + BSS, and RCS + hUTC P21 and P60 retinas on P95. Quantification of the number of synapses in the OPL revealed that hUTC transplantation protected (C) ribbon synapses. D, Particularly, α2δ-1-containing synapses were specifically preserved following hUTC treatment. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. ***p < 0.0001.

Article Snippet: Primary antibodies (mouse anti-Bassoon 1:500 [RRID: AB_10618753 , ADI-VAM-PS003-F, Enzo], rabbit anti-mGluR6 1:150 [RRID:not applicable, RA13105, Neuromics], guinea pig anti-VGlut1 1:750 [AB5905, Millipore], rabbit anti-PSD95 1:500 [RRID: AB_87705 , 51-6900, Invitrogen], mouse anti-Gephyrin 1:250 [RRID: AB_1279448 , 147-021, Synaptic Systems], goat anti-TSP1 1:200 [RRID: AB_2201958 , AF3074, R&D Systems], goat-anti-TSP2 1:200 [RRID: AB_2202068 , AF1635, R&D Systems], mouse anti-glutamine synthetase 1:1000 [RRID: AB_397879 , 610517, BD Biosciences], rabbit anti-SOX9 1:4000 [RRID: AB_2239761 , AB5535, Millipore], goat anti-ChAT[RRID: AB_11214092 , AB144P, Millipore], rabbit anti-α2δ-1 [RRID: AB_258885 , C5105, Sigma-Aldrich]), rabbit anti-α2δ-1 [RRID: AB_2039785 , ACC-015, Alomone Labs], and rabbit anti-GFAP [RRID: AB_10013382 , Z033429-2, Dako] were diluted in 5% NGS or 5% BSA containing PBST.

Techniques: Transplantation Assay, Labeling

Journal: The Journal of Neuroscience

Article Title: Subretinal Human Umbilical Tissue-Derived Cell Transplantation Preserves Retinal Synaptic Connectivity and Attenuates Müller Glial Reactivity

doi: 10.1523/JNEUROSCI.1532-17.2018

Figure Lengend Snippet: Subretinal hUTC transplantation preserves α2δ-1-containing synapses in the IPL of RCS rats. A, Representative images of the IPL labeled for VGluT1 (green) and PSD95 (red) from LE (control), RCS+BSS, and RCS+hUTC P21 and P60 retinas on P95. B, Quantification of excitatory synapses in the IPL revealed that synapse numbers did not differ between RCS+BSS and RCS+hUTC P21 and P60. C, Representative images of the IPL labeled for Bassoon (green) and α2δ-1 (red) from LE (control), RCS+BSS, and RCS+hUTC P21 and P60 retinas on P95. D, The α2δ-1-containing synapses were specifically preserved with hUTC transplantation. E, Representative images of the IPL stained for Bassoon (green) and Gephyrin (blue) from LE (control), RCS+BSS, and RCS+hUTC P21 and P60 retinas on P95. F, hUTC transplantation did not preserve inhibitory synapses. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. *p < 0.05.

Article Snippet: Primary antibodies (mouse anti-Bassoon 1:500 [RRID: AB_10618753 , ADI-VAM-PS003-F, Enzo], rabbit anti-mGluR6 1:150 [RRID:not applicable, RA13105, Neuromics], guinea pig anti-VGlut1 1:750 [AB5905, Millipore], rabbit anti-PSD95 1:500 [RRID: AB_87705 , 51-6900, Invitrogen], mouse anti-Gephyrin 1:250 [RRID: AB_1279448 , 147-021, Synaptic Systems], goat anti-TSP1 1:200 [RRID: AB_2201958 , AF3074, R&D Systems], goat-anti-TSP2 1:200 [RRID: AB_2202068 , AF1635, R&D Systems], mouse anti-glutamine synthetase 1:1000 [RRID: AB_397879 , 610517, BD Biosciences], rabbit anti-SOX9 1:4000 [RRID: AB_2239761 , AB5535, Millipore], goat anti-ChAT[RRID: AB_11214092 , AB144P, Millipore], rabbit anti-α2δ-1 [RRID: AB_258885 , C5105, Sigma-Aldrich]), rabbit anti-α2δ-1 [RRID: AB_2039785 , ACC-015, Alomone Labs], and rabbit anti-GFAP [RRID: AB_10013382 , Z033429-2, Dako] were diluted in 5% NGS or 5% BSA containing PBST.

Techniques: Transplantation Assay, Labeling, Staining

Journal: The Journal of Neuroscience

Article Title: Subretinal Human Umbilical Tissue-Derived Cell Transplantation Preserves Retinal Synaptic Connectivity and Attenuates Müller Glial Reactivity

doi: 10.1523/JNEUROSCI.1532-17.2018

Figure Lengend Snippet: KO of MERTK in MG results in impaired synapse development in vivo. A, Representative images of the OPL synapses labeled by Bassoon (green) and mGluR6 (red) from Control (CTR, right eyeballs) and MERTK KO (KO, left eyeballs) at P21 and P45. B, Quantification of synapses in the OPL revealed that synapse development is impaired in MG-specific MERTK-KO at both P21 and P45. C, IPL excitatory synapses are visualized by VGluT1 (green) and PSD95 (red). D, Quantification of synapse number demonstrates that early synaptic development at P21 is significantly affected by MERTK KO but not at P45. Representative images of the (E) OPL and (G) IPL synapses labeled for Bassoon (green) and α2δ-1 (red). Quantification of the number of α2δ-1-containing synapses in both (F) OPL and (H) IPL revealed that KO of MERTK in MG induces severely reduced number of α2δ-1-containing synapses at P21 and P45. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. *p < 0.05.

Article Snippet: Primary antibodies (mouse anti-Bassoon 1:500 [RRID: AB_10618753 , ADI-VAM-PS003-F, Enzo], rabbit anti-mGluR6 1:150 [RRID:not applicable, RA13105, Neuromics], guinea pig anti-VGlut1 1:750 [AB5905, Millipore], rabbit anti-PSD95 1:500 [RRID: AB_87705 , 51-6900, Invitrogen], mouse anti-Gephyrin 1:250 [RRID: AB_1279448 , 147-021, Synaptic Systems], goat anti-TSP1 1:200 [RRID: AB_2201958 , AF3074, R&D Systems], goat-anti-TSP2 1:200 [RRID: AB_2202068 , AF1635, R&D Systems], mouse anti-glutamine synthetase 1:1000 [RRID: AB_397879 , 610517, BD Biosciences], rabbit anti-SOX9 1:4000 [RRID: AB_2239761 , AB5535, Millipore], goat anti-ChAT[RRID: AB_11214092 , AB144P, Millipore], rabbit anti-α2δ-1 [RRID: AB_258885 , C5105, Sigma-Aldrich]), rabbit anti-α2δ-1 [RRID: AB_2039785 , ACC-015, Alomone Labs], and rabbit anti-GFAP [RRID: AB_10013382 , Z033429-2, Dako] were diluted in 5% NGS or 5% BSA containing PBST.

Techniques: In Vivo, Labeling

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Enhanced large conductance K + channel activity contributes to the impaired myogenic response in the cerebral vasculature of Fawn Hooded Hypertensive rats

doi: 10.1152/ajpheart.00636.2013

Figure Lengend Snippet: Comparison of BK-α- and BK-β-subunit expression in cerebral vessel homogenates isolated from FHH and FHH.1BN rats. BK-α- and BK-β-subunits were probed on different blots. The same blots were then reprobed for β-actin. A: representative bands corresponding to the molecular weight of the BK-α-subunit (∼100 kDa), BK-β-subunit (∼25 kDa), and β-actin (∼42 kDa) in cerebral vessels obtained from FHH and FHH.1BN rats (n = 8 animals each; 3 sets of experiments). B: a comparison of the expression of α- and β-subunits. Numbers in parentheses indicate the number of samples studied.

Article Snippet: After transfer, the membrane was blocked with TBS-T buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, 0.05% Tween, and a 5% blocking powder (Bio-Rad) at 4°C for 1 h. The blot was probed with primary antibody against BK α- and β-subunit [1:500 and 1:200, respectively; polyclonal rabbit Anti-K Ca 1.1, to amino acids 1184–1200 and Anti-sloβ1 (KCNMB1); Alomone Labs, Jerusalem, Israel] overnight at 4°C.

Techniques: Expressing, Isolation, Molecular Weight

Journal: Biocell

Article Title: Stretch Enhances Secretion of Extracellular Vehicles from Airway Smooth Muscle Cells via Endoplasmic Reticulum Stress Signaling in Relation to Ventilator-Induced Lung Injury

doi: 10.32604/biocell.2025.063869

Figure Lengend Snippet: Figure 2: The effect of stretch on EVs and endoplasmic reticulum (ER) stress in ASMCs. (A) Left to right: representative immunofluorescence images of CD63/HSPA5 in ASMCs acquired with confocal microscopy (×100 objective), the quantified CD63/HSPA5 puncta number inside ASMCs (ImageJ), and Pearson coefficient of the pixel-intensity correlation of CD63/HSPA5 in ASMCs (ImageJ, scale bar = 200 μm); (B) Protein expression of CD63/HSPA5 in ASMCs (WB); (C) Size distribution and quantified counts, as well as protein concentration of EVs in ASMCs (NTA and BCA). ER: endoplasmic reticulum; HSPA5: biomarker protein of ER stress; TUDCA: tauroursodeoxycholic acid, ER stress inhibitor; NTA, nanoparticle tracking analysis; BCA: bicinchoninic acid assay; data present as means ± SD, experiments were repeated three times (n = 3), **p < 0.01 compared with Static, ##p < 0.01 compared with Stretch

Article Snippet: Afterwards it was immersed in 10% horse serum (Sigma, #H0146) in PBS as a blocking solution for 1 h at RT, washed 3 times in PBS for 5 min, and then incubated with HSPA5 antibody (1:100, #BA2042, BOSTER) in PBS containing 5% horse serum for 1 h at RT.

Techniques: Immunofluorescence, Confocal Microscopy, Expressing, Protein Concentration, Biomarker Discovery, Acid Assay

Journal: Biocell

Article Title: Stretch Enhances Secretion of Extracellular Vehicles from Airway Smooth Muscle Cells via Endoplasmic Reticulum Stress Signaling in Relation to Ventilator-Induced Lung Injury

doi: 10.32604/biocell.2025.063869

Figure Lengend Snippet: Figure 6: The effect of mechanical ventilation on secretion of EVs, ER stress, airway inflammation and injury in mouse models of VILI. (A) Representative images of lung tissue slides with hematoxylin and eosin (HE) or immunohisto- chemistry (IHC, HSPA5) staining (scale bar = 100 μm, arrows indicate alveolar collapse, arrowhead indicate HSPA5 expression). Healthy, MV, MV + TUDCA/GW4869 indicates mice breathed spontaneously, under 18 mL/kg mechanical ventilation (MV), MV with pretreatment of TUDCA/GW4869, respectively; (B) Protein expression of HSPA5 in lung tissue with IHC staining from C57BL/6 mice treated with different conditions, respectively; (C–E) Size distribution and quantified count, HSPA5, as well as protein concentration of EVs isolated from bronchoalveolar lavage fluid (BALF) of mice treated with different conditions; (F) Relative TGF-β1 and IL-10 secretion in BALF from mice treated with different conditions. Data present as means ± SD, experiments were repeated six times (n = 6), ** p < 0.01 compared with Healthy, #p < 0.05, ##p < 0.01 compared with MV

Article Snippet: Afterwards it was immersed in 10% horse serum (Sigma, #H0146) in PBS as a blocking solution for 1 h at RT, washed 3 times in PBS for 5 min, and then incubated with HSPA5 antibody (1:100, #BA2042, BOSTER) in PBS containing 5% horse serum for 1 h at RT.

Techniques: Immunohistochemistry, Staining, Expressing, Protein Concentration, Isolation