Journal: Poultry Science

Article Title: Synergistic effect of genistein and adiponectin reduces fat deposition in chicken hepatocytes by activating the ERβ-mediated SIRT1-AMPK signaling pathway

doi: 10.1016/j.psj.2024.103734

Figure Lengend Snippet: Effect of genistein on the protein levels of estrogen receptors and Erk-PPARγ signaling pathway related factors in chicken adipocytes. (A) Immunoblot of ERα, ERβ, GPER1, and Tublin β protein; (B) The protein levels of ERα, ERβ, and GPER1; (C) Immunoblot of p-Erk, t-Erk, PPARγ, and Tublin β protein; (D) The protein levels of p-Erk/t-Erk and PPARγ. Data are expressed as means ± SEM (n = 3). ** P < 0.01, compared with the control group.

Article Snippet: After that, the membranes were incubated overnight with the rabbit antibodies against GPER1 (Cat#A10217; 1:1000 dilution; ABclonal Technology, Wuhan, China), Nampt (Cat#A0256; 1:1500 dilution; ABclonal Technology, Wuhan, China), ERβ (Cat#A2546; 1:1500 dilution; ABclonal Technology, Wuhan, China), PPARγ (Cat#A19676; 1:1500 dilution; ABclonal Technology, Wuhan, China), ACTB (Cat#AC026; 1:20000 dilution; ABclonal Technology, Wuhan, China), ERα (Cat#8644; 1:2000 dilution; Cell Signaling Technology, Boston, USA), Erk (Cat#4695; 1:2000 dilution; Cell Signaling Technology, Boston, MA), p-Erk (Cat#4370; 1:2000 dilution; Cell Signaling Technology), AMPK (Cat#5831; 1:2000 dilution; Cell Signaling Technology), p-AMPK (Cat#2535; 1:1000 dilution; Cell Signaling Technology), ACC (Cat#3662; 1:2000 dilution; Cell Signaling Technology), p-ACC (Cat#3661; 1:1000 dilution; Cell Signaling Technology) and SIRT1 (Cat#BS6494; 1:1000 dilution; Bioworld Technology, Nanjing, China).

Techniques: Western Blot, Control

Journal: Poultry Science

Article Title: Synergistic effect of genistein and adiponectin reduces fat deposition in chicken hepatocytes by activating the ERβ-mediated SIRT1-AMPK signaling pathway

doi: 10.1016/j.psj.2024.103734

Figure Lengend Snippet: Genistein promotes adiponectin secretion through binding with ERβ in adipocytes. ICP-1 cells were pretreated with vehicle, GPER1 inhibitor G15 (1 μM; 12h), GPER1 activator G1 (1 μM; 1h), ER antagonist Fulvestrant (1 μM; 12h) or ERβ antagonist PHTPP (10 μM; 1h); and then the cells were treated with 0 or 40 μM GEN for another 24 h. (A) Immunoblot of p-Erk, t-Erk, PPARγ, and Tublin β protein; (B–C) The protein levels of p-Erk/t-Erk and PPARγ; (D) APN mRNA expression; (E) Immunoblot of p-Erk, t-Erk, PPARγ, and Tublin β protein; (F–G) The protein levels of p-Erk/t-Erk and PPARγ; (H) APN mRNA expression. (I) Immunoblot of p-Erk, t-Erk, PPARγ, and Tublin β protein; (J–K) The protein levels of p-Erk/t-Erk and PPARγ; L: APN mRNA expression; Data are expressed as means ± SEM (n = 3). ** P < 0.01, comparison with the respective control groups; NS, no significant difference between the indicated groups.

Article Snippet: After that, the membranes were incubated overnight with the rabbit antibodies against GPER1 (Cat#A10217; 1:1000 dilution; ABclonal Technology, Wuhan, China), Nampt (Cat#A0256; 1:1500 dilution; ABclonal Technology, Wuhan, China), ERβ (Cat#A2546; 1:1500 dilution; ABclonal Technology, Wuhan, China), PPARγ (Cat#A19676; 1:1500 dilution; ABclonal Technology, Wuhan, China), ACTB (Cat#AC026; 1:20000 dilution; ABclonal Technology, Wuhan, China), ERα (Cat#8644; 1:2000 dilution; Cell Signaling Technology, Boston, USA), Erk (Cat#4695; 1:2000 dilution; Cell Signaling Technology, Boston, MA), p-Erk (Cat#4370; 1:2000 dilution; Cell Signaling Technology), AMPK (Cat#5831; 1:2000 dilution; Cell Signaling Technology), p-AMPK (Cat#2535; 1:1000 dilution; Cell Signaling Technology), ACC (Cat#3662; 1:2000 dilution; Cell Signaling Technology), p-ACC (Cat#3661; 1:1000 dilution; Cell Signaling Technology) and SIRT1 (Cat#BS6494; 1:1000 dilution; Bioworld Technology, Nanjing, China).

Techniques: Binding Assay, Western Blot, Expressing, Comparison, Control

Journal: Materials Today Bio

Article Title: Polydopamine-cladded montmorillonite micro-sheets as therapeutic platform repair the gut mucosal barrier of murine colitis through inhibiting oxidative stress

doi: 10.1016/j.mtbio.2023.100654

Figure Lengend Snippet: PDA/MMT recovered the intestinal mucosal barrier in vivo . ( A ) Immunofluorescent staining of Occluding-1 (OCLN-1), claudin-5 (CLDN-5), and zonula occludens-1 (ZO-1) and ( B-D ) corresponding quantitative analysis of the fluorescent area ratio of tight junctions (blue: DAPI, cell nucleus; red: tight junction proteins). ( E-F ) Blood fluorescent intensity of mice after enema FITC-dextran. ( G ) Western Blot of tight junctions and ( H-J ) semi-quantification of protein content. (∗∗∗P ​< ​0.001, ∗∗P ​< ​0.01, ∗P ​< ​0.05, n ​≥ ​3). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For immunofluorescence staining, the colon sections were stained with rabbit polyclonal occludin (ab216327, 1:200, Abcam®), rabbit polyclonal claudin-5 (A10207, 1:100, ABclonal®), and rabbit polyclonal ZO-1 (A0659, 1:200, ABclonal®) for 12 ​h in 4 ​°C followed by secondary antibody conjugated with CoraLite488 (SA00013-1, 1:200, Proteintech®) or Rhodamine (BS10250, 1:200, bioworlde®).

Techniques: In Vivo, Staining, Western Blot

Journal: Biomolecules

Article Title: The σ1 Receptor and the HINT1 Protein Control α2δ1 Binding to Glutamate NMDA Receptors: Implications in Neuropathic Pain

doi: 10.3390/biom11111681

Figure Lengend Snippet: Expression of α2(δ1), (α2)δ1, and (α2)δ2 in the cerebral cortex, PAG, pons-medulla, and SC of CD1 and 129 mice. ( A ) CD1 WT mice ( n = 5). ( B ) CD1 WT ( n = 5) and CD1 σ1R −/− mice ( n = 6). ( C ) 129 WT (n = 6) and 129 HINT1 −/− ( n = 6) mice. Mouse brain and SC structures were collected and P2 fractions enriched in synaptosomes were obtained. About 60 μg protein/lane was resolved by SDS-PAGE and examined in Western blots probed with antibodies against α2(δ) proteins and (α2)δ peptides, as described in the Methods. Further details in . The 3D structure of α2(δ1) and (α2)δ1 were generated with NovaFold v. 17 (DNASTAR), in which α2-Arg217 is indicated as a pink tube, and α2-Cys380 and δ1-Cys61 are shown as red tubes.

Article Snippet: The primary antibodies used in Western blotting were raised against: NMDAR NR1 (#MAB1586, Merck-Millipore, Burlington, MA, USA); NMDAR NR1 C1 (#AB5046, Merck-Millipore, Burlington, MA, USA); NMDAR NR2A (#AB1555P, Merck-Millipore, Burlington, MA, USA); NR2B (#MA1-2014, ThermoScientific, Waltham, MA, USA); α2(δ1) Nt (#C5105, Sigma Aldrich, St. Louis, MO, USA); α2(δ1) inner sequence (#SAB2107922, Sigma Aldrich, St. Louis, MO, USA); (α2)δ1 (#HPA008621, Sigma Aldrich, St. Louis, MO, USA); α2(δ2) (#A10267, Abclonal, Woburn, MA, USA); (α2)δ2 (#HPA071829, Sigma Aldrich, St. Louis, MO, USA), α-tubulin (#ab7291, Abcam, Cambridge, UK).

Techniques: Expressing, SDS Page, Western Blot, Generated

Journal: Biomolecules

Article Title: The σ1 Receptor and the HINT1 Protein Control α2δ1 Binding to Glutamate NMDA Receptors: Implications in Neuropathic Pain

doi: 10.3390/biom11111681

Figure Lengend Snippet: Expression of NR1, NR2A, and NR2B subunits of glutamate NMDARs in mouse PAG: Co-precipitation with α2(δ1), α2(δ2), and (α2)δ1 proteins. ( A ) PAG P2 fractions enriched in synaptosomes from the CD1 WT, CD1 σ1R −/− , 129 WT, and 129 HINT1 −/− mice of B,C, were used. About 60 μg protein/lane was resolved by SDS-PAGE and examined in Western blots that were probed with antibodies against NR1, NR1 C1, NR2A, and NR2B NMDAR subunits, as described in Methods. The assay was repeated twice with comparable outcomes. Immunoprecipitation assays on CD1 WT mice ( n = 12): PAG and SC membranes from CD1 WT mice were solubilized with 1% NP-40 and incubated overnight at 4 °C with affinity-purified biotinylated IgGs raised against the ( B ) NR1, ( C ) NR2A, or ( D ) NR2B subunits. Protein complexes were immunoprecipitated (IP) with streptavidin agarose, resolved by SDS-PAGE and visualized in Western blots. The expected size of the α2(δ1) and α2(δ2) is approximately 100 kDa; however, the bands detected usually appeared as a doublet of 100–140 kDa. ( C , D ), The material associated with the NR2A or NR2B subunits was subjected to deglycosylation with PNGase F, which depleted the α2(δ) 140 kDa band in favor of a 100 kDa band. In SC synaptosomes, (α2)δ1-2 immunosignals were enriched at NR2A/B subunits (each lane was loaded with solubilized spinal cord tissue from a single CD1 WT mouse, for more details see ). Inset: Diagram of the proposed association of α2δ1 proteins with NMDARs. The α2(δ1) protein and the (α2)δ1 peptide are bridged by a disulfide bond, and both bind to the NR1-NR2A/B dimer. The heavily glycosylated α2(δ1) protein remains outside the membrane interacting with external sequences of the NMDAR, while the (α2)δ1 peptide contains a transmembrane region followed by the C terminal region, which interacts with the cytosolic regions of the NR1 C0-C1-C2(2’) subunits. Stimuli like nerve injury promote changes in the NR1 C1 subunit that augment the stability of its association with the (α2)δ1 peptide. Consequently, calcium permeation increases and persists, causing NMDAR over activation and allodynia. Plasma membrane in yellow; NR1 subunits in brown; NR2 subunits in blue; α2δ1 proteins in pink; spheres indicate calcium ions.

Article Snippet: The primary antibodies used in Western blotting were raised against: NMDAR NR1 (#MAB1586, Merck-Millipore, Burlington, MA, USA); NMDAR NR1 C1 (#AB5046, Merck-Millipore, Burlington, MA, USA); NMDAR NR2A (#AB1555P, Merck-Millipore, Burlington, MA, USA); NR2B (#MA1-2014, ThermoScientific, Waltham, MA, USA); α2(δ1) Nt (#C5105, Sigma Aldrich, St. Louis, MO, USA); α2(δ1) inner sequence (#SAB2107922, Sigma Aldrich, St. Louis, MO, USA); (α2)δ1 (#HPA008621, Sigma Aldrich, St. Louis, MO, USA); α2(δ2) (#A10267, Abclonal, Woburn, MA, USA); (α2)δ2 (#HPA071829, Sigma Aldrich, St. Louis, MO, USA), α-tubulin (#ab7291, Abcam, Cambridge, UK).

Techniques: Expressing, SDS Page, Western Blot, Immunoprecipitation, Incubation, Affinity Purification, Activation Assay

Journal: Biomolecules

Article Title: The σ1 Receptor and the HINT1 Protein Control α2δ1 Binding to Glutamate NMDA Receptors: Implications in Neuropathic Pain

doi: 10.3390/biom11111681

Figure Lengend Snippet: CCI promotes σ1R-mediated associations of α2δ1-2 proteins with NR1 subunits. ( A ) CD1 WT sham-operated ( n = 4), CD1 WT CCI ( n = 4), CD1 σ1R −/− sham ( n = 4) and CD1 σ1R −/− CCI ( n = 4) were sacrificed 7 days after surgery and PAG synaptosomal fractions were prepared. NR1 subunits were immunoprecipitated (IP) from the solubilized membrane preparations, and the presence of co-precipitated ( A ) NR1, ( B ) NR2A, ( C ) NR2B, ( D ) α2(δ1), ( E ) (α2)δ1, and ( F ) (α2)δ2 proteins was assessed in Western blots (WB). The bars represent the mean ± SD of three measurements. Data are computed relative to the WT mice and in the absence of CCI (assigned the arbitrary value of 1). The arrows refer to the comparison and * indicates significant difference of the CCI group relative to the CD1 WT or CD1 σ1R −/− group: p < 0.05. Details as in . ( G ) CD1 WT mice: effect of S1RA on the association of α2δ1-2 proteins with NR1 subunits promoted by CCI ( n = 6). S1RA (3 nM) was injected icv 7 days after CCI surgery in three CD1 WT mice. The animals were sacrificed 30 min later to obtain the synaptosomal fraction from the PAG. The NR1 subunits were immunoprecipitated from the solubilized membrane preparations, and the co-precipitated α2(δ1), α2(δ2), (α2)δ1, and (α2)δ2 were analyzed in Western blots: * indicating significant difference relative to the CCI group, p < 0.05. Sham operated CD1 WT mice ( n = 3) served as control to the effect of CCI (for further details see ).

Article Snippet: The primary antibodies used in Western blotting were raised against: NMDAR NR1 (#MAB1586, Merck-Millipore, Burlington, MA, USA); NMDAR NR1 C1 (#AB5046, Merck-Millipore, Burlington, MA, USA); NMDAR NR2A (#AB1555P, Merck-Millipore, Burlington, MA, USA); NR2B (#MA1-2014, ThermoScientific, Waltham, MA, USA); α2(δ1) Nt (#C5105, Sigma Aldrich, St. Louis, MO, USA); α2(δ1) inner sequence (#SAB2107922, Sigma Aldrich, St. Louis, MO, USA); (α2)δ1 (#HPA008621, Sigma Aldrich, St. Louis, MO, USA); α2(δ2) (#A10267, Abclonal, Woburn, MA, USA); (α2)δ2 (#HPA071829, Sigma Aldrich, St. Louis, MO, USA), α-tubulin (#ab7291, Abcam, Cambridge, UK).

Techniques: Immunoprecipitation, Western Blot, Injection