Journal: Molecules

Article Title: Astragalus membranaceus Extract Activates Immune Response in Macrophages via Heparanase

doi: 10.3390/molecules17067232

Figure Lengend Snippet: Effects of anti-HPA antibody on the expression of IL-1β and TNF-α in AME-treated macrophages. Cells were cultured with AME, pre-treated with anti-HPA antibody for 1 h before exposed to AME for 24 h. The secretion of IL-1β and TNF-α were measured by using ELISA ( A ); The mRNA levels of IL-1β and TNF-α were measured by RT-PCR ( B ). * p < 0.05 compared to control and # p < 0.05 compared to AME.

Article Snippet: Heparan degrading enzyme assay kit was from Takara Bio Inc. Antibody against HPA, ELISA kits for mouse TNF-α and IL-1β were from Wuhan Boster Bio-engineering Limited Company (Wuhan, China).

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Inhibition of the NMDA receptor protects the rat sciatic nerve against ischemia/reperfusion injury

doi: 10.3892/etm.2016.3148

Figure Lengend Snippet: Effects of MK-801 on tumor necrosis factor (TNF)-α protein expression levels in the rat sciatic nerve (SN) following ischemia/reperfusion (I/R) injury. Protein expression levels of TNF-α were determined using immunohistochemistry (magnification, ×400). (A) No TNF-α expression was detected in the Schwann cells derived from the SN of a sham-operated rat. (B) Moderate protein expression levels of TNF-α were detected in the Schwann cells derived from the SN fiber of a rat in the 12 h post-reperfusion I/R subgroup. (C) Higher protein expression levels of TNF-α were detected in the Schwann cells derived from the SN of a rat in the 24 h post-reperfusion I/R subgroup. (D) Numerous inflammatory cells had infiltrated the area surrounding the Schwann cells and moderate protein expression levels of TNF-α were detected in the SN of a rat in the 72 h post-reperfusion I/R subgroup. (E) Widespread demyelination and mild-to-moderate TNF-α protein expression levels in Schwann cells were detected in the SN derived from a rat in the 7 days post-reperfusion I/R subgroup. (F) A SN from a rat in the I/R + MK-801 group at 12 h post-reperfusion exhibited mild-to-moderate TNF-α protein expression levels in Schwann cells. (G) A SN from a rat in the I/R + MK-801 group at 24 h post-reperfusion exhibited markedly fewer infiltrating cells, as compared with the SN derived from I/R rats at the same time point post-reperfusion. Moderate protein expression levels of TNF-α expression were observed. (H) A SN derived from a rat in the I/R + MK-801 group at 7 days post-reperfusion. As compared with the SNs derived from the I/R rats, the extent of demyelination was markedly reduced and Schwann cells exhibited only low protein expression levels of TNF-α.

Article Snippet: Tissue slices were incubated with rabbit anti-mouse TNF-α polyclonal antibody (1:100; BA14901; Wuhan Boster Bio-Engineering Co., Ltd.) at 4°C overnight.

Techniques: Expressing, Immunohistochemistry, Derivative Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Inhibition of the NMDA receptor protects the rat sciatic nerve against ischemia/reperfusion injury

doi: 10.3892/etm.2016.3148

Figure Lengend Snippet: Protein expression levels of tumor necrosis factor-α in the various treatment subgroups were quantified using the integrated optical density method, and are presented as the mean ± standard deviation (n=6). Δ P<0.05, ΔΔ P<0.01 vs. the I/R group. I/R, ischemia reperfusion.

Article Snippet: Tissue slices were incubated with rabbit anti-mouse TNF-α polyclonal antibody (1:100; BA14901; Wuhan Boster Bio-Engineering Co., Ltd.) at 4°C overnight.

Techniques: Expressing, Standard Deviation

Journal: Experimental and Therapeutic Medicine

Article Title: Inhibition of the NMDA receptor protects the rat sciatic nerve against ischemia/reperfusion injury

doi: 10.3892/etm.2016.3148

Figure Lengend Snippet: Effects of MK-801 on TNF-α and TACE mRNA expression levels in the rat sciatic nerve (SN) following ischemia/reperfusion (I/R) injury. TNF-α and TACE mRNA expression levels were determined using reverse transcription-quantitative polymerase chain reaction and are expressed relative to β-actin. Agarose gel images showing TNF-α and TACE mRNA expression levels in the SN homogenates at (A) 0, (B) 6, (C) 12, (D) 24 and (E) 72 h and (F) 7 days post-reperfusion. β-actin=280 bp; TNF-α=402 bp; TACE=624 bp. Relative (G) TNF-α and (H) TACE mRNA expression levels are presented as the mean ± standard deviation (n=6). *P<0.05, **P<0.01 vs. the sham-operated group; Δ P<0.05, ΔΔ P<0.01 vs. the I/R group. TNF-α, tumor necrosis factor-α; TACE, TNF-α-converting enzyme.

Article Snippet: Tissue slices were incubated with rabbit anti-mouse TNF-α polyclonal antibody (1:100; BA14901; Wuhan Boster Bio-Engineering Co., Ltd.) at 4°C overnight.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Standard Deviation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Expression of activated Notch3 in transgenic mice enhances generation of T regulatory cells and protects against experimental autoimmune diabetes.

doi: 10.4049/jimmunol.171.9.4504

Figure Lengend Snippet: FIGURE 1. T-reg cell distribution and character- ization in lymphoid organs of 4-wk-old Notch3-IC Tg mice compared with wt littermates. Three-color anal- ysis of CD25 vs CD4 vs CD8 expression in thymo- cytes: CD4CD8low (R1 5.8 and 4.4% in Tg and wt, respectively) and CD4CD8 (R2 10.9 and 10.5% in Tg and wt, respectively, A) and in splenocytes: CD4CD8 (R3 26.4 and 36.2% in Tg and wt, respectively, B). Numbers indicate the percentages of CD25 cells. Left panels, The T lymphocyte subset distribution. C, Expression of IL-4 and IL-10 mRNAs assessed by RT-PCR in lymphocytes from thymus (THY), spleen (SPL), and mesenteric lymph node (LN) of wt and Tg animals. Cytokine expression was monitored along the exponential phase of the amplifi- cation and normalized to -actin (-act) expression. The results are representative of three independent ex- periments. D, Three-color analysis of CD4 vs CD25 vs intracellular IL-10 expression in Tg T splenocytes. Numbers indicate percentages of IL-10 cells. Left panel, The distribution of CD4 splenocytes (R2 CD4CD25; R3 CD4CD25). E, Expression of TGF- and GITR mRNAs assessed by RT-PCR in lymphocytes from thymus (THY) and splenic T cells (SPL) of wt and Tg animals. mRNAs expression was monitored along the exponential phase of the amplifi- cation and normalized to -actin (-act) expression. The results are representative of three independent ex- periments. F, Total cell number (left panel) and per- centage (right panel) of CTLA-4CD25 T lympho- cytes from thymus (THY) of wt () and Tg (f) mice (detected by CD25 vs intracellular CTLA-4 two-color FCA).

Article Snippet: For intracellular staining with anti-CTLA-4, anti-Notch3 and anti-IL-10 cells were treated as previously described (21, 19) and stained with PE-conjugated hamster anti-mouse CD152 (CTLA-4, UC10-4F10-11), PE-conjugated rat anti-mouse IL-10 (JES5-16E3; both from BD PharMingen), or rabbit polyclonal Ab against the intracellular domain of Notch3 protein (sc-7424; Santa Cruz Biotechnology, Santa Cruz, CA), respectively.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Expression of activated Notch3 in transgenic mice enhances generation of T regulatory cells and protects against experimental autoimmune diabetes.

doi: 10.4049/jimmunol.171.9.4504

Figure Lengend Snippet: FIGURE 2. Expression of Notch signaling components in T cell com- partments of 4-wk-old Notch3-IC Tg mice and wt littermates. A, RT-PCR analysis of Notch1, Jagged2, HES-1, and Deltex mRNA expression in lym- phocytes from thymus (THY) and spleen (SPL) of wt and Tg animals, as monitored along the exponential phase of the amplification and normalized to -actin (-act) expression. B, Three-color analysis of Notch3 vs CD4 vs CD25 expression in T splenic lymphocytes of wt mice: CD4CD25 (R4: 15,8%) and CD4CD25 (R5: 4,8%). Numbers indicate percentages of Notch3 cells. Left panel, The T lymphocyte subset distribution.

Article Snippet: For intracellular staining with anti-CTLA-4, anti-Notch3 and anti-IL-10 cells were treated as previously described (21, 19) and stained with PE-conjugated hamster anti-mouse CD152 (CTLA-4, UC10-4F10-11), PE-conjugated rat anti-mouse IL-10 (JES5-16E3; both from BD PharMingen), or rabbit polyclonal Ab against the intracellular domain of Notch3 protein (sc-7424; Santa Cruz Biotechnology, Santa Cruz, CA), respectively.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Expression of activated Notch3 in transgenic mice enhances generation of T regulatory cells and protects against experimental autoimmune diabetes.

doi: 10.4049/jimmunol.171.9.4504

Figure Lengend Snippet: FIGURE 3. Protection from STZ-induced diabetes in Notch3-IC Tg mice. A, Blood glucose levels (milli- grams per deciliter) determined at 0, 9, 15, and 22 days after STZ administration (T0, T9, T15, and T22, re- spectively) in wt and Notch3-IC Tg mice. Values are reported as mean of three independent experiments. B, Percentages of islets affected by insulitis in pancreata from wt () and Notch3-IC Tg mice (u) at 9 (T9) and 22 (T22) days after drug treatment, evaluated as de- scribed in Materials and Methods. C, The same af- fected islets determined in B were assorted in scoring categories as described in Materials and Methods. D, Representative islets stained with H&E from wt mouse at 9 and 22 days after induction of diabetes (a and b, respectively) and from Notch3-IC Tg animal at the same time (d and e, respectively) are shown. The func- tion of cells was evaluated with the confocal micro- scope by immunostaining with an anti-insulin Ab in pancreata from wt and Tg mice at 22 days after STZ treatment (c and f, respectively).

Article Snippet: For intracellular staining with anti-CTLA-4, anti-Notch3 and anti-IL-10 cells were treated as previously described (21, 19) and stained with PE-conjugated hamster anti-mouse CD152 (CTLA-4, UC10-4F10-11), PE-conjugated rat anti-mouse IL-10 (JES5-16E3; both from BD PharMingen), or rabbit polyclonal Ab against the intracellular domain of Notch3 protein (sc-7424; Santa Cruz Biotechnology, Santa Cruz, CA), respectively.

Techniques: Staining, Immunostaining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Expression of activated Notch3 in transgenic mice enhances generation of T regulatory cells and protects against experimental autoimmune diabetes.

doi: 10.4049/jimmunol.171.9.4504

Figure Lengend Snippet: FIGURE 4. Recruitment of T-reg cells in lymphoid organs and pancre- ata of ld-STZ-treated Notch3-IC Tg mice. A, Islets immunostained with anti-CTLA-4 from pancreata of wt and Tg mice at 22 days after diabetes induction. B, Percentages of CD25CD4 cells in splenic lymphocytes from wt and Notch3-IC Tg at 0 (T0), 9 (T9), and 22 (T22) days after drug administration, as assessed by two-color FCA (data represent the mean of three independent experiments). C, Total cell number of CTLA-4CD25

Article Snippet: For intracellular staining with anti-CTLA-4, anti-Notch3 and anti-IL-10 cells were treated as previously described (21, 19) and stained with PE-conjugated hamster anti-mouse CD152 (CTLA-4, UC10-4F10-11), PE-conjugated rat anti-mouse IL-10 (JES5-16E3; both from BD PharMingen), or rabbit polyclonal Ab against the intracellular domain of Notch3 protein (sc-7424; Santa Cruz Biotechnology, Santa Cruz, CA), respectively.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Expression of activated Notch3 in transgenic mice enhances generation of T regulatory cells and protects against experimental autoimmune diabetes.

doi: 10.4049/jimmunol.171.9.4504

Figure Lengend Snippet: FIGURE 5. Enhanced expression of IL-4 and IL-10 regulatory cyto- kines in lymphoid organs and pancreata of Notch3-IC Tg ld-STZ-treated mice compared with wt littermates. A, Expression of IL-4 and IL-10 mRNA in splenic lymphocytes, analyzed by semiquantitative RT-PCR in representative cases of untreated (T0) and 9 and 22 days postinduction (T9 and T22, respectively) Notch3-IC Tg compared with wt animals. mRNA of -actin (-act) was also investigated as a control. B, RT-PCR analysis of IL-4 and IL-10 expression in total RNA from pancreata of the same ani- mals as in A. C, Anti-IL-10 immunostaining of pancreatic section from wt (upper panel) and Tg (lower panel) mice at 22 days after drug adminis- tration. The results are representative of three independent experiments.

Article Snippet: For intracellular staining with anti-CTLA-4, anti-Notch3 and anti-IL-10 cells were treated as previously described (21, 19) and stained with PE-conjugated hamster anti-mouse CD152 (CTLA-4, UC10-4F10-11), PE-conjugated rat anti-mouse IL-10 (JES5-16E3; both from BD PharMingen), or rabbit polyclonal Ab against the intracellular domain of Notch3 protein (sc-7424; Santa Cruz Biotechnology, Santa Cruz, CA), respectively.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Immunostaining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Expression of activated Notch3 in transgenic mice enhances generation of T regulatory cells and protects against experimental autoimmune diabetes.

doi: 10.4049/jimmunol.171.9.4504

Figure Lengend Snippet: FIGURE 6. Enhanced regulatory functions of CD4 T cells from Notch3-IC Tg mice. A, Blood glu- cose levels (milligrams per deciliter) determined at 0, 9, 15, 22, and 25 days after ld-STZ treatment (T0, T9, T15, T22, and T25, respectively) in wt mice injected with 6 106 CD4 thymocytes from wt (F) or Tg (f) mice or with vehicle alone (Œ) 4 days before the drug induction. Values are reported as the mean of three independent experiments. B, Percentage of insulitis-af- fected islets, assorted in scoring categories, as de- scribed in Materials and Methods, in pancreata from the animals receiving the same treatment as in A sac- rificed at 25 days. C, Representative islets immuno- stained with anti-insulin Ab in a wt-untreated mouse (a) and in wt mice at 25 days after induction of diabetes and previously injected, as indicated in A, with 6 106

Article Snippet: For intracellular staining with anti-CTLA-4, anti-Notch3 and anti-IL-10 cells were treated as previously described (21, 19) and stained with PE-conjugated hamster anti-mouse CD152 (CTLA-4, UC10-4F10-11), PE-conjugated rat anti-mouse IL-10 (JES5-16E3; both from BD PharMingen), or rabbit polyclonal Ab against the intracellular domain of Notch3 protein (sc-7424; Santa Cruz Biotechnology, Santa Cruz, CA), respectively.

Techniques: Injection, Staining

Journal: bioRxiv

Article Title: Activation of Nedd4L Ubiquitin Ligase by FCHO2-generated Membrane Curvature

doi: 10.1101/2022.02.21.481372

Figure Lengend Snippet: a, b, Doxycycline (Dox)-induced expression of αENaC. αβγENaC-HeLa cells were cultured overnight in the presence or absence of Dox. The lysates of parental HeLa and αβγENaC-HeLa cells were subjected to immunoblotting (IB). (a) 7.5% gel. (b) Gradient gel (5-20%). Anti-γENaC antibody cross-reacted with two bands of endogenous proteins in parental HeLa cells, the lower band of which overlapped with γENaC (arrow) in αβγENaC-HeLa cells. Upon Dox-induced αENaC expression, an additional 70-kDa γENaC band (asterisk) was detected. It has been shown that co-expression of all three subunits induces ENaC maturation, including proteolytic cleavage of αENaC and γENaC . The 70-kDa γENaC band is likely a cleavage product comprising the C-terminal region. The 20-kDa αENaC band (arrowhead) is likely a cleavage product comprising the N-terminal region. c, Association of the α, β, and γ subunits. When αENaC was immunoprecipitated with anti-FLAG antibody from αβγENaC-HeLa cells treated with Dox, βENaC and γENaC were co-precipitated.

Article Snippet: Human UbcH7 cDNA was obtained by RT-PCR and cloned in pGEX-6P. pET21d-Ube1 was a gift from Cynthia Wolberger (Addgene plasmid # 34965 ; http://n2t.net/addgene:34965 ; RRID:Addgene_34965) . pRSET-mSA was a gift from Sheldon Park (Addgene plasmid # 39860 ; http://n2t.net/addgene:39860 ; RRID:Addgene_39860) . mSA-αENaC was constructed in pRSET-mSA to express a fusion protein of the N-terminal (aa 7–40) and C-terminal (aa 625–659) intracellular regions of αENaC (see ).

Techniques: Expressing, Cell Culture, Western Blot, Immunoprecipitation

Journal: bioRxiv

Article Title: Activation of Nedd4L Ubiquitin Ligase by FCHO2-generated Membrane Curvature

doi: 10.1101/2022.02.21.481372

Figure Lengend Snippet: FCHO2 binds to the plasma membrane and generates a specific degree of membrane curvature according to the intrinsic curvature of its BAR domain. Nedd4L exists in a catalytically autoinhibited state due to an intramolecular interaction between the N-terminal C2 and C-terminal HECT domains. Nedd4L is recruited to FCHO2-generated membrane curvature at the rim of nascent CCPs that ENaC enters. Membrane binding of the C2 domain causes catalytic activation by relieving autoinhibition. ENaC is ubiquitinated and captured by adaptor proteins with Ub-interacting motifs, such as Eps15.

Article Snippet: Human UbcH7 cDNA was obtained by RT-PCR and cloned in pGEX-6P. pET21d-Ube1 was a gift from Cynthia Wolberger (Addgene plasmid # 34965 ; http://n2t.net/addgene:34965 ; RRID:Addgene_34965) . pRSET-mSA was a gift from Sheldon Park (Addgene plasmid # 39860 ; http://n2t.net/addgene:39860 ; RRID:Addgene_39860) . mSA-αENaC was constructed in pRSET-mSA to express a fusion protein of the N-terminal (aa 7–40) and C-terminal (aa 625–659) intracellular regions of αENaC (see ).

Techniques: Generated, Binding Assay, Activation Assay