Journal: Oncology Letters

Article Title: Significance of interstitial tumor-associated macrophages in the progression of lung adenocarcinoma

doi: 10.3892/ol.2016.5270

Figure Lengend Snippet: Typical expression images of immunochemistry staining in tissue microarrays. (A and B) Low expression of CD68 in MIA tissues. Original magnification, (A) ×100 and (B) ×200. (C and D) High expression of CD68 in LPA tissues. Original magnification, (C) ×100 and (D) ×200. (E) Negative expression of IL-6 in MIA tissues. Original magnification, ×200. (F) Positive expression of IL-6 in LPA tissues. Original magnification, ×200. (G) Negative expression of CSF-1 in MIA tissues. Original magnification, ×200. (H) Positive expression of CSF-1 in LPA tissues. Original magnification, ×200. (I) High E-cadherin expression in MIA tissues. Original magnification, ×100. (J) Low E-cadherin expression in LPA tissues. Original magnification, ×100. (K) High Snail expression in LPA tissues. Original magnification, ×100. (L) High MMP-2 expression in LPA tissues. Original magnification, ×100. CD, cluster of differentiation; MIA, minimally invasive adenocarcinoma; LPA, lepidic predominant adenocarcinoma; CSF, colony-stimulating factor; IL, interleukin; MMP, metalloproteinase.

Article Snippet: Immunohistochemistry was performed with mouse anti-human CD68 monoclonal antibody at 1:5 dilution (clone KP1; Abcam, Cambridge, UK), rabbit anti-human colony-stimulating factor (CSF)-1 polyclonal antibody at 1:100 dilution (BA0750; Wuhan Boster Biological Technology, Ltd., Wuhan, China), anti-interleukin (IL)-6 at 1:100 dilution (BS0781R; BIOSS, Beijing, China), anti-matrix metalloproteinase (MMP)-2 at 1:400 dilution (ab37150; Abcam), anti-E-cadherin at 1:200 dilution (sc-7,870; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and anti-Snail at 1:25 dilution (AP2054a; Abgent, Inc., San Diego, CA, USA).

Techniques: Expressing, Staining

Journal: Oncology Letters

Article Title: Significance of interstitial tumor-associated macrophages in the progression of lung adenocarcinoma

doi: 10.3892/ol.2016.5270

Figure Lengend Snippet: (A) Kaplan-Meier analysis of overall survival for the combination of CD68, CSF-1 and IL-6. (B) Kaplan-Meier analysis of disease-free survival for the combination of CD68, CSF-1 and IL-6. CSF-1, colony-stimulating factor-1; CD, cluster of differentiation; IL, interleukin,

Article Snippet: Immunohistochemistry was performed with mouse anti-human CD68 monoclonal antibody at 1:5 dilution (clone KP1; Abcam, Cambridge, UK), rabbit anti-human colony-stimulating factor (CSF)-1 polyclonal antibody at 1:100 dilution (BA0750; Wuhan Boster Biological Technology, Ltd., Wuhan, China), anti-interleukin (IL)-6 at 1:100 dilution (BS0781R; BIOSS, Beijing, China), anti-matrix metalloproteinase (MMP)-2 at 1:400 dilution (ab37150; Abcam), anti-E-cadherin at 1:200 dilution (sc-7,870; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and anti-Snail at 1:25 dilution (AP2054a; Abgent, Inc., San Diego, CA, USA).

Techniques:

Journal: Oncology Letters

Article Title: Significance of interstitial tumor-associated macrophages in the progression of lung adenocarcinoma

doi: 10.3892/ol.2016.5270

Figure Lengend Snippet: (A) OS in patients with AIS/MIA in the CD68+CSF-1+IL-6+ group compared with other groups, including the CD68-, CD68+CSF-1+IL-6-, CD68+CSF-1-IL-6+ and CD68+CSF-1-IL-6- groups. (B) DFS in patients with AIS/MIA in the CD68+ CSF-1+ IL-6+ group compared with other groups (C) Comparison of OS in AIS/MIA patients with CD68+CSF-1+IL-6+ and in LPA patients with CD68+CSF-1+IL-6+. (D) Comparison of DFS in AIS/MIA patients with CD68+CSF-1+IL-6+ and in LPA patients with CD68+CSF-1+IL-6+. *Others include the CD68+CSF-1+IL-6- group, the CD68+CSF-1-IL-6+ group, the CD68+CSF-1-IL-6- group and the CD68- group. AIS, adenocarcinoma in situ ; MIA, minimally invasive adenocarcinoma; CD, cluster of differentiation; CSF, colony-stimulating factor; IL, interleukin; OS, overall survival; DFS, disease-free survival.

Article Snippet: Immunohistochemistry was performed with mouse anti-human CD68 monoclonal antibody at 1:5 dilution (clone KP1; Abcam, Cambridge, UK), rabbit anti-human colony-stimulating factor (CSF)-1 polyclonal antibody at 1:100 dilution (BA0750; Wuhan Boster Biological Technology, Ltd., Wuhan, China), anti-interleukin (IL)-6 at 1:100 dilution (BS0781R; BIOSS, Beijing, China), anti-matrix metalloproteinase (MMP)-2 at 1:400 dilution (ab37150; Abcam), anti-E-cadherin at 1:200 dilution (sc-7,870; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and anti-Snail at 1:25 dilution (AP2054a; Abgent, Inc., San Diego, CA, USA).

Techniques: In Situ

Journal: Frontiers in Immunology

Article Title: MSC therapy ameliorates experimental gouty arthritis hinting an early COX-2 induction

doi: 10.3389/fimmu.2023.1193179

Figure Lengend Snippet: Ad-MSC modulates pro- and anti-inflammatory cytokine profile in synovial membranes of arthritic rabbits. Representative western blot of pro-inflammatory cytokines (A) COX-2, TNF, IL-6 and M2 anti-inflammatory cytokines levels (B) IL-10 and TGF-β. EZ blue staining was used as protein loading control and to normalize the results, which are expressed as a fold-change of the Control group. Bars show the mean and SEM. COX-2, Ciclooxygenase-2; IL, interleukin; TGF-β, tumor growth factor-β, TNF-α, tumor necrosis factor α; MSC, mesenchymal stem cells; MSU, monosodium urate.

Article Snippet: The following primary antibodies were applied overnight at 4°C: anti-human COX-2 (Santa Cruz Biotechnology, Dallas TX, USA), anti-rabbit IL-6, anti-rabbit TNF, anti-rabbit IL-10, anti-rabbit TGF-β (all from Cloud-Clone Corp; 1/250 dilution); anti-human NLRP3 (AdipoGen, Liestal, Switzerland; 1/1000 dilution); anti-human Caspase-1 (Thermo Fisher Scientific, IL, USA; 1/500 dilution), anti-rabbit IL-18 and IL-1β antibodies (Cloud-Clone Corp, Houston TX, USA; 1/250 dilution).

Techniques: Western Blot, Staining

Journal: The Journal of Experimental Medicine

Article Title: Assembly and Regulation of the CD40 Receptor Complex in Human B Cells

doi:

Figure Lengend Snippet: Stimulation with sCD154 induces recruitment of TRAF2 and TRAF3 to CD40. ( A ) DND39 cells (2 × 10 7 cells/lane) were either left unstimulated (lanes 1 , 2 , 5 , and 7 ), or stimulated with sCD154 (4 nM) (lanes 3 , 4 , 6 , and 8 ) for 15 min before lysis and immunoprecipitation with either irrelevant mouse IgG1 (lanes 1 , 3 ) and or with anti-human CD40 mouse IgG1 monoclonal BE-1 ( 2 , 4 ) or S2C6 (lanes 5 , 6 , and 7 , 8 ), respectively. The immunoprecipitated samples were immunoblotted for TRAF2 ( arrowhead ) (lanes 1–6 ) or for TRAF3 (indicated by asterisk) (lanes 7 , 8 ). This experiment is representative of 12 experiments.

Article Snippet: Precipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and coprecipitated TRAF molecules detected with polyclonal rabbit anti–human TRAF2 (C20) ( Santa Cruz Biotech nologies, Santa Cruz, CA) or anti–human TRAF3 (N) produced against a peptide corresponding to residues 9–32 of the human TRAF3 sequence.

Techniques: Lysis, Immunoprecipitation

Journal: The Journal of Experimental Medicine

Article Title: Assembly and Regulation of the CD40 Receptor Complex in Human B Cells

doi:

Figure Lengend Snippet: Anti-μ cross-linking reduces TRAF2 association and Fas upregulation induced by CD40 signaling. DND39 cells (2 × 10 7 cells/lane) were incubated in media or with 10 μg/ml goat anti–human IgM for 24 h and both groups were either left unstimulated ( minus ) or stimulated with sCD154 (4 nM) ( plus ) for 15 min. Lysates were immunoprecipitated for CD40 and immunoblotted for TRAF2 ( A ) or TRAF3 ( B ). ( C and D ) Immunoblot analysis of cytosolic TRAF content after GST–CD40cyt immunoprecipitations. The lysates used for the anti-CD40 precipitations described in A and B were further precipitated with GST–CD40cyt. 5 × 10 6 cells/lane were loaded for the TRAF2 blot, while the TRAF3 blot received 2 × 10 7 cell equivalents per lane. ( E ) Fluorescence-activated cell sorting analysis of DND39 cells after treatment with anti-IgM and sCD154. DND39 cells were incubated for 24 h with media ( minus ), 10 μg/ml anti-IgM, sCD154, or both (as indicated). Fas expression was detected with anti-CD95 monoclonal antibody ( open profile ) or a control mouse IgG1 monoclonal ( closed profile ). The mean fluorescent intensity of the cells is indicated in the upper righthand corner. This experiment is representative of four experiments.

Article Snippet: Precipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and coprecipitated TRAF molecules detected with polyclonal rabbit anti–human TRAF2 (C20) ( Santa Cruz Biotech nologies, Santa Cruz, CA) or anti–human TRAF3 (N) produced against a peptide corresponding to residues 9–32 of the human TRAF3 sequence.

Techniques: Incubation, Immunoprecipitation, Western Blot, Fluorescence, FACS, Expressing, Control

Journal: The Journal of Experimental Medicine

Article Title: Assembly and Regulation of the CD40 Receptor Complex in Human B Cells

doi:

Figure Lengend Snippet: Engagement of CD40 results in the loss of immunoprecipitable TRAF2 and TRAF3 from DND39 cells. DND39 cells (2 × 10 6 cells/ml) were left untreated ( minus ) or treated with 4 nM sCD154 ( plus ) for 15 min. After treatment, lysates were immunoprecipitated with either anti-TRAF2 or anti-TRAF3 antibodies. 5 × 10 6 cell equivalents were loaded per lane.

Article Snippet: Precipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and coprecipitated TRAF molecules detected with polyclonal rabbit anti–human TRAF2 (C20) ( Santa Cruz Biotech nologies, Santa Cruz, CA) or anti–human TRAF3 (N) produced against a peptide corresponding to residues 9–32 of the human TRAF3 sequence.

Techniques: Immunoprecipitation

Journal: The Journal of Experimental Medicine

Article Title: Assembly and Regulation of the CD40 Receptor Complex in Human B Cells

doi:

Figure Lengend Snippet: Pretreatment with IL-4 increases TRAF2, but not TRAF3, recruited to CD40 and increases cell surface Fas expression. DND39 cells (2 × 10 7 cells/lane) were either left untreated or pretreated for 10 min with human (h)IL-4 (2 ng/ml) (Genzyme, Cambridge, MA); and both groups were either left unstimulated ( minus ) or stimulated with sCD154 (4 nM) ( plus ) for 15 min. Lysates were immunoprecipitated for CD40 and immunoblotted for TRAF2 ( A ) or TRAF3 ( B ). ( C ) Fluorescence-activated cell sorting analysis of DND39 cells after treatment with hIL-4 and sCD154. DND39 cells were incubated for 24 h with media ( minus ), 2 ng/ml hIL-4, sCD154, or both (as indicated). Fas expression was detected with anti-CD95 monoclonal antibody ( open profile ) or a control mouse IgG1 monoclonal ( closed profile ). The mean fluorescent intensity of the cells is indicated in the upper righthand corner. This experiment is representative of four experiments.

Article Snippet: Precipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and coprecipitated TRAF molecules detected with polyclonal rabbit anti–human TRAF2 (C20) ( Santa Cruz Biotech nologies, Santa Cruz, CA) or anti–human TRAF3 (N) produced against a peptide corresponding to residues 9–32 of the human TRAF3 sequence.

Techniques: Expressing, Immunoprecipitation, Fluorescence, FACS, Incubation, Control

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

Article Title: A virus-like particle-based anti-nerve growth factor vaccine reduces inflammatory hyperalgesia: potential long-term therapy for chronic pain.

doi: 10.4049/jimmunol.1000030

Figure Lengend Snippet: FIGURE 1. Production and functional analysis of the rNGFQb vaccine. A, Production of rNGF. Murine NGF was expressed in HEK293T cells. Antipentahistidine immunoblot analysis of HEK293T lysates (first lane) and cell-culture supernatants (second lane) revealed expression of the NGF proprotein by HEK293T cells and secretion of the processed 14.7-kDa mature form of NGF into the cell-culture supernatant. Coomassie staining of NGF purified from supernatants by affinity chromatography confirmed homogeneity (third lane). B, Recognition of NGF by anti-NGF Abs. Recombinantly expressed NGF (white circles) was compared with ex vivo- purified NGF (black squares). Different dilutions of both proteins were applied to ELISA plates coated with anti-NGF mAb. Bound NGF was detected with polyclonal anti-NGF Abs. Averages of triplicates 6 SEM. C, NGF receptor binding. The NGF-specific receptor TrkA was coated onto ELISA plates and descending concentrations of both proteins applied. Receptor bound NGF was detected with polyclonal NGF Abs as in B. Averages of triplicates 6 SEM. D, Bioactivity of NGF. The NGF re- sponsive cell line TF-1 was stimulated with descending concentrations of NGF and proliferation determined by BrdU incorporation during DNA synthesis. Averages of triplicates 6 SEM. E, Analysis of NGFQb vaccine. NGF was cross-linked to VLPs with a heterobifunctional cross-linker. An immunoblot of NGF (left lane) or the NGFQb conjugate vaccine (right lane) analyzed with an anti-pentahistidine–specific Ab showed a dominant band appearing at 29 kDa corresponding to a Qb monomer (14.2 kDa) conjugated to one molecule NGF (14.7 kDa). Higher molecular mass bands correspond to Qb-oligomers linked to one NGF molecule. F, Binding of TrkA to NGFQb. Ascending concentrations of Qb (filled squares) or NGFQb (open circles) were immobilized on an ELISA plate coated with anti-Qb mAb. Bound vaccine was incubated with TrkA re- ceptor containing a human Fc domain. Binding of receptor to NGF dis- played on VLP was detected with peroxidase-conjugated goat anti-human Abs. Averages of triplicates 6 SEM. LY, lysates; SN, supernatants.

Article Snippet: Then, plates were washed six times with PBS-T and incubated with a 1:500 dilution of a sheep anti-NGF polyclonal Ab (AbD Serotec) for 1 h at RT.

Techniques: Functional Assay, Western Blot, Cell Culture, Expressing, Staining, Chromatography, Ex Vivo, Enzyme-linked Immunosorbent Assay, Binding Assay, BrdU Incorporation Assay, DNA Synthesis, Incubation