Journal: Cell Reports

Article Title: Enhanced eosinophil-mediated inflammation associated with antibody and complement-dependent pneumonic insults in critical COVID-19

doi: 10.1016/j.celrep.2021.109798

Figure Lengend Snippet: Kinetic changes of SARS-CoV-2 N-specific antibodies and complement activation in respiratory tracts and plasma (A and B) Kinetic changes in specific antibody responses against viral N protein (A) and C3a (B) in respiratory samples. Blue indicates non-critical (n = 13 for antibodies and 20 for C3a); red indicates critical (n = 28 for antibodies and 20 for C3a); solid lines indicate non-linear regression with 95% confidential intervals. (C) Correlation of C3a levels with N-specific IgM and IgG. Solid lines indicate linear regression with 95% confidential intervals. p values were determined by a Spearman’s rank test. n = 27. (D and E) Kinetic changes in specific antibody responses against viral N protein (D), C3a, and C5a (E) in plasma samples. Blue indicates non-critical (n = 30); red indicates critical (n = 18); solid lines indicate non-linear regression with 95% confidential intervals. Dashed lines (A and D) indicate data points from individual patients. Violin plots show levels in NC and C cases. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (F) Correlation of C5a levels with N-specific IgG1 and IgG3. Solid lines indicate linear regression. p values were determined by a Spearman’s rank test. n = 61.

Article Snippet: The antibodies used to detect specific antigens for immunostaining processes were rabbit polyclonal anti-SARA-CoV-2 N antibody was obtained from Sino biological (Beijing, China) and anti-CD31 and pan-cytokeratin (CK) antibodies purchased from DAKO (Carpinteria, CA, USA).

Techniques: Activation Assay

Journal: Cell Reports

Article Title: Enhanced eosinophil-mediated inflammation associated with antibody and complement-dependent pneumonic insults in critical COVID-19

doi: 10.1016/j.celrep.2021.109798

Figure Lengend Snippet: Pathologic association of immune complex deposition in lung biopsies from six fatal COVID-19 cases (A) Depositions of IgG immune complexes (white) in airways and blood vessels identified by immunofluorescence analysis of lung tissue from a fatal case, P15, at the indicated times after symptom onset. CK, pan-cytokeratin (green); N, SARS-CoV-2 N antigens (red); DIC, differential interference contrast. White arrows indicate depositions of IgG immune complexes in the luminal spaces; white asterisks indicate blood vessels. Nuclei are shown in blue. Scale bar, 100 μm. (B) Depositions of IgG immune complexes in airways and blood vessels identified by immunofluorescence analysis of lung tissues obtained from five fatal cases, P71–P75 (collection day after symptom onset indicated). Scale bars, 100 μm.

Article Snippet: The antibodies used to detect specific antigens for immunostaining processes were rabbit polyclonal anti-SARA-CoV-2 N antibody was obtained from Sino biological (Beijing, China) and anti-CD31 and pan-cytokeratin (CK) antibodies purchased from DAKO (Carpinteria, CA, USA).

Techniques: Immunofluorescence

Journal: Bioactive Materials

Article Title: Recruited CD68 + CD206 + macrophages orchestrate graft immune tolerance to prompt xenogeneic-dentin matrix-based tooth root regeneration

doi: 10.1016/j.bioactmat.2020.09.029

Figure Lengend Snippet: M2γ macrophages suppressed the Th1-type CTL response triggered by xeno-complex in vitro . (A) The hypothesis that polarized M2 macrophages perform anti-inflammatory effects on cellular response triggered by xeno-complex. (B) Incubation and identification of PPARγ-primed macrophages (M2γ). Human peripheral monocytes were primed towards M2γ macrophages via the addition of rosiglitazone (RSG) in the presence of IL-4. The M2γ macrophages were obtained after incubation for 7 days. The phenotype marker CD206 was identified by Flow cytometry analysis (FACS). Data are means ± SEM (n ≥ 3). * p < 0.05. (C) Identification of M2γ conditional medium (M2γ medium). The anti-inflammatory and pro-inflammatory cytokines were analyzed using Immunology Multiplex MAP. Data are means ± SEM (n ≥ 3). * p < 0.05, *** p < 0.001. (D) M2γ medium or RSG inhibited proliferated CD3 + CD8 + cells triggered by xeno-complex. The cellular response to xeno-complex was quantified by FCS using CFSE-based proliferation assay. The proliferated CD3 + , CD3 + CD4 + , and CD3 + CD8 + lymphocytes were graphed as an overlay (black line) in each FACS histogram covering hPBMCs (gray shaded histogram). hPBMCs without stimulus was the negative group, and PHA stimulation was the positive group. The % value on the left defined the level of proliferated cells within 5 days. Data are means ± SEM (n ≥ 3). * p < 0.05, ** p < 0.01. (E) hPBMCs in the six coculture systems were measured for mRNA expression of pro-inflammatory TNF-α, INF-γ. The data were normalized to those of GAPDH mRNA and are presented relative to those of hPBMCs without any stimulus, set as 1. Data are means ± SEM (n ≥ 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; NS, no significance. (F) The adherent monocytes/macrophages differentiation in the six coculture systems were co-immunostained with antibodies against CD68/CCR7 or CD68/CD206, DAPI was used for nuclear staining. Scale bars, 50 μm.

Article Snippet: The following primary antibodies were used: the pan-macrophage marker, mouse anti-rat CD68 (Abcam, Cambridge, MA, UK), the M1 macrophage marker, rabbit anti-rat CCR7 (Abcam, Cambridge, MA, UK), and the M2 macrophage marker, goat anti-rat CD206 (Santa Cruz).

Techniques: In Vitro, Incubation, Marker, Flow Cytometry, Multiplex Assay, Proliferation Assay, Expressing, Staining

Journal: Bioactive Materials

Article Title: Recruited CD68 + CD206 + macrophages orchestrate graft immune tolerance to prompt xenogeneic-dentin matrix-based tooth root regeneration

doi: 10.1016/j.bioactmat.2020.09.029

Figure Lengend Snippet: PPARγ activation locally modulated M1-to-M2 tissue macrophage polarization. (A) The schematic diagram illustrated the correlation between M1/M2 macrophage phenotypes and the fate of implanted ECM. Macrophage polarization in response to xeno-complex were sequentially observation at postoperative 3 days, 1 week, 3 weeks, 6 weeks, and 12 weeks. Xeno-complex with RSG treatment served as experimental groups, while with no treatment (Control) or with vehicle (Vehicle control) served as controls. Macrophage phenotypes (B) at periphery sites and (C) in the intracavity sites of xeno-complex were shown by representative confocal immunofluorescence images. Co-immunostaining of pan-macrophage cell surface marker CD68 (red), the M1 marker CCR7 (orange), and the M2 marker CD206 (green) were merged with nuclear staining DAPI (blue). Scale bars, 50 μm. (D) A ratio was calculated as to the percentage of M2 cells to M1 cells (M2/M1 radio). The gray dotted line represented M2/M1 radio as 1.0, with over 1.0 indicating predominant M2 vs. below 1.0 representing predominant M1 cells. Data are mean ± SEM (n ≥ 3), * p < 0.05, ** p < 0.01, **** p < 0.0001. NS, no significance. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The following primary antibodies were used: the pan-macrophage marker, mouse anti-rat CD68 (Abcam, Cambridge, MA, UK), the M1 macrophage marker, rabbit anti-rat CCR7 (Abcam, Cambridge, MA, UK), and the M2 macrophage marker, goat anti-rat CD206 (Santa Cruz).

Techniques: Activation Assay, Control, Immunofluorescence, Immunostaining, Marker, Staining

Journal: Molecular Cancer

Article Title: S6K1 amplification confers innate resistance to CDK4/6 inhibitors through activating c-Myc pathway in patients with estrogen receptor-positive breast cancer

doi: 10.1186/s12943-022-01642-5

Figure Lengend Snippet: The key genes related to the innate resistance of CDK4/6 inhibitors were explored by analysis of ctDNA derived from patients with breast cancer. A - D Schematic overview of the study design and analytical workflow. PFS: progression-free survival. ctDNA: circulating tumour DNA. ddPCR: droplet digital PCR. IHC: immunohistochemistry. Multi-IF: multiplex immunofluorescence. E The landscape of high-frequency altered genes in plasma from the discovery patient cohort. The plot shows patients with innate resistance or clinical benefit following palbociclib treatment, with individual information about numbers of metastases, treatment lines, and endocrine therapy drugs used in combination with palbociclib. S6K1 (i.e. RPS6KB1) amplification was observed in three patients with innate resistance to palbociclib but not in those with clinical benefit

Article Snippet: The primary antibodies used in this study were as follows: S6K1 (Proteintech, Cat# 14485-1-AP), cyclin D1 (Proteintech, Cat# 60186-1-1 g), cyclin E1 (Abcam, Cat# ab33911), phospho-Rb (Abcam, Cat# ab109399), CDK2 (Proteintech, Cat# 10122-1-AP), CDK4 (Santa Cruz Biotechnology, Cat# sc-260), CDK6 (CST, Cat# 3136S), c-Myc (CST, Cat# 5605S), β-Actin (CST, Cat# 3700S), and GAPDH (Proteintech, Cat# 60004-1-Ig).

Techniques: Derivative Assay, Digital PCR, Immunohistochemistry, Multiplex Assay, Immunofluorescence, Clinical Proteomics, Amplification

Journal: Molecular Cancer

Article Title: S6K1 amplification confers innate resistance to CDK4/6 inhibitors through activating c-Myc pathway in patients with estrogen receptor-positive breast cancer

doi: 10.1186/s12943-022-01642-5

Figure Lengend Snippet: S6K1 promotes cell proliferation and palbociclib resistance via cell cycle progression. A Cell-cycle distribution was measured using PI staining followed by flow cytometry for MCF-7 cells transfected with S6K1 siRNA pool or non-sense control siRNA as well as exogenous S6K1-expressed T47D cells or control cells, respectively. P value was calculated by Student’s t -test. *, P < 0.05, **, P < 0.01. B , C KEGG pathway enrichment analysis ( B ) and GO functional analysis ( C ) of RNA-seq data obtained from S6K1-depleted MCF-7 cells. D GSEA showing an enrichment of E2F-targets and Myc-targets signatures in S6K1-depleted MCF-7 cells. E - G MCF-7 cells transfected with the indicated siRNAs as well as exogenous S6K1-expressed T47D cells or control cells with 70-80% confluent were harvested. Proteins were then subjected to western blotting with the antibodies against S6K1, cyclin D1, cyclin E1, CDK2, CDK4, CDK6, p-Rb, and c-Myc proteins. GAPDH/β-Actin was used as the loading control. H Dual luciferase reporter assay showing the transcriptional activity of CCNE1 promoter in S6K1-depleted MCF-7 cells. P value was calculated by Student’s t -test. **, P < 0.01. I c-Myc was re-expressed into S6K-depleted MCF-7 cells and the levels of cyclin E1 and p-Rb were measured by western blotting

Article Snippet: The primary antibodies used in this study were as follows: S6K1 (Proteintech, Cat# 14485-1-AP), cyclin D1 (Proteintech, Cat# 60186-1-1 g), cyclin E1 (Abcam, Cat# ab33911), phospho-Rb (Abcam, Cat# ab109399), CDK2 (Proteintech, Cat# 10122-1-AP), CDK4 (Santa Cruz Biotechnology, Cat# sc-260), CDK6 (CST, Cat# 3136S), c-Myc (CST, Cat# 5605S), β-Actin (CST, Cat# 3700S), and GAPDH (Proteintech, Cat# 60004-1-Ig).

Techniques: Staining, Flow Cytometry, Transfection, Control, Functional Assay, RNA Sequencing, Western Blot, Luciferase, Reporter Assay, Activity Assay

Journal: Parasites & Vectors

Article Title: Toxoplasma gondii -induced adverse pregnancy outcomes: insight into the inhibitory role of Trem2 on TLR4/TRAF6/JNK signaling pathway

doi: 10.1186/s13071-025-07000-w

Figure Lengend Snippet: The absence of Trem2 further promoted the T. gondii -triggered upregulation of TLR4, TRAF6, and P-JNK in mouse placentas. A The docking models of TLR4 and Trem2 were predicted using the GRAMM docking software and visualized with PyMol. The calculated binding energy between TLR4 (green) and Trem2 (pink) was −15 kcal/mol. B Endogenous immunoprecipitation was conducted to assess the interaction between Trem2 and TLR4. RAW 264.7 cell lysate was immunoprecipitated with Trem2 antibody. Expression of Trem2 and TLR4 was assayed by western blotting. C Multiplex immunofluorescence analysis was conducted to examine the colocalization of TLR4, Trem2, and TRAF6. RAW 264.7 cells were incubated with Trem2 antibody (green), TLR4 antibody (red), TRAF6 antibody (magenta), and DAPI dye (blue). Fluorescence intensity and co-localization of the three proteins were analyzed using ImageJ software. Scale bar: 10 μm. D Expression of Trem2 in the placentas from WT pregnant mice challenged with/without T. gondii , along with corresponding quantitative analysis. Placental tissue was extracted from WT pregnant mice on G17.5, and western blotting was employed to assay Trem2 expression ( n = 6). E Expression of TLR4, JNK, P-JNK, and TRAF6 in placental tissues from Trem2 −/− and WT pregnant mice challenged with/without T. gondii , along with corresponding quantitative analysis. Placental tissue was extracted from WT and Trem2 −/− pregnant mice on G17.5. Western blotting was employed to assay the level of TLR4, JNK, P-JNK, and TRAF6 ( n = 6). WT: Wild type, TI: T. gondii infection. Significant changes were determined with a two-tailed unpaired Student t -test ( D ) and two-way ANOVA with Sidak’s multiple comparisons test ( E ) at P < 0.05. * P < 0.05

Article Snippet: The following antibodies were utilized in the experiments: anti-TRAF6 antibody (1:2000, rabbit monoclonal, ab40675, Abcam), anti-TLR4 antibody (1:1000, mouse monoclonal, 66350-1-Ig, Proteintech), anti-Phospho-SAPK/JNK antibody (Thr183/Tyr185; 1:1000, rabbit monoclonal, 4668, Cell Signaling Technology), anti-GAPDH (1:50,000, mouse monoclonal, 60004-1-Ig, Proteintech), anti-mouse HRP-IgG (1:5000, SA00001-1, Proteintech), anti-rabbit HRP-IgG (1:5000, SA00001-2, Proteintech), anti-Trem2 antibody (1:3000, sheep monoclonal, AF1729, R&D Systems), and anti-sheep HRP-IgG (1:5000; sc2473, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Software, Binding Assay, Immunoprecipitation, Expressing, Western Blot, Multiplex Assay, Immunofluorescence, Incubation, Fluorescence, Infection, Two Tailed Test

Journal: Parasites & Vectors

Article Title: Toxoplasma gondii -induced adverse pregnancy outcomes: insight into the inhibitory role of Trem2 on TLR4/TRAF6/JNK signaling pathway

doi: 10.1186/s13071-025-07000-w

Figure Lengend Snippet: Toxoplasma gondii antigens suppressed Trem2 expression but activated the downstream TLR4 signaling pathway in RAW 264.7 cells. A Immunoblot analysis of RAW 264.7 cells stimulated with/without Tg Ag. Western blotting was conducted to measure the expression of TLR4, P-JNK, JNK, TRAF6, and Trem2 in RAW 264.7 cells that were treated with/without Tg Ag (5 µg/ml) for 24 h ( n = 3). B – E Representative immunofluorescence photograph and quantitative analysis of the expression of TLR4, TRAF6, and P-JNK in RAW 264.7 cells stimulated with/without Tg Ag. Immunofluorescence was used to assess the fluorescence intensity of TLR4 (green), TRAF6 (magenta), P-JNK (red), and Trem2 (green) in RAW 264.7 cells stimulated with/without Tg Ag (5 μg/ml) for 24 h. Scale bar: 20 μm. Fluorescence intensity was quantified by using ImageJ software ( n = 3). F Endogenous immunoprecipitation was employed to measure the interaction between TLR4 and Trem2. RAW 264.7 cells were stimulated with Tg Ag (5 μg/ml) for 24 h, which was followed by immunoprecipitation with Trem2 antibody. TLR4 expression was measured by western blotting ( n = 3). Tg Ag: T. gondii antigens, Con: untreated group. Significant changes were determined with a two-tailed unpaired Student t -test ( A – F ) at P < 0.05. * P < 0.05

Article Snippet: The following antibodies were utilized in the experiments: anti-TRAF6 antibody (1:2000, rabbit monoclonal, ab40675, Abcam), anti-TLR4 antibody (1:1000, mouse monoclonal, 66350-1-Ig, Proteintech), anti-Phospho-SAPK/JNK antibody (Thr183/Tyr185; 1:1000, rabbit monoclonal, 4668, Cell Signaling Technology), anti-GAPDH (1:50,000, mouse monoclonal, 60004-1-Ig, Proteintech), anti-mouse HRP-IgG (1:5000, SA00001-1, Proteintech), anti-rabbit HRP-IgG (1:5000, SA00001-2, Proteintech), anti-Trem2 antibody (1:3000, sheep monoclonal, AF1729, R&D Systems), and anti-sheep HRP-IgG (1:5000; sc2473, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Immunofluorescence, Fluorescence, Software, Immunoprecipitation, Two Tailed Test

Journal: Parasites & Vectors

Article Title: Toxoplasma gondii -induced adverse pregnancy outcomes: insight into the inhibitory role of Trem2 on TLR4/TRAF6/JNK signaling pathway

doi: 10.1186/s13071-025-07000-w

Figure Lengend Snippet: Toxoplasma gondii antigens inhibited Trem2 to activate the TLR4/TRAF6/JNK axis. A Schematic representation of the treatment of TLR4 blocking antibody on RAW 264.7 cells. The cells, primed with TLR4 blocking antibody (20 μg/ml) or isotype control for 30 min, were stimulated with/without Tg Ag (5 μg/ml) for 24 h. Expression of P-JNK, JNK, Trem2, and TRAF6 was assayed by western blotting ( n = 3). B Schematic diagram of Trem2 knockdown in RAW 264.7 cells. Cells that were transfected with si- Trem2 or negative control (si-NC) for 6 h were stimulated with/without Tg Ag (5 μg/ml) for 24 h. Expression of P-JNK, JNK, TLR4, and TRAF6 was measured by western blotting ( n = 3). C Schematic diagram of Trem2 overexpression in RAW 264.7 cells. Cells were stimulated with or without Tg Ag for an additional 24 h. Expression of P-JNK, JNK, TLR4, Trem2, and TRAF6 was assayed by western blotting ( n = 3). D Schematic illustration of the treatment with the TRAF6 inhibitor on RAW 264.7 cells. Cells, primed with 20 µM C25-140 (TRAF6 inhibitor) for 2 h, were exposed to Tg Ag (5 μg/ml) for 24 h. Expression of P-JNK, JNK, TLR4, Trem2, and TRAF6 was assayed by western blotting ( n = 3). Created in BioRender. Cao, Y. (2025) https://BioRender.com/mwqhkc7 . Tg Ag: T. gondii antigens, Con: untreated group. Significant changes were determined with two-way ANOVA with Sidak’s multiple comparisons test ( A – D ) at P < 0.05. * P < 0.05

Article Snippet: The following antibodies were utilized in the experiments: anti-TRAF6 antibody (1:2000, rabbit monoclonal, ab40675, Abcam), anti-TLR4 antibody (1:1000, mouse monoclonal, 66350-1-Ig, Proteintech), anti-Phospho-SAPK/JNK antibody (Thr183/Tyr185; 1:1000, rabbit monoclonal, 4668, Cell Signaling Technology), anti-GAPDH (1:50,000, mouse monoclonal, 60004-1-Ig, Proteintech), anti-mouse HRP-IgG (1:5000, SA00001-1, Proteintech), anti-rabbit HRP-IgG (1:5000, SA00001-2, Proteintech), anti-Trem2 antibody (1:3000, sheep monoclonal, AF1729, R&D Systems), and anti-sheep HRP-IgG (1:5000; sc2473, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Blocking Assay, Control, Expressing, Western Blot, Knockdown, Transfection, Negative Control, Over Expression

Journal: Parasites & Vectors

Article Title: Toxoplasma gondii -induced adverse pregnancy outcomes: insight into the inhibitory role of Trem2 on TLR4/TRAF6/JNK signaling pathway

doi: 10.1186/s13071-025-07000-w

Figure Lengend Snippet: Trem2 deficiency in macrophages further activated the TLR4/TRAF6/JNK signaling cascade. A Flowchart depicting the extraction process of BMDMs derived from WT and Trem2 −/− mice. Created in BioRender. Cao, Y. (2025) https://BioRender.com/mwqhkc7 . B Expression of P-JNK, JNK, TLR4, and TRAF6 in BMDMs was assayed by western blotting. BMDMs derived from either WT or Trem2 −/− mice were stimulated with/without Tg Ag (5 μg/ml) for 24 h ( n = 3). C Expression of P-JNK, JNK, Trem2, and TRAF6 in BMDMs was assayed by western blotting. BMDMs isolated from WT mice, primed with TLR4 blocking antibody (20 μg/ml) or isotype control for 30 min, were stimulated with/without Tg Ag (5 μg/ml) for 24 h ( n = 3). D The messenger RNA (mRNA) levels of inflammatory factors in WT or Trem2 −/− mouse BMDMs. BMDMs isolated from either WT or Trem2 −/− mice were exposed to Tg Ag (5 μg/ml) for 24 h. Then, the mRNA levels of TNF-α , IL-10 , IFN-γ , and TGF-β were assayed by real-time PCR ( n = 5). Tg Ag: T. gondii antigens, Con: untreated group; anti-TLR4: TLR4/MD-2 Complex Antibody. Significant changes were determined with two-way ANOVA with Sidak’s multiple comparisons test ( B – D ) at P < 0.05. * P < 0.05

Article Snippet: The following antibodies were utilized in the experiments: anti-TRAF6 antibody (1:2000, rabbit monoclonal, ab40675, Abcam), anti-TLR4 antibody (1:1000, mouse monoclonal, 66350-1-Ig, Proteintech), anti-Phospho-SAPK/JNK antibody (Thr183/Tyr185; 1:1000, rabbit monoclonal, 4668, Cell Signaling Technology), anti-GAPDH (1:50,000, mouse monoclonal, 60004-1-Ig, Proteintech), anti-mouse HRP-IgG (1:5000, SA00001-1, Proteintech), anti-rabbit HRP-IgG (1:5000, SA00001-2, Proteintech), anti-Trem2 antibody (1:3000, sheep monoclonal, AF1729, R&D Systems), and anti-sheep HRP-IgG (1:5000; sc2473, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Extraction, Derivative Assay, Expressing, Western Blot, Isolation, Blocking Assay, Control, Real-time Polymerase Chain Reaction

Journal: Cancers

Article Title: Increased Expression and Activation of FAK in Small-Cell Lung Cancer Compared to Non-Small-Cell Lung Cancer

doi: 10.3390/cancers11101526

Figure Lengend Snippet: Illustrations of FAK and phospho-FAK (Y397) expression evaluated by multiplex immunofluorescence (IF) immunohistochemistry (IHC) in lung cancer and normal lung tissues. ( A ) Lung adenocarcinoma with the absence of phospho-FAK expression but homogenous cytoplasmic FAK staining (orange) in the tumor core, adjacent non-tumoral bronchi, and some stromal cells (including vessels and lymphoid structures). ( B ) Lung adenocarcinoma with nuclear phospho-FAK staining (red) and homogenous cytoplasmic FAK staining (orange). ( C ) Lung squamous carcinoma with the absence of phospho-FAK expression but weak cytoplasmic FAK staining. ( D ) Lung squamous carcinoma with nuclear phospho-FAK staining (red) and homogenous cytoplasmic FAK staining (orange). ( E ) Small-cell lung cancer with nuclear phospho-FAK staining (red) and cytoplasmic FAK staining (orange). ( F ) Normal lung with cytoplasmic FAK staining in bronchi and some stromal cells (including vessels and lymphoid structures). ( G ) Lung squamous carcinoma used as a negative control, showing the absence of phospho-FAK and FAK staining. Original magnification: 20×; scale bar: 50 µm.

Article Snippet: After blocking 1 h with 5% W/V BSA (Sigma, Saint-Louis, MO, USA) in TBS with 0.1% Tween 20 (Sigma), the membrane was incubated overnight at 4 °C with phospho-FAK Y397 rabbit antibody (1/1000 Cell Signaling Technology, Danvers, MA, USA) or total FAK mouse antibody (1/250, Santa Cruz Biotechnology, Dallas, TX, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rabbit antibody (1/5000, Sigma).

Techniques: Expressing, Multiplex Assay, Immunofluorescence, Immunohistochemistry, Staining, Negative Control

Journal: Cancers

Article Title: Increased Expression and Activation of FAK in Small-Cell Lung Cancer Compared to Non-Small-Cell Lung Cancer

doi: 10.3390/cancers11101526

Figure Lengend Snippet: Quantification of FAK and phospho-FAK (Y397) expression evaluated by multiplex immunofluorescence immunohistochemistry in 37 normal lungs, 95 non-small-cell lung cancer (NSCLC), and 105 small-cell lung cancer (SCLC) tissues: ( A ) FAK staining score: Percentage (%) of FAK-stained tumor area multiplied by (x) FAK mean intensity, ( B ) phospho-FAK (Y397) staining score: (% of phospho-FAK-stained tumor area of low intensity × 1) + (% of phospho-FAK-stained tumor area of medium intensity × 2) + (% of phospho-FAK-stained tumor area of high intensity × 3), and ( C ) ratio between phospho-FAK and FAK staining scores. Each dot represents one sample. Data presented as the mean ± S.D. p -values were obtained using linear models and adjusted for multiple testing using the Bonferroni method.

Article Snippet: After blocking 1 h with 5% W/V BSA (Sigma, Saint-Louis, MO, USA) in TBS with 0.1% Tween 20 (Sigma), the membrane was incubated overnight at 4 °C with phospho-FAK Y397 rabbit antibody (1/1000 Cell Signaling Technology, Danvers, MA, USA) or total FAK mouse antibody (1/250, Santa Cruz Biotechnology, Dallas, TX, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rabbit antibody (1/5000, Sigma).

Techniques: Expressing, Multiplex Assay, Immunofluorescence, Immunohistochemistry, Staining

Journal: Cancers

Article Title: Increased Expression and Activation of FAK in Small-Cell Lung Cancer Compared to Non-Small-Cell Lung Cancer

doi: 10.3390/cancers11101526

Figure Lengend Snippet: Quantification of nuclear FAK and nuclear phospho-FAK (Y397) expression evaluated by multiplex immunofluorescence immunohistochemistry in 37 normal lung, 95 non-small-cell lung cancer (NSCLC), and 105 small-cell lung cancer (SCLC) tissues: ( A ) Nuclear FAK staining score: Percentage (%) of FAK-stained nucleus area multiplied by (×) nuclear FAK mean intensity, ( B ) nuclear phospho-FAK (Y397) staining score: (% of phospho-FAK-stained nucleus area of low intensity × 1) + (% of phospho-FAK-stained nucleus area of medium intensity × 2) + (% of phospho-FAK-stained nucleus area of high intensity × 3). Each dot represents one sample. Data presented as the mean ± S.D. p -values were obtained using linear models and adjusted for multiple testing using the Bonferroni method.

Article Snippet: After blocking 1 h with 5% W/V BSA (Sigma, Saint-Louis, MO, USA) in TBS with 0.1% Tween 20 (Sigma), the membrane was incubated overnight at 4 °C with phospho-FAK Y397 rabbit antibody (1/1000 Cell Signaling Technology, Danvers, MA, USA) or total FAK mouse antibody (1/250, Santa Cruz Biotechnology, Dallas, TX, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rabbit antibody (1/5000, Sigma).

Techniques: Expressing, Multiplex Assay, Immunofluorescence, Immunohistochemistry, Staining

Journal: Cancers

Article Title: Increased Expression and Activation of FAK in Small-Cell Lung Cancer Compared to Non-Small-Cell Lung Cancer

doi: 10.3390/cancers11101526

Figure Lengend Snippet: Quantification of ( A ) FAK and ( B ) phospho-FAK expression evaluated by Western blot (WB), with normalization to glyceraldehyde 3-phosphate dehydrogenase (GAPDH ) expression, in nine normal lungs, 30 non-small-cell lung cancer (NSCLC), and 10 small-cell lung cancer (SCLC) tissue lysates. Each dot represents one sample. Data presented as the mean ± S.D. Significance determined by the Kruskal-Wallis test. ( C ) Illustration of a representative WB of FAK and phospho-FAK (Y397) expression in normal lung, NSCLC, and SCLC tissue lysates. All the WB are represented in .

Article Snippet: After blocking 1 h with 5% W/V BSA (Sigma, Saint-Louis, MO, USA) in TBS with 0.1% Tween 20 (Sigma), the membrane was incubated overnight at 4 °C with phospho-FAK Y397 rabbit antibody (1/1000 Cell Signaling Technology, Danvers, MA, USA) or total FAK mouse antibody (1/250, Santa Cruz Biotechnology, Dallas, TX, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rabbit antibody (1/5000, Sigma).

Techniques: Expressing, Western Blot

Journal: Frontiers in Immunology

Article Title: HEV-associated dendritic cells are observed in metastatic tumor-draining lymph nodes of cutaneous melanoma patients with longer distant metastasis-free survival after adjuvant immunotherapy

doi: 10.3389/fimmu.2023.1231734

Figure Lengend Snippet: Favoring Antigen-Presenting Structures (FAPS) are present in mTDLN of GO patients. Biomarkers were determined by IHC or multiplex IF, as described under Methods; representative images are shown. (A) Patient#17 is enriched in peri- and intra-tumoral CD11c + cells. (B) Patient#17 CD11c + cells (green) contain melanin and tyrosinase (orange, white arrow). (C) A FAPS example is shown (Patient#13). CD11c + cells (white arrow) are shown surrounding PNAd + HEV (black arrow). (D) CD11c + cells (green) surrounding HEV (white) contained melanin and tyrosinase (orange) (Patient#17). (E) DC-LAMP + cells (white arrow) surrounding HEV with cuboidal epithelium (black arrow) are shown (Patient#13). (F) FAPS structure showing PNAd + (white) HEV interactions between CD62L + cells (red) and CD11c + cells (green) are indicated by arrows (Patient#13). Original magnifications: (A) 400X; (B, D, F) 200X; (C, E) 1000X. Scale bars: (A) 50µm; (B, D, F) 10µm; (C, E) 20µm.

Article Snippet: Primary mAbs: CD11c (clone EP 1347Y, 1:200, Abcam); MART-1 conjugated to AF647 [IgG1, κ, clone 2A9 ( )]; TYR conjugated to AF647 (clone T311, Santa Cruz Biotechnology); CD62L mAb conjugated to AlexaFluor-647 (clone DREG56, 1:50, IgG1 κ, Santa Cruz Biotechnology).

Techniques: Multiplex Assay

Journal: Frontiers in Immunology

Article Title: HEV-associated dendritic cells are observed in metastatic tumor-draining lymph nodes of cutaneous melanoma patients with longer distant metastasis-free survival after adjuvant immunotherapy

doi: 10.3389/fimmu.2023.1231734

Figure Lengend Snippet: Multiplex immunofluorescence analysis of FAPS. Multiplex staining of a mTDLN peritumoral area of one GO patient (#13) with anti-CD3 (cyan), anti-CD20 (blue), anti-CD62L (pink), anti-PNAd (red), anti-CD11c (yellow), and anti-DC-LAMP (white) antibodies was performed as described under Methods. DC-LAMP + and CD11c + cells surround PNAd + HEV. In the lower part area of the figure, marked with orange arrows, sprouts of PNAd + HEV (red), and CD62L + naïve lymphocytes (pink) making contacts with HEV and DC-LAMP + dendritic cells (white) may be observed.

Article Snippet: Primary mAbs: CD11c (clone EP 1347Y, 1:200, Abcam); MART-1 conjugated to AF647 [IgG1, κ, clone 2A9 ( )]; TYR conjugated to AF647 (clone T311, Santa Cruz Biotechnology); CD62L mAb conjugated to AlexaFluor-647 (clone DREG56, 1:50, IgG1 κ, Santa Cruz Biotechnology).

Techniques: Multiplex Assay, Immunofluorescence, Staining

Journal: Cell Reports

Article Title: Enhanced eosinophil-mediated inflammation associated with antibody and complement-dependent pneumonic insults in critical COVID-19

doi: 10.1016/j.celrep.2021.109798

Figure Lengend Snippet: Kinetic changes of SARS-CoV-2 N-specific antibodies and complement activation in respiratory tracts and plasma (A and B) Kinetic changes in specific antibody responses against viral N protein (A) and C3a (B) in respiratory samples. Blue indicates non-critical (n = 13 for antibodies and 20 for C3a); red indicates critical (n = 28 for antibodies and 20 for C3a); solid lines indicate non-linear regression with 95% confidential intervals. (C) Correlation of C3a levels with N-specific IgM and IgG. Solid lines indicate linear regression with 95% confidential intervals. p values were determined by a Spearman’s rank test. n = 27. (D and E) Kinetic changes in specific antibody responses against viral N protein (D), C3a, and C5a (E) in plasma samples. Blue indicates non-critical (n = 30); red indicates critical (n = 18); solid lines indicate non-linear regression with 95% confidential intervals. Dashed lines (A and D) indicate data points from individual patients. Violin plots show levels in NC and C cases. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (F) Correlation of C5a levels with N-specific IgG1 and IgG3. Solid lines indicate linear regression. p values were determined by a Spearman’s rank test. n = 61.

Article Snippet: Mouse anti-Human IgG2 Fc, HRP conjugate , Southern Biotech , Cat# 9070-05, RRID: AB_2796633.

Techniques: Activation Assay, Clinical Proteomics

Journal: Cell Reports

Article Title: Enhanced eosinophil-mediated inflammation associated with antibody and complement-dependent pneumonic insults in critical COVID-19

doi: 10.1016/j.celrep.2021.109798

Figure Lengend Snippet: Pathologic association of immune complex deposition in lung biopsies from six fatal COVID-19 cases (A) Depositions of IgG immune complexes (white) in airways and blood vessels identified by immunofluorescence analysis of lung tissue from a fatal case, P15, at the indicated times after symptom onset. CK, pan-cytokeratin (green); N, SARS-CoV-2 N antigens (red); DIC, differential interference contrast. White arrows indicate depositions of IgG immune complexes in the luminal spaces; white asterisks indicate blood vessels. Nuclei are shown in blue. Scale bar, 100 μm. (B) Depositions of IgG immune complexes in airways and blood vessels identified by immunofluorescence analysis of lung tissues obtained from five fatal cases, P71–P75 (collection day after symptom onset indicated). Scale bars, 100 μm.

Article Snippet: Mouse anti-Human IgG2 Fc, HRP conjugate , Southern Biotech , Cat# 9070-05, RRID: AB_2796633.

Techniques: Immunofluorescence

Journal: Cell Reports

Article Title: Enhanced eosinophil-mediated inflammation associated with antibody and complement-dependent pneumonic insults in critical COVID-19

doi: 10.1016/j.celrep.2021.109798

Figure Lengend Snippet:

Article Snippet: Mouse anti-Human IgG2 Fc, HRP conjugate , Southern Biotech , Cat# 9070-05, RRID: AB_2796633.

Techniques: Control, Clinical Proteomics, Recombinant, Protease Inhibitor, Blocking Assay, Plasmid Preparation, Extraction, Real-time Polymerase Chain Reaction, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Amplification, Polymer, Staining, RNA Sequencing, Activation Assay, Software, Luminex

Journal: Nucleic Acids Research

Article Title: Selection-free precise gene repair using high-capacity adenovector delivery of advanced prime editing systems rescues dystrophin synthesis in DMD muscle cells

doi: 10.1093/nar/gkae057

Figure Lengend Snippet: Gene correction through AdVP-based prime editing in DMD defective myoblasts. ( A ) Genome structures of AdVPs assembled for DMD prime editing. DMD target sequences before and after prime editing are depicted. The hybrid CAG promoter drives PEmax expression whilst the human U6 promoter controls the synthesis of the indicated epegRNAs and gRNAs. Spacer, primer binding site (PBS) and reverse transcriptase template (RTT) sequences of epegRNAs are marked in cyan, orange and magenta, respectively, with encoded and installed edits labelled in green. Protospacer adjacent motifs (NGG) are boxed, and nicking positions are marked by open arrowheads. ITR and Ψ, adenovirus type-5 cis -acting inverted terminal repeats and packaging signal, respectively. ( B ) AdVP-based prime editing in DMD myoblasts. Human myoblasts with a Δ48-50 genotype (DMD.Δ48-50) were transduced with different multiplicities-of-infection (MOI) of AdVP.PE2 DMD.INS+1 , AdVP.PE3 DMD.DEL-2 and AdVP.PE3 DMD.INS+1 ; TU cell −1 , transducing units per cell. Prime edits and unwarranted byproducts (i.e. indels and scaffold-derived insertions) were quantified by next-generation deep sequencing at 10 days post-transduction (50,000 paired-end reads per sample). Bars and error bars denote mean ± SEM, respectively, of three biological replicates. ( C ) Prime-editing precision indexes upon AdVP transduction. Precision indexes corresponding to the cumulative ratios of precise edits to byproduct frequencies measured in AdVP-transduced myoblasts DMD.Δ48-50 (AB1098) are plotted as mean ± SEM of the independent datapoints. Significances were calculated with one-way ANOVA followed by Dunnett's multiple comparison tests; ***0.0001 < P < 0.001, **0.001 < P < 0.01. ( D ) Detection of dystrophin and β-dystroglycan in DMD.Δ48-50 (AB1098) muscle cells prime-edited using AdVPs. Dual-color immunofluorescence microscopy for dystrophin and β-dystroglycan was done on myotubes differentiated from DMD.Δ48–50 myoblasts transduced with the indicated DMD prime-editing AdVPs. Co-localization of dystrophin and β-dystroglycan at the plasma membrane of prime-edited DMD myotubes was assessed by image merging and dystrophin- plus β-dystroglycan-specific fluorescence signal measurements (boxed areas). Nuclei are labeled with DAPI in the merged images. ( E ) Dystrophin-β-dystroglycan interaction analysis in DMD muscle cells prime-edited using AdVPs. Proximity ligation assay detection of endogenous dystrophin-β-dystroglycan interactions was carried out on myotubes differentiated from DMD.Δ48–50 myoblasts transduced with the indicated DMD prime-editing AdVPs (red foci). Healthy donor (wild-type) and untreated DMD patient-derived myotubes served as positive and negative controls, respectively. Nuclei were labelled by DAPI staining.

Article Snippet: Subsequently, the specimens were blocked with Duolink ® Blocking Solution (Sigma-Aldrich; Cat. No.: DUO82007) for 1 h at 37°C in a heated humidity chamber and were then incubated overnight at 4°C with primary antibodies against the C-terminus of dystrophin (1:100 dilution; Abcam; Cat. No.: ab15277) and β-dystroglycan (1:100 dilution; Santa Cruz Biotechnologies; Cat. No.: sc-33702) diluted in Duolink ® Antibody Diluent (Sigma-Aldrich; Cat. No.: DUO82008).

Techniques: Expressing, Binding Assay, Reverse Transcription, Transduction, Infection, Derivative Assay, Sequencing, Comparison, Immunofluorescence, Microscopy, Clinical Proteomics, Membrane, Fluorescence, Labeling, Proximity Ligation Assay, Staining

Journal: Nucleic Acids Research

Article Title: Selection-free precise gene repair using high-capacity adenovector delivery of advanced prime editing systems rescues dystrophin synthesis in DMD muscle cells

doi: 10.1093/nar/gkae057

Figure Lengend Snippet: Gene correction through AdVP-based multiplexing prime editing in DMD defective myoblasts. ( A ) Genome structure of AdVP assembled for DMD gene correction using multiplexing prime editing complexes. ITR and Ψ, adenovirus type-5 cis -acting inverted terminal repeats and packaging signal, respectively. The hybrid CAG promoter drives PEmax synthesis whilst human U6 promoters drive the expression of an epegRNA pair (i.e. epegRNA IN50 and epegRNA IN51 ) for DMD reading frame repair in muscle cells amenable to exon 51 excision (e.g. DMD.Δ48-50). ( B ) Schematics of DMD exon 51 excision through twin prime editing. Spacer, primer binding site (PBS) and reverse transcriptase template (RTT) sequences of epegRNA IN50 and epegRNA IN51 are highlighted in green, orange and magenta, respectively. The latter sequence encodes exogenous genetic information in the form of the serine recombinase Bxb1 attB recognition site. Protospacer adjacent motifs (NGG) are boxed, and nicking positions are marked by open arrowheads. Twin prime editors engage offset protospacer sequences on opposite DNA strands generating nicks that lead to the hybridization of the released single-stranded DNA strands to each PBS. The resulting free 3’ hydroxyl groups prime the synthesis of 3’ DNA flaps over RTT sequences by the reverse transcriptases. After the annealing of 3’ and 5’ DNA flaps containing edited and original DNA sequences (not shown), respectively, removal of the 5’ flaps followed by ligation of the 3’ flaps to the respective DNA excising nicks yields the intended gene-editing product, i.e. replacement of genomic DNA encompassing DMD exon 51 by the Bxb1 attB recognition site. ( C ) Testing AdVP delivery of functional prime-editing multiplexes. Human myoblasts with a Δ48-50 genotype (AB1098) were transduced with AdVP.TwinPE ΔEX51 or AdVP.PE3 DMD.DEL-2 at 50, 100, 200 and 400 TU cell −1 . Twin PE- and PE3-derived prime edits were traced at 3 days post-transduction by DNA densitometry and sequencing of target amplicons, respectively. ( D ) Dystrophin detection in DMD muscle cells corrected via AdVP delivery of prime-editing multiplexes. Western blotting was performed on myotubes differentiated from DMD.Δ48-50 myoblasts previously transduced with the indicated DMD prime-editing AdVPs (sixty micrograms of total protein loaded per lane; 6% SDS-PAGE gel). Detection of vinculin and tubulin provided for independent protein loading controls. ( E ) Dystrophin-β-dystroglycan interaction analysis in DMD muscle cells after AdVP transfer of single and dual prime-editing complexes. Detection of endogenous dystrophin-β-dystroglycan interactions by proximity ligation assays on myotubes differentiated from DMD.Δ48–50 myoblasts transduced with the indicated DMD prime-editing AdVPs (red foci). Parallel cultures of untreated DMD.Δ48–50 myotubes (Mock) served as negative controls. Nuclei were labelled by DAPI staining.

Article Snippet: Subsequently, the specimens were blocked with Duolink ® Blocking Solution (Sigma-Aldrich; Cat. No.: DUO82007) for 1 h at 37°C in a heated humidity chamber and were then incubated overnight at 4°C with primary antibodies against the C-terminus of dystrophin (1:100 dilution; Abcam; Cat. No.: ab15277) and β-dystroglycan (1:100 dilution; Santa Cruz Biotechnologies; Cat. No.: sc-33702) diluted in Duolink ® Antibody Diluent (Sigma-Aldrich; Cat. No.: DUO82008).

Techniques: Multiplexing, Expressing, Binding Assay, Reverse Transcription, Sequencing, Hybridization, Ligation, Functional Assay, Transduction, Derivative Assay, Western Blot, SDS Page, Staining

Journal: Cell Death & Disease

Article Title: BCLAF1 is a radiation-induced H2AX-interacting partner involved in γ H2AX-mediated regulation of apoptosis and DNA repair

doi: 10.1038/cddis.2012.76

Figure Lengend Snippet: Proteomic identification of IR-induced dose-dependent γ H2AX-interacting partners including BCLAF1. ( a ) Workflow of AACT-based multiplex dual-tagging quantitative proteomic approach. The 293T cells stably expressing FLAG-tagged H2AX grown in either 12 C 6 -arginine (L), or 13 C 6 -arginine (M), or 13 C 6 15 N 4 -arginine (H)-containing medium were subjected to IR at the dose level of 0, 7.5 cGy, and 10 Gy, respectively. The immunoprecipitates (IP) originated from each of the three cell pools were mixed at 1 : 1 : 1 ratio based on the amount of FLAG-tagged H2AX in each IP elute as measured by immunoblotting (IB) prior to mixing. The equally combined IP elutes were then separated by SDS-PAGE, digested with trypsin, and analyzed by nanoLC-MS/MS. ( b ) MS spectra of AACT-containing peptides of the representative proteins detected in the IP mixture. Peptide sequences include IDISPSTLR (BCLAF1), SILQER (Ku86), SDSFENPVLQQHFR (Ku70), LLEEALLR (DNA-PKcs). The relative abundance of the bait protein, H2AX, in each immunoprecipitate, was measured based on the ratio of isotope peak intensity, and the ratio was calibrated to 1 : 1 : 1 for the normalization of those of other H2AX-interacting proteins. ( c ) Immunoblot analysis of the immunoprecipitates pulled down from non-irradiated or 7.5-cGy- or 10-Gy-irradiated cells by anti- γ H2AX antibody. A mouse IgG was used as the control. ( d ) Immunofluorescence analysis of IR dose-dependent BCLAF1- γ H2AX association in situ. The 293T cells were irradiated at indicated dose level and were then incubated for 1 h prior to fixing and staining with indicated antibodies (lower panel) with both anti-mouse and -rabbit IgG antibodies as the control. 4,6-diamidino-2-phenylindole (DAPI) was used for DNA staining (top panel). The scale bar is 24 μ m

Article Snippet: Nuclear extracts and immunoprecipitates were obtained, separated, and transferred as described previously, The PVDF membranes (Bio-Rad, Hercules, CA, USA) were blotted respectively with a rabbit polyclonal antibody against BCLAF1 (A300-608A, BETHYL laboratories, Inc., Montgomery, TX, USA), and Histone H3S10ph (ab47297, Abcam, Cambridge, MA, USA), and DNA-PKcs (4602), cleaved Caspase-3 (Asp175) (9664), Caspase 8 (4927) and Bax (2772) (Cell Signaling Technology, Danvers, MA, USA) and Cyclin D1 (H-295) (sc-753) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); goat-polyclonal antibody against β -actin (sc-1615) (Santa Cruz Biotechnology, Inc.) and GAPDH (IMG-3073) (Imgenex Inc., San Diego, CA, USA); mouse monoclonal antibody against FLAG M2 (F1804), BrdU (B8434) and p21 (P1484) (Sigma), γ H2AX (ab22551) (Abcam) and, PARP1 (sc-8007), p53 (DO-1) (sc-126), Bcl-2 (C-2) (sc-7382) and Ku70 (N3H10) (sc-56129) (Santa Cruz Biotechnology, Inc.) and Caspase-9 (9508) (Cell Signaling Technology) and Cyclin E (Ab-2) (MS-870-P0) (Thermo Scientific, Inc., Waltham, MA, USA) and 53BP1 (612522) (BD, San Jose, CA, USA).

Techniques: Multiplex Assay, Stable Transfection, Expressing, Western Blot, SDS Page, Tandem Mass Spectroscopy, Irradiation, Control, Immunofluorescence, In Situ, Incubation, Staining

Journal: Cell Death & Disease

Article Title: BCLAF1 is a radiation-induced H2AX-interacting partner involved in γ H2AX-mediated regulation of apoptosis and DNA repair

doi: 10.1038/cddis.2012.76

Figure Lengend Snippet: BCLAF1 regulates the subcellular-specific feedback loop in balancing apoptosis and cell cycle progression and DNA-PKcs regulates the formation of BCLAF1 foci. ( a ) Effect of BCLAF1 on the formation of DSB-indicative γ H2AX and 53BP1 foci. A549 cells were harvested at 1 h after a defined IR and the percentage of γ H2AX and 53BP1 foci-positive cells was determined by analyzing 100 randomly chosen cells and the number of γ H2AX and 53BP1 foci per cell was determined on a cell-to-cell basis by the quantitative analysis of at least 30 cells randomly chosen as previously described. Error bars represent S.D. of a technical duplicate. ** P <0.05, * P <0.01. ( b ) Immunoblot analysis of the effect of BCLAF1 on Ku70 associations with DNA-PKcs in differentially irradiated A549 cells. Relative intensities representing the binding strength between DNA-PKcs and Ku70 measured by ImageJ (version 1.4.5q) are indicated as italic numbers. Data represent the average of two independent experiments. P <0.05. ( c ) The Ku70 immunoprecipitates were isolated from cytosolic and nuclear fractions of non-irradiated, IR-responsive, -resistant cells by using an agarose-conjugated Ku70 antibody. Individual complex components were immunoblotted with indicated antibodies. Relative intensities of interaction strength between DNA-PKcs, p18-cyclin E, Bax and Bcl-2, and Ku70 measured by ImageJ (version 1.4.5q) are indicated as italic numbers and separately measured for either nuclear or Cytosolic fraction. Data are from two independent experiments. P <0.01. ( d ) For each radiation condition without or with BCLAF1 knockdown, the cells were harvested after 1- or 8-h recovery time. Quantitative measurements of γ H2AX were performed as described in the Materials and Methods section. One experiment containing a technical duplicate representative of two is shown with the data precision indicated by the error bar based on mean±S.D. of duplicate samples. ** P <0.05, * P <0.01. ( e ) Immunostaining of IR-induced BCLAF1 foci in the DNA-PKcs wild-type ( DNA-PKcs +/− ) and null ( DNA-PKcs −/− ) MEF cells under either IR-responsive or -resistant condition. DAPI was used to stain DNA. More than 200 cells from each condition were examined (scale bar is 8 μ m)

Article Snippet: Nuclear extracts and immunoprecipitates were obtained, separated, and transferred as described previously, The PVDF membranes (Bio-Rad, Hercules, CA, USA) were blotted respectively with a rabbit polyclonal antibody against BCLAF1 (A300-608A, BETHYL laboratories, Inc., Montgomery, TX, USA), and Histone H3S10ph (ab47297, Abcam, Cambridge, MA, USA), and DNA-PKcs (4602), cleaved Caspase-3 (Asp175) (9664), Caspase 8 (4927) and Bax (2772) (Cell Signaling Technology, Danvers, MA, USA) and Cyclin D1 (H-295) (sc-753) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); goat-polyclonal antibody against β -actin (sc-1615) (Santa Cruz Biotechnology, Inc.) and GAPDH (IMG-3073) (Imgenex Inc., San Diego, CA, USA); mouse monoclonal antibody against FLAG M2 (F1804), BrdU (B8434) and p21 (P1484) (Sigma), γ H2AX (ab22551) (Abcam) and, PARP1 (sc-8007), p53 (DO-1) (sc-126), Bcl-2 (C-2) (sc-7382) and Ku70 (N3H10) (sc-56129) (Santa Cruz Biotechnology, Inc.) and Caspase-9 (9508) (Cell Signaling Technology) and Cyclin E (Ab-2) (MS-870-P0) (Thermo Scientific, Inc., Waltham, MA, USA) and 53BP1 (612522) (BD, San Jose, CA, USA).

Techniques: Western Blot, Irradiation, Binding Assay, Isolation, Knockdown, Immunostaining, Staining

Journal: Cell Death & Disease

Article Title: BCLAF1 is a radiation-induced H2AX-interacting partner involved in γ H2AX-mediated regulation of apoptosis and DNA repair

doi: 10.1038/cddis.2012.76

Figure Lengend Snippet: A mechanistic model illustrating the multiple roles of BCLAF1 in regulation of apoptosis, cell cycle progression, and NHEJ DBS repair. In IR-responsive cells exposed to a high-dose radiation that activates DNA-PKcs, BCLAF1 re-localizes to the nuclear envelope through site-specific phosphorylation by DNA-PKcs and then emerges with γ H2AX foci, which in turn stabilizes the Ku70/DNA-PKcs complex for facilitating NHEJ DSB repair. Meanwhile, BCLAF1 regulates both cyclin E and p53/p21-dependent mitochondria-mediated pathway(s) for promoting pro-apoptotic activity of Caspase-3, leading to the generation of an apoptotic amplifier, p18-cyclin E, followed by Ku70-mediated liberation of active Bax. In contrast, in the IR-resistant carcinoma cells BCLAF1 is intrinsically suppressed, leading to weakened Ku70/DNA-PKcs complex but enhanced associations of Ku70/BCL-2/p18-Cyclin E instead, subsequently reducing the activity of DSB repair. In the cytosol, suppressed BCLAF1 could result in more Ku70-bound Bax, which inhibits the formation of pro-apoptotic Ku70-p18-Cyclin E complex

Article Snippet: Nuclear extracts and immunoprecipitates were obtained, separated, and transferred as described previously, The PVDF membranes (Bio-Rad, Hercules, CA, USA) were blotted respectively with a rabbit polyclonal antibody against BCLAF1 (A300-608A, BETHYL laboratories, Inc., Montgomery, TX, USA), and Histone H3S10ph (ab47297, Abcam, Cambridge, MA, USA), and DNA-PKcs (4602), cleaved Caspase-3 (Asp175) (9664), Caspase 8 (4927) and Bax (2772) (Cell Signaling Technology, Danvers, MA, USA) and Cyclin D1 (H-295) (sc-753) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); goat-polyclonal antibody against β -actin (sc-1615) (Santa Cruz Biotechnology, Inc.) and GAPDH (IMG-3073) (Imgenex Inc., San Diego, CA, USA); mouse monoclonal antibody against FLAG M2 (F1804), BrdU (B8434) and p21 (P1484) (Sigma), γ H2AX (ab22551) (Abcam) and, PARP1 (sc-8007), p53 (DO-1) (sc-126), Bcl-2 (C-2) (sc-7382) and Ku70 (N3H10) (sc-56129) (Santa Cruz Biotechnology, Inc.) and Caspase-9 (9508) (Cell Signaling Technology) and Cyclin E (Ab-2) (MS-870-P0) (Thermo Scientific, Inc., Waltham, MA, USA) and 53BP1 (612522) (BD, San Jose, CA, USA).

Techniques: Phospho-proteomics, Activity Assay

Journal: World Journal of Biological Chemistry

Article Title: Current understanding of glucose transporter 4 expression and functional mechanisms

doi: 10.4331/wjbc.v11.i3.76

Figure Lengend Snippet: Summary of glucose transporter family members

Article Snippet: Western blot. , Whole cell and cell fractions from rat L6 and mouse C2C12 muscle cells, and soleus muscle of hind limb from mice. Anti-GLUT4 from Santa Cruz. Biotechnology (1:1000). , Whole cell lysate vs membrane fractions. Treatments without or with insulin or AICAR. , GLUT4 translocation occurs in L6 myotubes and 3T3-L1 adipocytes stimulated by insulin and AICAR. GLUT4 translocation occurs in muscle at 15 to 30 minutes and in adipose tissue at 15 minutes after glucose treatment. , [ ] .

Techniques:

Journal: World Journal of Biological Chemistry

Article Title: Current understanding of glucose transporter 4 expression and functional mechanisms

doi: 10.4331/wjbc.v11.i3.76

Figure Lengend Snippet: Schematic of insulin-induced translocation of glucose transporter 4 from cytosol to the cell membrane. The binding of insulin to its receptors initiates a signal transduction cascade, which results in the activation of Akt. Akt acts on the glucose transporter 4 (GLUT4) containing vesicles in the cytosol to facilitate their fusion with the cell membrane. When more GLUT4 molecules are present in the membrane, the rate of glucose uptake is elevated. GLUT4: Glucose transporter 4.

Article Snippet: Western blot. , Whole cell and cell fractions from rat L6 and mouse C2C12 muscle cells, and soleus muscle of hind limb from mice. Anti-GLUT4 from Santa Cruz. Biotechnology (1:1000). , Whole cell lysate vs membrane fractions. Treatments without or with insulin or AICAR. , GLUT4 translocation occurs in L6 myotubes and 3T3-L1 adipocytes stimulated by insulin and AICAR. GLUT4 translocation occurs in muscle at 15 to 30 minutes and in adipose tissue at 15 minutes after glucose treatment. , [ ] .

Techniques: Translocation Assay, Binding Assay, Transduction, Activation Assay

Journal: World Journal of Biological Chemistry

Article Title: Current understanding of glucose transporter 4 expression and functional mechanisms

doi: 10.4331/wjbc.v11.i3.76

Figure Lengend Snippet: Recent studies of glucose transporter 4 expression and translocation in the skeletal muscle

Article Snippet: Western blot. , Whole cell and cell fractions from rat L6 and mouse C2C12 muscle cells, and soleus muscle of hind limb from mice. Anti-GLUT4 from Santa Cruz. Biotechnology (1:1000). , Whole cell lysate vs membrane fractions. Treatments without or with insulin or AICAR. , GLUT4 translocation occurs in L6 myotubes and 3T3-L1 adipocytes stimulated by insulin and AICAR. GLUT4 translocation occurs in muscle at 15 to 30 minutes and in adipose tissue at 15 minutes after glucose treatment. , [ ] .

Techniques: Expressing, Translocation Assay, Western Blot, Real-time Polymerase Chain Reaction, Negative Control, Positive Control, Immunoprecipitation, Isolation, Transgenic Assay, Immunofluorescence, Microscopy, Electron Microscopy, Injection, In Vitro, Activation Assay, Labeling, Protease Inhibitor

Journal: World Journal of Biological Chemistry

Article Title: Current understanding of glucose transporter 4 expression and functional mechanisms

doi: 10.4331/wjbc.v11.i3.76

Figure Lengend Snippet: The movement of glucose transporter 4 in adipocytes. Adipose tissue is made of adipocytes. In adipocytes, glucose transporter 4 (GLUT4) can be found in the cell membrane and in the cytosol. The translocation of GLUT4 from cytosolic vesicles to the cell membrane leads to elevated glucose uptake, whereas endocytosis brings GLUT4 back to the cytosol. ( 1): In unstimulated cells, GLUT4 containing membrane portions are internalized in an endocytosis manner to generate vesicles containing GLUT4. GLUT4 vesicles are internalized into early (or sorted) endosomes. They can enter the recovery endoplasmic body, and follow the retrograde pathway to the trans-Golgi network and endoplasmic reticulum-Golgi intermediate compartment or other donor membrane compartments. (2): GLUT4 vesicles derived from the donor membrane structures are secured by tether containing a UBX domain for GLUT4 (TUG) protein. (3): During insulin signal stimulation, GLUT4 vesicles are released and loaded onto the microtubule motor to be transferred to the plasma membrane. The continuous presence of insulin leads to the direct movement of these vesicles to the plasma membrane. (4): GLUT4 vesicles are tethered to motor protein kinesin and other proteins. A stable ternary SNARE complex forms when this occurs. (5): The stable ternary SNARE complex is docked on the target membrane. (6): The docked vesicles rely on SNARE to move to and fuse with the target membrane[ , , ]. GLUT4: Glucose transporter 4.

Article Snippet: Western blot. , Whole cell and cell fractions from rat L6 and mouse C2C12 muscle cells, and soleus muscle of hind limb from mice. Anti-GLUT4 from Santa Cruz. Biotechnology (1:1000). , Whole cell lysate vs membrane fractions. Treatments without or with insulin or AICAR. , GLUT4 translocation occurs in L6 myotubes and 3T3-L1 adipocytes stimulated by insulin and AICAR. GLUT4 translocation occurs in muscle at 15 to 30 minutes and in adipose tissue at 15 minutes after glucose treatment. , [ ] .

Techniques: Translocation Assay, Derivative Assay

Journal: World Journal of Biological Chemistry

Article Title: Current understanding of glucose transporter 4 expression and functional mechanisms

doi: 10.4331/wjbc.v11.i3.76

Figure Lengend Snippet: Recent studies of effects of bioactive compounds and chemical drugs on glucose transporter 4 expression and translocation in adipocytes

Article Snippet: Western blot. , Whole cell and cell fractions from rat L6 and mouse C2C12 muscle cells, and soleus muscle of hind limb from mice. Anti-GLUT4 from Santa Cruz. Biotechnology (1:1000). , Whole cell lysate vs membrane fractions. Treatments without or with insulin or AICAR. , GLUT4 translocation occurs in L6 myotubes and 3T3-L1 adipocytes stimulated by insulin and AICAR. GLUT4 translocation occurs in muscle at 15 to 30 minutes and in adipose tissue at 15 minutes after glucose treatment. , [ ] .

Techniques: Expressing, Translocation Assay, Fluorescence, Immunostaining, Western Blot, Inhibition, Immunoprecipitation, Real-time Polymerase Chain Reaction, Electrophoretic Mobility Shift Assay, Immunofluorescence, In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Activity Assay, Produced, Microscopy, Plasmid Preparation, Flow Cytometry

Journal: World Journal of Biological Chemistry

Article Title: Current understanding of glucose transporter 4 expression and functional mechanisms

doi: 10.4331/wjbc.v11.i3.76

Figure Lengend Snippet: Recent studies of mechanisms of glucose transporter 4 expression and translocation in adipocytes

Article Snippet: Western blot. , Whole cell and cell fractions from rat L6 and mouse C2C12 muscle cells, and soleus muscle of hind limb from mice. Anti-GLUT4 from Santa Cruz. Biotechnology (1:1000). , Whole cell lysate vs membrane fractions. Treatments without or with insulin or AICAR. , GLUT4 translocation occurs in L6 myotubes and 3T3-L1 adipocytes stimulated by insulin and AICAR. GLUT4 translocation occurs in muscle at 15 to 30 minutes and in adipose tissue at 15 minutes after glucose treatment. , [ ] .

Techniques: Expressing, Translocation Assay, Real-time Polymerase Chain Reaction, Electrophoretic Mobility Shift Assay, Immunohistochemistry, Western Blot, Transfection, Northern Blot, Nuclear Run-on Assay, Plasmid Preparation, Flow Cytometry, Fluorescence, Microscopy, Over Expression, Isolation, Concentration Assay, Immunofluorescence

Journal: World Journal of Biological Chemistry

Article Title: Current understanding of glucose transporter 4 expression and functional mechanisms

doi: 10.4331/wjbc.v11.i3.76

Figure Lengend Snippet: Recent studies of glucose transporter 4 expression and translocation in the heart

Article Snippet: Western blot. , Whole cell and cell fractions from rat L6 and mouse C2C12 muscle cells, and soleus muscle of hind limb from mice. Anti-GLUT4 from Santa Cruz. Biotechnology (1:1000). , Whole cell lysate vs membrane fractions. Treatments without or with insulin or AICAR. , GLUT4 translocation occurs in L6 myotubes and 3T3-L1 adipocytes stimulated by insulin and AICAR. GLUT4 translocation occurs in muscle at 15 to 30 minutes and in adipose tissue at 15 minutes after glucose treatment. , [ ] .

Techniques: Expressing, Translocation Assay, Positive Control, Inhibition, Activation Assay, Isolation, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Immunohistochemistry

Journal: World Journal of Biological Chemistry

Article Title: Current understanding of glucose transporter 4 expression and functional mechanisms

doi: 10.4331/wjbc.v11.i3.76

Figure Lengend Snippet: Recent studies of glucose transporter 4 expression and translocation in the brain

Article Snippet: Western blot. , Whole cell and cell fractions from rat L6 and mouse C2C12 muscle cells, and soleus muscle of hind limb from mice. Anti-GLUT4 from Santa Cruz. Biotechnology (1:1000). , Whole cell lysate vs membrane fractions. Treatments without or with insulin or AICAR. , GLUT4 translocation occurs in L6 myotubes and 3T3-L1 adipocytes stimulated by insulin and AICAR. GLUT4 translocation occurs in muscle at 15 to 30 minutes and in adipose tissue at 15 minutes after glucose treatment. , [ ] .

Techniques: Expressing, Translocation Assay, Knock-Out, Western Blot, Cell Culture, Activity Assay, Immunocytochemistry, Real-time Polymerase Chain Reaction, Immunofluorescence, Immunohistochemistry, Positive Control, Negative Control, Microscopy, Transgenic Assay