Journal: Cancers

Article Title: Role of Calcium Signaling in GA101-Induced Cell Death in Malignant Human B Cells

doi: 10.3390/cancers11030291

Figure Lengend Snippet: Effect of GA101 on intracellular Ca 2+ concentration in SU-DHL-4 ( A ) and BL2 ( B ) cell lines. Ca 2+ responses to GA101 (10 µg/mL) were measured using Fluo2-Leak Resistant-Acetoxy Methyl ester (Fluo2-LR-AM) Ca 2+ dye and recorded by videomicroscopy (Zeiss LSM 510) using a 25× objective. Black arrows indicate GA101 addition. Each trace represents the response of one cell and data are representative of at least three independent experiments. Data were processed using OriginPro 7.5 (Origin Lab) or GraphPad prism. Cells were recorded in extracellular Hank’s Balanced Salt Solution (HBSS) containing 2 mM Ca 2+ (2Ca) or in Ca 2+ -free HBSS (0Ca). Cells were preincubated with 100 nM thapsigargin (TG) for 45 min and recorded in Ca 2+ -free HBSS (0Ca + TG) or with 10 µM Ned-19 for 1 h and recorded in Ca 2+ -free HBSS (0Ca + Ned19). Calcium responses to GA101 in cells expressing Non Targeting shRNA (sh NT) or sh Orai1 were recorded in HBSS containing 2 mM Ca 2+ . Histograms represent areas under curves (AUC) calculated, under various recording conditions, between the application time of GA101 and t = 2000 s; * p < 0.05.

Article Snippet: Anti-human Orai1 rabbit polyclonal antibody was from Alomone Labs (Jerusalem, Israel).

Techniques: Concentration Assay, Expressing, shRNA

Journal: Cancers

Article Title: Role of Calcium Signaling in GA101-Induced Cell Death in Malignant Human B Cells

doi: 10.3390/cancers11030291

Figure Lengend Snippet: Involvement of store-operated Ca 2+ entry (SOCE) in GA101-induced cell death. ( A ) BL2 cells. ( B ) SU-DHL-4 cells. Left panels: Cells were incubated with GA101 in the presence or absence of BTP2 (10 µM) for 24 h. Right panels: Cells expressing sh NT or sh Orai1 were treated with GA101 for 24 h. Cell death was assessed by measuring the loss of mitochondrial membrane potential (Δψm), using tetramethylrhodamine methyl ester (TMRM) as a fluorescent dye, or by caspase 3 activation, measured by the FAM-FLICA in vitro caspase detection kit and both analyzed by flow cytometry; * p < 0.05.

Article Snippet: Anti-human Orai1 rabbit polyclonal antibody was from Alomone Labs (Jerusalem, Israel).

Techniques: Incubation, Expressing, Activation Assay, In Vitro, Flow Cytometry

Journal: Cancers

Article Title: Role of Calcium Signaling in GA101-Induced Cell Death in Malignant Human B Cells

doi: 10.3390/cancers11030291

Figure Lengend Snippet: Differential activation of endoplasmic reticulum (ER) stress in SU-DHL-4 and BL2 cell lines. ( A ) Cells expressing sh NT or sh Orai1 were treated with GA101 (1 µg/mL) for varying lengths of time. After lysis, phosphorylated eukaryotic translation initiation factor alpha (P-eIF2α), eIF2α, and BIM expression levels were assessed by immunoblot analysis. GAPDH was used as a loading control. ( B ) Quantification of Western blots is given as means ± SE of three to five independent experiments; * p < 0.05.

Article Snippet: Anti-human Orai1 rabbit polyclonal antibody was from Alomone Labs (Jerusalem, Israel).

Techniques: Activation Assay, Expressing, Lysis, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Oxytocin Protects Against Isoproterenol-Induced Cardiac Hypertrophy by Inhibiting PI3K/AKT Pathway via a lncRNA GAS5/miR-375-3p/KLF4-Dependent Mechanism

doi: 10.3389/fphar.2021.766024

Figure Lengend Snippet: Antihypertrophic effects of OT in vitro . Neonatal rat cardiomyocytes stimulated with ISO for 24 h in the presence or absence of OT. (A) The cell morphology was evaluated by H&E staining. (B) Statistical results of measurement of cell surface areas. (C,D) Effects of OT on the protein expressions of BNP and β-MHC. (E) Effects of OT on the expression of lncRNA GAS5. (F) Effects of OT on the expression of miR-375-3p. (G,H) Effects of OT on the mRNA and protein expressions of KLF4. (I) Effects of OT on the p-PI3K/PI3K ratio. (J) Effects of OT on the p-AKT1/AKT1 ratio. (K) Western blot images of BNP, β-MHC, KLF4, p-PI3K, PI3K, AKT1, p-AKT1, and β-actin levels. Data are shown as the mean ± sd of three independent experiments. *, p < 0.05; **, p < 0.01.

Article Snippet: The blots were blocked with 5% non-fat milk and incubated overnight with the primary antibodies against BNP (1:500, rabbit #ab19645 abcam), β-MHC (1:1,000, rabbit #ab172967 abcam), KLF4 (1:1,000, rabbit #bs-1064R BIOSS), PI3K (1:1,000, rabbit #ab182651 abcam), p-PI3K (1:1,000, rabbit #205841-1-AP Proteintech), AKT1 (1:1,000, rabbit # bs-0115M BIOSS), p-AKT1 (1:1,000, rabbit # bs-0876R BIOSS), β-actin (1:1,000, rabbit #abmart P30002) and GAPDH (1:2,000, rabbit #2118S CST).

Techniques: In Vitro, Staining, Expressing, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Oxytocin Protects Against Isoproterenol-Induced Cardiac Hypertrophy by Inhibiting PI3K/AKT Pathway via a lncRNA GAS5/miR-375-3p/KLF4-Dependent Mechanism

doi: 10.3389/fphar.2021.766024

Figure Lengend Snippet: Over-expression of miR-375-3p blunted anti-hypertrophic effects of oxytocin via KLF4 and modulated the PI3K/AKT signaling pathway. (A) Expression of miR-375-3p in cardiomyocytes following transfection of miR-375-3p mimics and miR-375-3p mimics NC. (B) Expression of miR-375-3p was detected by qRT-PCR. (C,D) Expressions of BNP and β-MHC proteins were measured by Western blot analysis. (E) Expression of KLF4 mRNA was detected by qRT-PCR. (F) Expression of KLF4 protein was measured by Western blot analysis. (G) p-PI3K/PI3K ratio. (H) p-AKT1/AKT1 ratio. (I) Statistical results of measurement of cell surface areas. (J) Representative western blot images of KLF4, PI3K, p-PI3K, AKT1, p-AKT1, BNP, β-MHC and GAPDH levels. (K) Cardiomyocyte surface areas were measured by immunofluorescent staining. Scale bars represent 20 µm. Images were captured at ×400 magnification. (a) ISO+OT+miR-375-3p mimics NC group. (b) ISO+OT+miR-375-3p mimics group. (c) ISO+OT+miR-375-3p mimics+pcDNA-NC group. (d) ISO+OT+miR-375-3p mimics+pcDNA-KLF4 group. Data are shown as the mean ± sd of three independent experiments. *, p < 0.05; **, p < 0.01.

Article Snippet: The blots were blocked with 5% non-fat milk and incubated overnight with the primary antibodies against BNP (1:500, rabbit #ab19645 abcam), β-MHC (1:1,000, rabbit #ab172967 abcam), KLF4 (1:1,000, rabbit #bs-1064R BIOSS), PI3K (1:1,000, rabbit #ab182651 abcam), p-PI3K (1:1,000, rabbit #205841-1-AP Proteintech), AKT1 (1:1,000, rabbit # bs-0115M BIOSS), p-AKT1 (1:1,000, rabbit # bs-0876R BIOSS), β-actin (1:1,000, rabbit #abmart P30002) and GAPDH (1:2,000, rabbit #2118S CST).

Techniques: Over Expression, Expressing, Transfection, Quantitative RT-PCR, Western Blot, Staining

Journal: Frontiers in Pharmacology

Article Title: Oxytocin Protects Against Isoproterenol-Induced Cardiac Hypertrophy by Inhibiting PI3K/AKT Pathway via a lncRNA GAS5/miR-375-3p/KLF4-Dependent Mechanism

doi: 10.3389/fphar.2021.766024

Figure Lengend Snippet: knock-down of KLF4 blunted anti-hypertrophic effects of oxytocin via PI3K/AKT pathway. (A–C) Detection of relative shRNA-KLF4 interference effects. The changes in KLF4 mRNA and protein levels were detected by qRT-PCR and Western blotting after transfection of shRNA-KLF4 #1, shRNA-KLF4 #2, shRNA-KLF4 #3 in primary cardiomyocytes. (D) Expression of KLF4 mRNA was determined by qRT-PCR. (E) Expression of KLF4 protein was measured by Western blot analysis. (F,G) Expressions of BNP, β-MHC proteins were measured by Western blot analysis. (H) p-PI3K/PI3K ratio. (I) p-AKT1/AKT1 ratio. (J) Statistical results of measurement of cell surface areas. (K) Cardiomyocyte surface areas were measured by immunofluorescent staining. Scale bars represent 20 µm. Images were captured at ×400 magnification. (L) Western blot images of KLF4, PI3K, p-PI3K, AKT1, p-AKT1, BNP, β-MHC and GAPDH levels. (a) ISO+OT+shRNA-NC group. (b) ISO+OT+shRNA-KLF4 group. (c) ISO+OT+shRNA-KLF4+LY194002 group. Data are shown as the mean ± sd of three independent experiments. *, p < 0.05; **, p < 0.01.

Article Snippet: The blots were blocked with 5% non-fat milk and incubated overnight with the primary antibodies against BNP (1:500, rabbit #ab19645 abcam), β-MHC (1:1,000, rabbit #ab172967 abcam), KLF4 (1:1,000, rabbit #bs-1064R BIOSS), PI3K (1:1,000, rabbit #ab182651 abcam), p-PI3K (1:1,000, rabbit #205841-1-AP Proteintech), AKT1 (1:1,000, rabbit # bs-0115M BIOSS), p-AKT1 (1:1,000, rabbit # bs-0876R BIOSS), β-actin (1:1,000, rabbit #abmart P30002) and GAPDH (1:2,000, rabbit #2118S CST).

Techniques: shRNA, Quantitative RT-PCR, Western Blot, Transfection, Expressing, Staining

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) Identification of RNF168-associated proteins. A representative SDS–polyacrylamide gel electrophoresis of Flag-RNF168-associated proteins. Flag-tagged RNF168 was transfected in HEK293T cells and pull-down analysis was performed 48 h later. Protein bands were detected by silver staining. Protein bands were identified by mass spectrometry analysis following in-gel protease digestion. ( b ) HEK293T cells were transfected as indicated with HA-tagged RNF168 and Flag-TOP2α expression vectors. Cells were lysed and IP was performed using anti-Flag antibody. The resulting precipitates were subjected to IB analysis with the indicated antibodies. WCL, whole-cell lysate. ( c ) TOP2α, RNF168 and IgG (control) immunoprecipitates from HEK293T cells were examined by IB as indicated. ( b , c ) Data are representative of three independent experiments. ( d ) Cells treated with EdU were used for detection of localization patterns of TOP2α (Alexa Fluor 488) and RNF168 (Alexa Fluor 594) using confocal microscopy. Scale bar, 20 μm.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Polyacrylamide Gel Electrophoresis, Transfection, Silver Staining, Mass Spectrometry, Expressing, Control, Confocal Microscopy

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) Representative agarose gel of in vitro kinetoplast DNA-based decatenation assays performed for 10 min with different amount of nuclear extracts from WT and Rnf168 −/− MEFs. Catenated and decatenated kDNA were separated by electrophoresis using 1% agarose gel. IB show Top2α's level in the total nuclear extracts used for this assay. ( b ) Representative data of the mitotic inhibition assay of decatenation G2 checkpoint in WT and Rnf168 −/− primary MEFs. Cells were treated with DMSO or ICRF-193 for 15 min and then incubated in culture media for an additional 2 h. The fraction of mitotic cells (pHH3 + ) was determined by flow cytometry. ( c ) Bar graphs represent the mean inhibition of mitotic index 2 h post ICRF-193 treatment of passage 1 primary MEFs (% pHH3 + cells post ICRF-193 treatment compared with DMSO-treated controls). * P <0.05. ( d ) Bar graphs represent the mean inhibition of mitotic index 2 h post ICRF-193 treatment of RIDDLE cells reconstituted with either HA-RNF168 or HA-empty vector as in c . * P <0.05. ( e ) Analysis of decatenation G2 checkpoint of Rnf168 −/− and WT 3T3 MEFs using the mitotic entry assay. Percentage of pHH3 + cells is shown at the indicated time post-treatment with colcemid in the presence of DMSO or ICRF-193. * P <0.05; Rnf168 −/− MEFs compared with WT MEFs 6 h post-ICRF-193 treatment. ( f ) Bar graphs represent the mean fraction of pHH3 + WT and Rnf168 −/− MEFs evading G2 arrest 6 h post-treatment with ICRF-193 compared with DMSO-treated cells as in e . * P <0.05. ( g ) Mitotic entry assay of decatenation G2 checkpoint in human RIDDLE cells reconstituted with HA-empty vector or HA-RNF168. Percentage of pHH3 + cells is shown at the indicated times post-treatment with colcemid with or without ICRF-193. * P <0.05, RIDDLE cells reconstituted with HA-empty vector compared with RIDDLE cells reconstituted with HA-RNF168 at 6 h post-ICRF-193 treatment. ( h ) Bar graphs represent the mean fraction of RIDDLE cells (reconstituted with HA-empty vector compared with those reconstituted with HA-RNF168) evading G2 arrest 6 h post ICRF-193 treatment compared with DMSO-treated controls as in f . * P <0.05. Three independent experiments in triplicates unless specified. Error bars in c , d , f and h represent mean±s.e.m.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Agarose Gel Electrophoresis, In Vitro, Electrophoresis, Inhibition, Incubation, Flow Cytometry, Plasmid Preparation

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a – d ) Sensitivity of WT and Rnf168 −/− MEFs to ICRF-193 ( a , b ) or etoposide ( c , d ) was determined using clonogenic assays. ( e , f ) Sensitivity of the human breast cancer cell lines T47D and MDA-MB-231 to etoposide was determined using clonogenic assays. ( a , c , e ) Data are presented as the mean±s.e.m. ( a , c , n >4; e , n =4). * P <0.05 for Rnf168 −/− MEFs compared with WT MEFs and T47D and MDA-MB-231 cells knocked down for RNF168 (sh.RNF168) compared with their respective controls (Sh.Ctr: ShRNA control). ( b , d ) Representative pictures of dishes showing surviving WT and Rnf168 −/− colonies post ICRF-193 ( b ) or etoposide ( d ) treatment. ( f ) Representative pictures of dishes showing surviving colonies of etoposide treated T47D and MDA-MB-231 cells.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: shRNA, Control

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) RIDDLE cells reconstituted with HA-RNF168 or HA-empty vector, and control HA-RNF168-reconstituted RIDDLE cells with TOP2α knock down were lysed and WCL subjected to IP with anti-TOP2α or IgG (control) antibodies. Immunoprecipitates were blotted with the indicated antibodies to detect ubiquitylated TOP2α. ( b ) Human breast cancer cell lines T47D and MDA-MB-231 knocked down for RNF168 (Sh.RNF168) and their control expressing ShRNA control (sh.Ctr) were examined for their level of ubiquitylated TOP2α as in a . ( c ) Rnf168 −/− , Brca1 −/− and WT MEFs were lysed and subjected to IP with anti-Top2α or IgG (control) antibodies. IPs from WCL were blotted with the indicated antibodies. ( d ) HEK293T cells were transfected with RNF168 (WT or mutant Rnf168-C21S), Flag-TOP2α and HA-Ub vectors as indicated. WCL were subjected to IP with anti-Flag, and IB analysis was performed using anti-HA antibody to detect ubiquitylated Flag-TOP2α. ( e ) Nuclear extracts from RIDDLE cells reconstituted with HA-RNF168 or HA-empty vector were subjected to IP with anti-TOP2α or IgG (control) antibodies. Immunoprecipitates were blotted with the indicated antibodies against K63- and K48-Ub linkages. ( f ) In vitro ubiquitylation of recombinant TOP2α in the presence of recombinant RNF168 (500 ng for lane 5, 1 μg for lanes 2, 3 and 6 and 2 μg for lane7), UBE1 (E1), UBE2E2 (E2) and Ub. Nuc, nuclear extract; WCL, whole-cell lysate.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Plasmid Preparation, Control, Knockdown, Expressing, shRNA, Transfection, Mutagenesis, In Vitro, Recombinant

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) WT and Rnf168 −/− MEFs, and Top2α knockdown control MEFs were fixed and stained with DAPI. Representative cells with defective chromosome segregation, as indicated by chromosome bridges (arrow head) and micronuclei (arrow) are shown. ( b ) Histograms show quantification of cells with chromosome bridges or micronuclei (mean±s.e.m., n =3). * P <0.05. ( c ) Histograms show the fraction of RIDDLE cells reconstituted with HA-RNF168 or HA-empty vector that display micronuclei (mean±s.e.m., n =3). * P <0.05. ( d ; upper panels) Representative metaphase spreads showing undercondensed chromosomes in WT MEFs 24 h post ICRF-193 treatment (positive control), and in Rnf168 −/− DMSO-treated MEFs. ( d , lower panels) Representative metaphase spreads of DMSO-treated Rnf168 −/− MEFs complemented with RNF168-WT (WT) or the E3 ligase deficient RNF168-C21S (C21S). ( e ) Histograms show quantification of abnormal metaphase spreads with entangled or undercondensed chromosomes from the indicated cells (mean±s.e.m., n =3). * P <0.05 compared with WT MEFs. ( f ) Histograms present the mean fraction of MEFs evading ICRF-193-induced G2 arrest as compared with DMSO-treated controls (mean±s.e.m., n =3). Data are shown for WT MEFs, mock infected Rnf168 −/− MEFs and Rnf168 −/− MEFs complemented with RNF168-WT or RNF168-C21S. 3T3 MEFs were used for these experiments. Data shown are for 6 h post-treatment with colcemid±4 μM ICRF-193. * P <0.05 compared with WT MEFs. ( g ) Clonogenic assay was used to determine sensitivity to ICRF-193 of mock infected WT and Rnf168 −/− MEFs, as well as Rnf168 −/− MEFs complemented with RNF168 (WT or C21S mutant). Data are presented as the mean±s.e.m. ( n =4). * P <0.05 compared with WT MEFs. Scale bar, 20 μm.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Knockdown, Control, Staining, Plasmid Preparation, Positive Control, Infection, Clonogenic Assay, Mutagenesis

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) Nuclear (Nuc) and chromatin (Chr) fractions prepared from WT, Rnf168 −/− and Brca1 −/− MEFs were analysed by IB for chromatin occupancy of Top2α. Additional IBs were performed with the indicated antibodies as controls. H4, histone H4. ( b ) Nuclear and chromatin fractions were prepared from the human breast cancer cell lines T47D and MDA-MB-231 knocked down for RNF168 (Sh.RNF168) and their controls (Sh.Ctr) and analysed by IB for the chromatin occupancy of TOP2α as in a . ( c ) HEK293T were transfected with Flag-TOP2α along with RNF168 (+) or empty vector (−) and their nuclear and chromatin fractions were prepared and examined by IB using the indicated antibodies. ( d ) A representative agarose gel showing decatenation activity of soluble nuclear and chromatin extracts from 2 WT and 2 Rnf168 −/− MEFs. In vitro kinetoplast DNA-based decatenation assay was performed for 20 min with different amounts of nuclear and chromatin extracts, and catenated and decatenated kDNA were separated by electrophoresis. IB using anti-Top2α was performed to show the level of Top2α present in each sample.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Transfection, Plasmid Preparation, Agarose Gel Electrophoresis, Activity Assay, In Vitro, Electrophoresis

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a , b ) HEK293T cells were transfected with Flag-RNF168 and HA-USP10 vectors ( a ) or Flag-TOP2α and HA-USP10 vectors ( b ) as indicated. Cells were lysed and IP was performed using anti-Flag and anti-HA antibodies. The resulting precipitates were subjected to IB analysis with the indicated antibodies. WCL, whole-cell lysate. ( c ) HEK293T cells were transfected with Flag-TOP2α, RNF168, HA-USP10 and Myc-Ub vectors, as indicated. IP using anti-Flag and WCL was subjected to anti-Ub IB analysis to detect TOP2α ubiquitylation. ( d ) WT MEFs with knockdown of Usp10 (Sh1 and Sh2) and WT controls were examined for the level of Top2α ubiquitylation. Top2α was immunoprecipitated from whole-cell extracts and examined by IB for its ubiquitylation level using anti-Ub. IP using IgG was used as a control. The indicated antibodies were used for IB. ( e ) HEK293T cells were transfected with Flag-TOP2α with or without RNF168, HA-Ub, USP10 and USP10-C424A as indicated. TOP2α chromatin occupancy in these cells was examined by IB using anti-Flag antibodies and chromatin fractions (Chr). IB analysis of the chromatin fractions is also shown for the indicated antibodies. ( f ) A simplified model of RNF168-mediated regulation of TOP2α ubiquitylation and decatenation function.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Transfection, Knockdown, Immunoprecipitation, Control

Journal: Molecular Medicine Reports

Article Title: Girdin regulates the migration and invasion of glioma cells via the PI3K-Akt signaling pathway

doi: 10.3892/mmr.2015.4049

Figure Lengend Snippet: Girdin silencing inhibits the expression and activity of MMP-2 and MMP-9. (A and B) Changes in the mRNA levels of MMP-2 and MMP-9 were measured using reverse transcription-quantitative polymerase chain reaction following transfection. The relative mRNA expression levels were calculated using the 2 −ΔΔCt method. (C and D) Following transfection, changes in the protein levels of MMP-2 and MMP-9 were detected using western blot analysis. (E and F) Following transfection, gelatin zymography was performed to detect changes in the activities of MMP-2 and MMP-9. Each experiment was repeated three times. The experimental results are presented as the mean ± standard deviation. ** P<0.01, compared with the NC group. shRNA, short hairpin RNA; NC, negative control.

Article Snippet: Following washing with Tris-buffered saline with 0.05% Tween-20 (TBST), the membranes were incubated with the following primary antibodies at 4°C overnight: Rabbit anti-human polyclonal antibody against girdin (1:500 diluted; cat. no. bs-5150R; Bioss, Beijing, China); rabbit anti-human polyclonal antibody against MMP-2 (1:1,000 diluted; cat. no. WL0657); rabbit anti-human polyclonal antibody against MMP-9 (1:1,000 diluted; cat. no. WL0884); rabbit anti-human polyclonal antibody against P85α (1:1,000 diluted; cat. no. WL0191); rabbit anti-human polyclonal antibody against P110α (1:1,000 diluted; cat. no. WL0339); rabbit anti-human polyclonal antibody against AKT (1:1,000 diluted; cat. no. WL0003); rabbit anti-human polyclonal antibody against p-AKT (1:1,000 diluted; cat. no. WLP001).

Techniques: Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Zymography, Standard Deviation, shRNA, Negative Control

Journal: PLoS ONE

Article Title: IGFBP2 promotes immunosuppression associated with its mesenchymal induction and FcγRIIB phosphorylation in glioblastoma

doi: 10.1371/journal.pone.0222999

Figure Lengend Snippet: The interfering RNA sequences used for IGFBP2 knockdown.

Article Snippet: For anti-IGFBP2 antibody treatment, 2.0 μg/ml of a rabbit anti-IGFBP2 monoclonal antibody (anti-IGFBP2mAb) (bs-1108R, Bioss) or isotype IgG control (bs-0295P, Bioss) was added to GL261 culture for 3 days and collected for western blotting.

Techniques: Sequencing

Journal: PLoS ONE

Article Title: IGFBP2 promotes immunosuppression associated with its mesenchymal induction and FcγRIIB phosphorylation in glioblastoma

doi: 10.1371/journal.pone.0222999

Figure Lengend Snippet: (A) An association of the enrichment score of the MES signature with the expression of IGFBP2 using the TCGA-gliomas database. R = Pearson’s correlation coefficient. (B-C) Effect of IGFBP2 knockdown with shRNA (IGFBP2KD) (B) or an anti-IGFBP2 antibody (anti-IGFBP2) (C) on mesenchymal marker protein levels in mouse GBM GL261 cells. (D) Effect of overexpressed IGFBP2 wildtype (IGFBP2OE) or mutant (IGFBP2mt) with the change of RGD to RGE on mesenchymal marker protein levels. The experiments were repeated at least three times. A statistical significance was calculated using an unpaired Welch’s t test. * P<0.05, ** P<0.01, *** P<0.001.

Article Snippet: For anti-IGFBP2 antibody treatment, 2.0 μg/ml of a rabbit anti-IGFBP2 monoclonal antibody (anti-IGFBP2mAb) (bs-1108R, Bioss) or isotype IgG control (bs-0295P, Bioss) was added to GL261 culture for 3 days and collected for western blotting.

Techniques: Expressing, shRNA, Marker, Mutagenesis

Journal: PLoS ONE

Article Title: IGFBP2 promotes immunosuppression associated with its mesenchymal induction and FcγRIIB phosphorylation in glioblastoma

doi: 10.1371/journal.pone.0222999

Figure Lengend Snippet: (A-C) The mouse splenocytes (SPCs) were co-cultured with GL261 cells for 6 days and then collected to analyze the number of CD8 + and CD4 + T cells by fluorescence activated cell sorting (FACS) under the setting of IGFBP2KD (A), anti-IGFBP2 (B) or overexpressed IGFBP2 and its mutant (C). The experiments were repeated at least three times. Representative IHC images of CD8 + T cells (D-E), CD163 + M2 macrophages (G-H), and pY513-CD19 + cells (J-K) in tumor-bearing brains of mice treated with anti-IGFBP2 or IgG. 100X (left), 400X (right). N = 3. (F, I) The percentage of tumor infiltrating CD8 + T cells (F) and CD163 + M2 macrophages in F4/80 + macrophages (I) analyzed by FACS after anti-IGFBP2 or IgG treatment. N = 5. The data are mean ± SD. A statistical significance was calculated by an unpaired Welch’s t test. * P<0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For anti-IGFBP2 antibody treatment, 2.0 μg/ml of a rabbit anti-IGFBP2 monoclonal antibody (anti-IGFBP2mAb) (bs-1108R, Bioss) or isotype IgG control (bs-0295P, Bioss) was added to GL261 culture for 3 days and collected for western blotting.

Techniques: Cell Culture, Fluorescence, FACS, Mutagenesis

Journal: PLoS ONE

Article Title: IGFBP2 promotes immunosuppression associated with its mesenchymal induction and FcγRIIB phosphorylation in glioblastoma

doi: 10.1371/journal.pone.0222999

Figure Lengend Snippet: ( A) An association of the enrichment score of the MES signature with FcγRIIB expression analyzed by using the TCGA-human glioma database. R = Pearson’s correlation coefficient. (B) Representative IHC images of IGFBP2 protein and phosphorylated FcγRIIB (p-FcγRIIB) levels in human GBMs (hGBMs) or normal brains stained in tissue microarrays. (C) An association of IGFBP2 protein and p-FcγRIIB levels in human GBMs. R, the Pearson correlation coefficient. (D-I), The percentage of p-FcγRIIB + cells in the SPCs after co-cultured with GL261 cells treated with sh1RNA-IGFBP2 (D-E), anti-IGFBP2 (F-G) or overexpressing IGFBP2 or IGFBP2mt (H-I) analyzed by FACS. (J-K), Representative IHC images of p-FcγRIIB staining in tumor-bearing brains treated with anti-IGFBP2 or IgG. N = 3. (L) The percentage of p-FcγRIIB + cells in infiltrating immune cells in GL261 tumors analyzed by FACS. N = 5. The data are mean ± SD. A statistical significance was computed by an unpaired Welch’s t test. * P < 0.05, ** p < 0.01.

Article Snippet: For anti-IGFBP2 antibody treatment, 2.0 μg/ml of a rabbit anti-IGFBP2 monoclonal antibody (anti-IGFBP2mAb) (bs-1108R, Bioss) or isotype IgG control (bs-0295P, Bioss) was added to GL261 culture for 3 days and collected for western blotting.

Techniques: Expressing, Staining, Cell Culture

Journal: PLoS ONE

Article Title: IGFBP2 promotes immunosuppression associated with its mesenchymal induction and FcγRIIB phosphorylation in glioblastoma

doi: 10.1371/journal.pone.0222999

Figure Lengend Snippet: (A-B) The time course induction of FcγRIIB phosphorylation on CD19 + B cells in SPCs co-cultured with IGFBP2OE GL261 or control cells. The percentage of CD19 L p-FcγRIIB H , CD19 L p-FcγRIIB - , and CD19 H p-FcγRIIB L B cell subsets was analyzed by FACS. L is low, H is high. (C-H) The percentage of p-FcγRIIB + cells in CD19 + B cells after co-cultured with GL261 cells treated with shRNA (C-D), anti-IGFBP2 (E-F) or overexpressing IGFBP2 or its mutant (G-H). (I-J) The percentage of CD19 + p-FcγRIIB + B cells in infiltrating immune cells (I) and p-FcγRIIB + cells in CD19 + B cells in anti-IGFBP2 or IgG tumors analyzed by FACS(J). (K) The ratio of CD19 + B cells versus CD19 + p-FcγRIIB + B cells. (L-M) The percentage of F4/80 + p-FcγRIIB + macrophages in infiltrating immune cells (L) and p-FcγRIIB + cells in F4/80 + macrophages (M) in anti-IGFBP2 or IgG tumors analyzed by FACS. N = 5. The data are mean ± SD. A statistical significance was calculated by an unpaired Welch's t test. * P < 0.05, ** P < 0.01.

Article Snippet: For anti-IGFBP2 antibody treatment, 2.0 μg/ml of a rabbit anti-IGFBP2 monoclonal antibody (anti-IGFBP2mAb) (bs-1108R, Bioss) or isotype IgG control (bs-0295P, Bioss) was added to GL261 culture for 3 days and collected for western blotting.

Techniques: Cell Culture, shRNA, Mutagenesis

Journal: PLoS ONE

Article Title: IGFBP2 promotes immunosuppression associated with its mesenchymal induction and FcγRIIB phosphorylation in glioblastoma

doi: 10.1371/journal.pone.0222999

Figure Lengend Snippet: P values in Fig 4C–4H .

Article Snippet: For anti-IGFBP2 antibody treatment, 2.0 μg/ml of a rabbit anti-IGFBP2 monoclonal antibody (anti-IGFBP2mAb) (bs-1108R, Bioss) or isotype IgG control (bs-0295P, Bioss) was added to GL261 culture for 3 days and collected for western blotting.

Techniques:

Journal: PLoS ONE

Article Title: IGFBP2 promotes immunosuppression associated with its mesenchymal induction and FcγRIIB phosphorylation in glioblastoma

doi: 10.1371/journal.pone.0222999

Figure Lengend Snippet: ( A) Representative T2-weighted MRI of GL261-bearing brains of mice treated with anti-IGFBP2 or IgG after 3 and 5 weeks of GL261cell injection. (B) The tumor growth curve in anti-IGFBP2 and IgG groups. (C) The Kaplan-Meier survival plot of GL261-bearing mice treated with anti-IGFBP2 or IgG. Log-rank test was used to compare the difference of the median survival time between the two groups. N = 5.

Article Snippet: For anti-IGFBP2 antibody treatment, 2.0 μg/ml of a rabbit anti-IGFBP2 monoclonal antibody (anti-IGFBP2mAb) (bs-1108R, Bioss) or isotype IgG control (bs-0295P, Bioss) was added to GL261 culture for 3 days and collected for western blotting.

Techniques: Injection

Journal: Journal of neurochemistry

Article Title: The mitochondrial intramembrane protease PARL cleaves human Pink1 to regulate Pink1 trafficking.

doi: 10.1111/j.1471-4159.2011.07253.x

Figure Lengend Snippet: Fig. 1 PARL over-expression leads to increased processing of Pink1-66. (a) Schematic representation of human Pink1. The predicted matrix targeting sequence (MTS), the transmembrane domain (TMD), the kinase domain and the putative PARL processing site are indicated. Comparison of the TMDs of human (Hs) and D. melanog- aster (Dm) Pink1 using the EMBOSS pair- wise alignment algorithm reveals significant sequence conservation. The hydrophobicity plot of the relevant region is shown [using the scale of Kyte and Doolittle (1982), with a window size of 7] indicating the potential TMD boundaries. (b) Co-expression of hu- man Pink1 with PARL leads to an increased processing of the full-length 66 kDa form (open triangle). A catalytically inactive PARL mutant (SA) shows no activity. Subcellular fractionation reveals that Pink1-66 and the processed Pink-55 (filled triangle) are found in mitochondria (m) and also the non-mito- chondrial soluble fraction (c). IRES GFP and the cellular markers VDAC, AIF and actin were used as transfection and loading con- trol. t, total cell extract (10% loaded). (c) Quantification of Pink1 66/55 kDa ratio in the mitochondrial fraction upon PARL wt and SA over-expression (n = 3, means ± SEM). Significant changes versus mock are indi- cated (**p < 0.01; One-way ANOVA with Bonferroni’s post-test). (d) Titration of PARL cDNA significantly increases processing of Pink1-66 (n = 3, means ± SEM). Significant changes versus mock are indicated (*p < 0.05; One-way ANOVA with Bonferroni’s post-test).

Article Snippet: The following antibodies were used at dilutions recommended by the manufacturer: polyclonal rabbit Pink1 (BC100-494) (Novus Biologicals, Littelton, CO, USA), polyclonal rabbit voltage-dependent anion-selective channel protein (VDAC) (Pierce, Rockford, IL, USA), monoclonal mouse apoptosis-inducing factor (AIF; Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal mouse FLAG (M2) (Sigma, St Louis, MO, USA), polyclonal rabbit PARL and a-actin (Abcam, Cambridge, MA, USA).

Techniques: Over Expression, Sequencing, Comparison, Expressing, Mutagenesis, Activity Assay, Fractionation, Transfection, Titration

Journal: Journal of neurochemistry

Article Title: The mitochondrial intramembrane protease PARL cleaves human Pink1 to regulate Pink1 trafficking.

doi: 10.1111/j.1471-4159.2011.07253.x

Figure Lengend Snippet: Fig. 2 PARL is required for Pink1 processing. (a) Left panel: PARL knockdown by two non-overlapping siRNAs (PARL-1 and -2) blocks Pink1-66 processing and reduces the level of Pink1-55, when com- pared to a non-targeting siRNA (nt). Other proteins like VDAC, AIF, actin and IRES GFP are not affected. t, total cell extracts; m, mito- chondrial pellet and c, non-mitochondrial fraction. Right panel: Quan- tification of the Pink1 66/55 kDa ratio in the mitochondrial fractions upon knockdown of endogenous PARL by siRNAs (means ± SEM, n = 3). Significant changes versus mock are indicated (**p < 0.01; One-way ANOVA with Bonferroni’s post-test). Right bottom panel: PARL knockdown was monitored in the mitochondrial fraction. (b) Left panel: Doxycyclin-induced expression of PARL-specific shRNA in Hek293- shRNA cells for 6 days diminishes Pink1-66 processing. For PARL

Article Snippet: The following antibodies were used at dilutions recommended by the manufacturer: polyclonal rabbit Pink1 (BC100-494) (Novus Biologicals, Littelton, CO, USA), polyclonal rabbit voltage-dependent anion-selective channel protein (VDAC) (Pierce, Rockford, IL, USA), monoclonal mouse apoptosis-inducing factor (AIF; Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal mouse FLAG (M2) (Sigma, St Louis, MO, USA), polyclonal rabbit PARL and a-actin (Abcam, Cambridge, MA, USA).

Techniques: Knockdown, Expressing, shRNA

Journal: Journal of neurochemistry

Article Title: The mitochondrial intramembrane protease PARL cleaves human Pink1 to regulate Pink1 trafficking.

doi: 10.1111/j.1471-4159.2011.07253.x

Figure Lengend Snippet: Fig. 4 Both uncoupling of the mitochondrial membrane potential and PARL ablation lead to the accumulation of Pink1-66 at the outer mitochondrial membrane. (a) Upper panel: Uncoupling of the mitochondrial membrane potential in Hek293-shRNA cells (by CCCP) leads to a Pink1 66/55 kDa ratio as observed upon PARL knockdown (shRNA). Total cell extracts (t), mitochondrial fractions (m) and non-mitochondrial soluble fractions (c) were analyzed. Lower panel: Quantification of Pink1 66/55 kDa ratio of the indicated fractions is shown (means ± SEM, n = 3). Mitochondrial Pink1 66/ 55 kDa ratio is significantly lower after PARL shRNA and CCCP treatment than after treatment either with PARL shRNA or CCCP alone (*p < 0.05; **p < 0.01; paired t-test). (b) Upper panel: Outline of the Pink1 construct with a juxtamembrane factor Xa protease cleavage site (Pink1Xa). Lower panel: Mitochondria isolated from cells expressing Pink1Xa were subjected to digestion by factor Xa protease. Factor Xa cleaves Pink1-66 (open triangle) but not Pink1-55 (filled triangle) generating a 52 kDa form (grey triangle). In mitochondria isolated from cells expressing the PARL-specific shRNA or CCCP-treated cells, the factor Xa-sensitive outer membrane form of Pink1-66 increased. Note that upon PARL knockdown Pink1Xa was cleaved by an alternative protease within the N-terminal MTS leading to a 64 kDa form (labeled with an asterisk), which is not observed upon expression of Pink1 wild type.

Article Snippet: The following antibodies were used at dilutions recommended by the manufacturer: polyclonal rabbit Pink1 (BC100-494) (Novus Biologicals, Littelton, CO, USA), polyclonal rabbit voltage-dependent anion-selective channel protein (VDAC) (Pierce, Rockford, IL, USA), monoclonal mouse apoptosis-inducing factor (AIF; Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal mouse FLAG (M2) (Sigma, St Louis, MO, USA), polyclonal rabbit PARL and a-actin (Abcam, Cambridge, MA, USA).

Techniques: Membrane, shRNA, Knockdown, Construct, Isolation, Expressing, Labeling

Journal: Journal of neurochemistry

Article Title: The mitochondrial intramembrane protease PARL cleaves human Pink1 to regulate Pink1 trafficking.

doi: 10.1111/j.1471-4159.2011.07253.x

Figure Lengend Snippet: Fig. 5 Pink1 TMD mutations block PARL-catalyzed processing. (a) TMD sequences of Pink1 wild type (wt), Pink1G107L, Pink1G109L, Pink1R98W and Pink1I111S are shown. Mutated residues are highlighted in bold. (b) and (c) Western analysis of Pink1 mutants expressed in Hek293-shRNA cells after doxycyclin-induced knockdown of PARL (shRNA) and CCCP treatment (total cell extracts are shown). Notably, for Pink1G107L, Pink1G109L and Pink1R98W, the Pink1-66 precursor was modified by an unknown post-translational modification leading to a 68 kDa species (labeled by an asterisk). Quantification of the Pink1 66/55 kDa ratio in the indicated fractions is shown (means ± SEM, n = 3). Pink1-55 levels are lower for all probed mutants tested. Pink1G107L and Pink1R98W processing is insensitive to PARL ablation showing highly reduced processing of these mutants by PARL.

Article Snippet: The following antibodies were used at dilutions recommended by the manufacturer: polyclonal rabbit Pink1 (BC100-494) (Novus Biologicals, Littelton, CO, USA), polyclonal rabbit voltage-dependent anion-selective channel protein (VDAC) (Pierce, Rockford, IL, USA), monoclonal mouse apoptosis-inducing factor (AIF; Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal mouse FLAG (M2) (Sigma, St Louis, MO, USA), polyclonal rabbit PARL and a-actin (Abcam, Cambridge, MA, USA).

Techniques: Blocking Assay, Western Blot, shRNA, Knockdown, Labeling

Journal: Gene therapy

Article Title: Targeted inhibition of platelet-derived growth factor receptor-beta subunit in hepatic stellate cells ameliorates hepatic fibrosis in rats.

doi: 10.1038/gt.2008.93

Figure Lengend Snippet: Figure 3 Localization of LacZ driven by CMV promoter and GFAP promoter in normal rats, acute liver injury rats and chronic liver injury rats. (a) Representative graphs of b-galactosidase (b-gal) immunofluorescence showed the distribution of b-gal-positive cells in normal rats, acute liver injury rats and chronic liver injury rats treated with pCMV-shRNA-LacZ or pGfa-shRNA-LacZ. Magnification of 20. The scale bar represents 80 mm. (b) Representative graphs of a-SMA and GFAP immunofluorescence showed that in the chronic injured liver, a-SMA and GFAP proteins distributed mainly around the portal area and the hyperplastic bile duct, whereas in the acute injured liver, both the proteins revealed a much more diffuse distribution in the hepatic lobule. In addition, the GFAP-positive cells were more than the a-SMA- positive cells both in the acute injured liver and in the chronic injured liver. Magnification of 20. The scale bar represents 80 mm. (c) Representative graphs of b-gal and a-SMA double-staining revealed that b-gal protein in pCMV-shRNA-LacZ-treated livers could be expressed in hepatocytes (blue arrow) and activated HSCs stained by a-SMA (white arrow), magnification of 63. The scale bar represents 40 mm. (d) Representative graphs of b-gal and GFAP double-staining showed that b-gal-positive cells were all GFAP-positive HSCs (pink arrow) in pGfa-shRNA-LacZ-treated livers, magnification of 63. The scale bar represents 40 mm. (e) Representative graphs of b-gal and a-SMA double-staining showed that some b-gal protein was expressed in activated HSCs stained by a-SMA (white arrow) in pGfa-shRNA- LacZ-treated livers, magnification of 63. The scale bar represents 40 mm. CMV, cytomegalovirus; GFAP, glial fibrillary acidic protein; HSCs, hepatic stellate cells; PDGFR-b, platelet-derived growth factor receptor-b subunit; a-SMA, a-smooth muscle actin; shRNA, short hairpin RNA; RT-PCR, reverse transcriptional-PCR.

Article Snippet: Frozen liver sections (8 mm in thickness) were incubated with mouse anti-b-galactosidase monoclonal antibody (Promega, Madison, WI, USA), rabbit anti-GFAP polyclonal antibody (Boster, Wuhan, China) or rabbit anti-a-SMA polyclonal antibody (Bios, Beijing, China) for 20 h at 4 1C, then washed in phosphate-buffered saline and incubated for 30 min with Cy5-labeled anti-rabbit IgG antiserum and FITC-labeled anti-mouse IgG antiserum (Jackson, West Grove, PA, USA) at 1:100 dilution in phosphate-buffered saline.

Techniques: shRNA, Double Staining, Staining, Derivative Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Neuron

Article Title: TREK-1 and TRAAK are principal K + channels at the nodes of Ranvier for rapid action potential conduction on mammalian myelinated afferent nerves

doi: 10.1016/j.neuron.2019.08.042

Figure Lengend Snippet: A) Left, TREK-1 immunoreactivity (TREK-1-ir) at a NR and MBP immunoreactivity (MBP-ir) on myelin sheath. Right, TREK-1-ir at a NR and CASPR-ir in paranodal regions. B) Left, TRAAK-ir at a NR and MBP-ir on myelin sheath. Right, TRAAK-ir at a NR and CASPR-ir in paranodal regions. C) Similar to A&B except TREK-2-ir was examined and was negative at NRs. In A-C, NRs are indicated by arrows. MBP, myelin basic protein. CASPR, contactin associated protein. D) Summary of immunoreactive nodes for experiments represented in A-C: 112/129 nodes were TREK-1-ir positive, 118/129 nodes were TRAAK-ir positive. 0/129 nodes were TREK-2-ir positive. E) HEK293 cells transfected with TREK-1/eGFP (left), TRAAK/mCherry (middle), and both TREK-1/EGFP and TRAAK/mCherry (right). F) Traces illustrate single channel currents recorded at −80 mV from an HEK293 cell transfected with TREK-1/eGFP (upper, homomeric TREK-1) or an HEK293 cell transfected with TRAAK/mCherry (lower, homomeric TRAAK). Bottom, I-V curves of single channel currents recorded at different transmembrane voltages for homomeric TREK-1 channels (open circles, n = 7) or homomeric TRAAK (solid circles, n = 6). G) Sample traces show two types of single channels recorded at −80 mV from a HEK293 cell co-transfected with TREK-1/eGFP and TRAAK/mCherry plasmids, one type (upper, TREK-1/TRAAK) has unitary currents apparently larger than homomeric channels and another type (lower, TREK-1-like) has unitary currents similar to homomeric TREK-1 channels shown in F. Bottom panel, I-V curves of the currents of TREK-1/TRAAK single channels (n = 12, solid triangles) and TREK-1-like single channels (n = 13, open triangles). H) Summary of single channel conductance at −80 mV (open bars) and 80 mV (closed bars) for homomeric TREK-1 (n = 5 at −80 mV, n = 6 at 80 mV), homomeric TRAAK (n = 6 at both voltages), TREK-1-like (n = 12 at −80 mV, n = 5 at 80 mV), and TREK-1/TRAAK channels (n = 8 at −80 mV, n = 5 at 80 mV) expressed in HEK293 cells. The single channel conductance of nodal K2P channels (n = 13 at both voltages) is also included in the graph for a comparison. The single channel conductance at −80 mV was used for comparison. All recordings were performed under the cell-attached mode. Data represent Mean ± SEM, ns, no significant difference, *p < 0.05, ***p < 0.001, one-way ANOVA with the Tukey post hoc test. See also Fig. S5–9

Article Snippet: Following primary antibodies were used: rabbit anti-K2P 2.1 ( TREK-1, 1:1000 or 1:500, Alomone labs, Jerusalem, Israel), rabbit anti-K2P 10.1 ( TREK-2, 1:1000, Alomone labs, Jerusalem, Israel), rabbit anti-K2P 4.1 ( TRAAK, 1:1000 or 1:500, Alomone labs, Jerusalem, Israel), chicken anti-Myelin basic protein (1:1000, Abcam, Cambridge, MA, USA), mouse anti-contactin associated protein 1 (CASPR, 1:1000 or 1:500, MilliporeSigma, Burlington, MA, USA), chicken anti-GFP (1:2000, Aves Labs Inc., Tigard, OR, USA) and mouse anti-mCherry (1:2000, Abcam, Cambridge, MA, USA).

Techniques: Transfection

Journal: Neuron

Article Title: TREK-1 and TRAAK are principal K + channels at the nodes of Ranvier for rapid action potential conduction on mammalian myelinated afferent nerves

doi: 10.1016/j.neuron.2019.08.042

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Following primary antibodies were used: rabbit anti-K2P 2.1 ( TREK-1, 1:1000 or 1:500, Alomone labs, Jerusalem, Israel), rabbit anti-K2P 10.1 ( TREK-2, 1:1000, Alomone labs, Jerusalem, Israel), rabbit anti-K2P 4.1 ( TRAAK, 1:1000 or 1:500, Alomone labs, Jerusalem, Israel), chicken anti-Myelin basic protein (1:1000, Abcam, Cambridge, MA, USA), mouse anti-contactin associated protein 1 (CASPR, 1:1000 or 1:500, MilliporeSigma, Burlington, MA, USA), chicken anti-GFP (1:2000, Aves Labs Inc., Tigard, OR, USA) and mouse anti-mCherry (1:2000, Abcam, Cambridge, MA, USA).

Techniques: Recombinant, shRNA, Software

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: VP treatment enhances apical AQP2 expression and its colocalization with ezrin. (A) VP treatment enhances apical AQP2 expression and its colocalization with ezrin in cultured renal epithelial cells. AQP2-MDCK cells were stained with antibodies against ezrin (green) and AQP2 (red) in the presence (VP) and absence (Control) of VP treatment (AVP 20 nM for 20 min). The larger panels represent confocal sections through the subapical regions of the cells above the nucleus. The smaller horizontal strips at the bottom of each panel are z-sections through the entire cell for direct comparison of the respective staining intensities of the apical and basolateral membranes, and the cytosol. Upper panels show that in the absence of VP stimulation, ezrin staining localized to the cytosol and basolateral region, while AQP2 staining was mainly detected in the subapical region. Lower panels show that after VP treatment, the ezrin signal was redistributed toward the apical and sub-apical regions and partially colocalized with the similarly apically redistributed AQP2. Scale bar: 10 μm. (B) Super-resolution Airyscan confocal microscopy imaging revealed that AQP2 and ezrin partially colocalize on the apical membrane in VP-treated MDCK cells. Left panels show no apparent colocalization of ezrin and AQP2, in the absence of VP stimulation. Right panels are cells treated with VP. Scale bar: 5 μm. (C) AQP2 and ezrin are co-expressed in principal cells of the Brattleboro rat collecting duct, and co-accumulate on the plasma membrane after vasopressin treatment. Without VP treatment (Control), ezrin was located in the cytosol and basal region, while AQP2 was detected mainly in the sub-apical region of the principal cells of the collecting ducts. After 7 days of VP treatment (VP), ezrin (red in the merge panel) colocalized with AQP2 (green in the merge panel) on the plasma membrane of the principal cells. Scale bar: 20 μm.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Expressing, Cell Culture, Staining, Confocal Microscopy, Imaging

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: Interaction of AQP2 with ezrin is detected in co-IP experiments. (A) List of ezrin peptides detected by mass spectrometry from the AQP2 co-IP complex. (B,C) By using an anti-ezrin antibody for co-IP, we were able to detect AQP2 in the co-IP complex from stable AQP2-expressing LLC-PK1 cell lysates and mouse kidney (B). Similarly, ezrin signal was detected in the co-IP complex using anti-AQP2 antibody (C). WB, western blotting.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Co-Immunoprecipitation Assay, Mass Spectrometry, Expressing, Western Blot

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: AQP2 interacts with ezrin via the ezrin N-terminal FERM-containing domain. (A) Recombinant His-tagged ezrin full-length (FL, amino acids 1–586), N-terminus (NT, amino acids 1–308), and C-terminus (CT, amino acids 285–586) were expressed in E. coli and purified to homogeneity as revealed by SDS-PAGE together with purified recombinant AQP2 C-terminus (AQP2CT). Schematic representation of each recombinant protein is shown in B. (C) The purified His-tagged ezrin full-length protein (FL) and the N-terminal FERM-containing recombinant protein (but not the C-terminal protein) were able to pull down AQP2 from both LLC-AQP2 cell lysates and mouse kidney lysates. Lanes 1–3, beads alone pulled down with PBS (lane 1), LLC-AQP2 cell lysate (lane 2) and kidney lysate (lane 3); lanes 4–6, ezrin FL pulled down with PBS (lane 4), LLC-AQP2 cell lysate (lane 5) and kidney lysate (lane 6); lanes 7–9, ezrin NT pulled down with PBS (lane 7), LLC-AQP2 cell lysate (lane 8) and kidney lysate (lane 9); lanes 10–12, ezrin CT pulled down with PBS (lane 10), LLC-AQP2 cell lysate (lane 11) and kidney lysate (lane 12). WB, western blotting.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Recombinant, Purification, SDS Page, Western Blot

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: AQP2 C-terminus directly interacts with ezrin N-terminal FERM-containing domain. The direct interaction of AQP2 and ezrin is revealed by pulldown experiments using purified recombinant ezrin and AQP2 proteins. Only the purified ezrin full-length (FL) and the N-terminus FERM domain-containing (NT) recombinant protein were able to pull down the purified AQP2 C-terminus. The ezrin C-terminal domain (CT) did not pull down the AQP2 C-terminal domain. WB, western blotting.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Purification, Recombinant, Western Blot

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: Downregulating ezrin causes membrane accumulation of AQP2. (A) Immunoblotting revealed a strong knockdown of endogenous ezrin in LLC-AQP2 cells by ezrin shRNA lentivirus. There was an ∼80% reduction of endogenous ezrin in ezrin shRNA knockdown LLC-AQP2 cells (graph shows the quantification of ezrin band intensity relative to actin). Results are mean±s.e.m. (n≥3). ***P<0.001 (one-way ANOVA). (B) Immunofluorescence staining of AQP2 in control LLC-AQP2 cells and cells infected with ezrin shRNA lentivirus. After knocking down ezrin in LLC-AQP2 cells, AQP2 was found to increasingly accumulate on the cell surface under baseline conditions (without any stimulation). VP-treated LLC-AQP2 cells were used for comparison. Scale bar: 10 μm. (C) Surface biotinylation experiment revealed a significantly increased accumulation of AQP2 signal on cell surface after knocking down ezrin expression in cells. Transferrin receptor 1 (TFR-1) was used as an internal control for stable membrane proteins. WB, western blotting.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Western Blot, shRNA, Immunofluorescence, Staining, Infection, Expressing

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: Phosphorylation of key residues in AQP2 was not altered in ezrin knockdown cells. (A) Representative western blot showing that there is no alteration of the levels of total AQP2 and AQP2 phosphorylated at residues 256 or 261 in ezrin shRNA lentivirus-infected LLC-AQP2 cells unlike in cells treated with VP. Immunoblot using anti-β-actin antibody was used as control. Quantification of western blotting results for AQP2 phosphorylated on S256 (p256), S261 (p261) and total AQP2. (B) Intracellular cAMP measurement in LLC-PK1 cells. The intracellular cAMP concentration was significantly increased by 30 min VP treatment (20 nM LVP) without clonal variation. After ezrin knockdown no significant difference in cAMP concentration was observed compared to control. Results in A and B are mean±s.e.m. (n≥3). **P<0.01, ***P<0.001 (one-way ANOVA).

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Western Blot, shRNA, Infection, Concentration Assay

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: Downregulating ezrin reduces clathrin-mediated endocytosis and causes concomitant membrane accumulation of AQP2 without affecting the overall exocytosis. (A) Ezrin knockdown does not affect the overall exocytosis in LLC-AQP2-ssYFP cells. LLC-AQP2 cells were stably transfected with ssYFP. The fluorescence signal in the extracellular medium was measured in LLC-AQP2-ssYFP cells with and without treatment with VP or ezrin shRNA lentivirus, respectively. The fluorescence intensity in the medium reflected the exocytotic activity of LLC-AQP2-ssYFP cells, and therefore the rate of exocytosis. No significant increase was seen in the overall exocytosis in LLC-AQP2-ssYFP cells treated with ezrin shRNA lentivirus compared to control. In contrast, a significant increase in exocytosis was observed in VP-treated LLC-AQP2-ssYFP cells, which is consistent with our previous reports (Nunes et al., 2008). (B) Endocytosis assay using Rhodamine-conjugated transferrin showed that Alexa Fluor 568-labeled transferrin accumulated on the apical membrane following ezrin knockdown (upper panel), and a simultaneous acute membrane accumulation of AQP2 (lower panel). Scale bar: 10 μm. (C) A bar graph showing that ezrin knockdown affects clathrin-mediated endocytosis. A block of endocytosis with MβCD was used as a positive control. Results in A and C are mean±s.e.m. (n≥3). **P<0.01, ***P<0.001 (one-way ANOVA).

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Stable Transfection, Transfection, Fluorescence, shRNA, Activity Assay, Endocytosis Assay, Labeling, Blocking Assay, Positive Control

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: AQP2 internalization was reduced after ezrin knockdown in a cold block experiment. (A) Dynamic distribution of AQP2 after a 20°C cold block was revealed by immunofluorescence staining of LLC-AQP2 cells. Cells were incubated at 20°C for 30, 60 and 120 min to block AQP2 release from the trans-Golgi network. AQP2 formed a ‘perinuclear patch’ in cells at 20°C. In the absence of protein synthesis (blocked by cycloheximide), the speed of formation and immunostaining intensity of the perinuclear patch reflect the speed and extent of AQP2 internalization from the cell surface over time. Scale bar: 10 μm. (B) Quantification of the growth of the AQP2-positive perinuclear patch after 20°C cold block over time. The fluorescence intensity of AQP2 staining in the perinuclear patch was measured using Volocity software as described previously (Arthur et al., 2015). Results are mean±s.e.m. (n≥13 for each data point). Experiments were repeated at least three times.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Blocking Assay, Immunofluorescence, Staining, Incubation, Immunostaining, Fluorescence, Software

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Spinal cord astrocyte identification and high mobility group box-1 (HMGB1) knockdown. a Spinal cord astrocytes were identified using immunofluorescence. The percentage of cells stained with the astrocytic marker S100β, which were identified as astrocytes, was more than 95% of the total cells (three replicates). b HMGB1 knockdown efficiency in the plasma membrane and cytoplasm of spinal cord astrocytes was evaluated using Western blot for HMGB1 protein levels. Results were obtained after 72 h of specific HMGB1 shRNA treatment. HMGB1 protein levels were decreased to approximately 30% of normal levels with shRNA multiplicity of infection 60 as compared to normal astrocytes. * P < 0.05 vs. normal group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Immunofluorescence, Staining, Marker, Western Blot, shRNA, Infection

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of oxygen-glucose deprivation/reoxygenation (OGD/R) on cellular swelling, high mobility group box-1 (HMGB1), and aquaporin-4 (AQP4) expression in cultured spinal cord astrocytes as well as levels of HMGB1 and interleukin-6 (IL-6) released into the surrounding medium. a Astrocyte volume measurement was performed using a Live Cell Imaging System. Cellular volume was calculated by the average value of four measured diameters of the largest compiled Z-slice image. Cellular volumes of spinal cord astrocytes were significantly increased at 2, 6, 12, 24, and 48 h during reoxygenation after OGD when compared with normal astrocytes. # P < 0.05 vs. normal group; * P < 0.05 vs. OGD6h/R24h group (three replicates). b Membrane and cytoplasmic HMGB1 expression was significantly increased in spinal cord astrocytes at different time points after OGD/R. # P < 0.05 vs. normal group; * P < 0.05 vs. OGD6h/R24h group (three replicates). c Membrane and cytoplasmic AQP4 expression was significantly increased in spinal cord astrocytes at different time points after OGD/R. # P < 0.05 vs. normal group; * P < 0.05 vs. OGD6h/R24h group (three replicates). d HMGB1 levels in the surrounding medium of spinal cord astrocytes were significantly increased at 6, 12, and 24 h during reoxygenation after OGD/R. # P < 0.05 vs. normal group; * P < 0.05 vs. OGD6h/R24h group (three replicates). e IL-6 levels in the surrounding medium of spinal cord astrocytes were significantly increased at 6, 12, and 24 h during reoxygenation after OGD/R. # P < 0.05 vs. normal group; * P < 0.05 vs. OGD6h/R24h group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Expressing, Cell Culture, Live Cell Imaging

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of inhibiting high mobility group box-1 (HMGB1) on cellular swelling in cultured spinal cord astrocytes after oxygen-glucose deprivation/reoxygenation (OGD/R). a Astrocyte volume analysis was performed using a Live Cell Imaging System, and cellular volume was calculated by the average value of four measured diameters. Inhibiting HMGB1 using either HMGB1 shRNA or ethyl pyruvate (EP) significantly blocked increases in cellular volume of spinal cord astrocytes at 6, 12, and 24 h during reoxygenation when compared with astrocytes of the OGD/R group. * P < 0.05 vs. OGD/R group (three replicates). b — d Effects of inhibiting HMGB1 on spinal cord astrocytic morphology and ultrastructure were evaluated using transmission electron microscopy at 6, 12, and 24 h during reoxygenation after OGD. After OGD/R, spinal cord astrocytes showed swelling at 6, 12, and 24 h during reoxygenation. The mitochondrial (M) swelling, endoplasmic reticulum (ER) swelling and fragmentation, and an increase in the number of lysosomes (L) were concurrent with this observation. However, astrocytic swelling, mitochondrial (M) swelling, endoplasmic reticulum (ER) swelling and fragmentation, and the increase in lysosome (L) number after OGD/R were reduced by HMGB1 inhibition using either HMGB1 shRNA or EP (× 50,000, bar equal to 1 μm, three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Cell Culture, Live Cell Imaging, shRNA, Transmission Assay, Electron Microscopy, Inhibition

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of inhibiting high mobility group box-1 (HMGB1) on HMGB1, aquaporin-4 (AQP4), and toll-like receptor-4 (TLR4) expression in cultured spinal cord astrocytes after oxygen-glucose deprivation/reoxygenation (OGD/R) as well as levels of HMGB1 and interleukin-6 (IL-6) release into the surrounding medium. a Inhibiting HMGB1 using either HMGB1 shRNA or ethyl pyruvate (EP) significantly suppressed the increased levels of HMGB1 in both the plasma membrane and cytoplasm of spinal cord astrocytes at 6, 12, and 24 h during reoxygenation after OGD. * P < 0.05 vs. OGD/R group (three replicates). b Inhibiting HMGB1 significantly suppressed the increased levels of AQP4 in both the plasma membrane and cytoplasm of spinal cord astrocytes at 6, 12, and 24 h during reoxygenation after OGD. * P < 0.05 vs. OGD/R group (three replicates). c Inhibiting HMGB1 significantly suppressed increased levels of TLR4 in both the plasma membrane and cytoplasm of spinal cord astrocytes at 6, 12, and 24 h during reoxygenation after OGD. * P < 0.05 vs. OGD/R group (three replicates). d HMGB1, AQP4, and TLR4 immunofluorescence on spinal cord astrocytes at 24 h into the reoxygenation process after OGD showed significantly increased membrane and cytoplasmic levels of HMGB1, AQP4, and TLR4 in the OGD/R group when compared with those in the normal group. These were markedly suppressed in both the OGD/R + HMGB1 shRNA and OGD/R + EP groups (× 200, bar equal to 100 μm). * P < 0.05 vs. OGD/R group (three replicates). e , f Inhibiting HMGB1 mitigated increases in levels of HMGB1 and IL-6 in the surrounding medium when compared with levels in the OGD/R group at 6, 12, and 24 h during reoxygenation after OGD. * P < 0.05 vs. OGD/R group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Expressing, Cell Culture, shRNA, Immunofluorescence

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of either inhibiting high mobility group box-1 (HMGB1) or toll-like receptor-4 (TLR4) on oxygen-glucose deprivation/reoxygenation (OGD/R)-induced astrocytic swelling, TLR4, myeloid differentiation primary response gene 88 (MyD88), aquaporin-4 (AQP4) upregulation, and nuclear factor-kappa B (NF-κB) activation as well as levels of interleukin-6 (IL-6) released into the surrounding medium. a Inhibiting HMGB1 (using either HMGB1 shRNA or ethyl pyruvate (EP)) or TLR4 (using CLI-095 or C34) significantly reduced the increase in cellular volume of spinal cord astrocytes at 24 h during the reoxygenation process after OGD when compared with those in the OGD/R group. * P < 0.05 vs. OGD/R group (three replicates). b Inhibiting HMGB1 or TLR4 significantly suppressed the increased levels of TLR4, MyD88, and AQP4 in both the plasma membrane and cytoplasm of spinal cord astrocytes at 24 h during the reoxygenation process after OGD. * P < 0.05 vs. OGD/R group (three replicates). c Inhibiting HMGB1 or TLR4 significantly suppressed the increased nuclear levels of NF-κB and the upregulation of cytoplasmic p-IκBα in spinal cord astrocytes after 24 h of the reoxygenation process after OGD. * P < 0.05 vs. OGD/R group (three replicates). d Immunofluorescence results showed that either inhibiting HMGB1 or TLR4 decreased membrane and cytoplasmic TLR4 and AQP4 upregulation and attenuated the increases of nuclear NF-κB when compared with the OGD/R group at 24 h during reoxygenation (× 200, bar equal to 100 μm). * P < 0.05 vs. OGD/R group (three replicates). e Inhibiting HMGB1 or TLR4 reduced increased levels of IL-6 in the surrounding medium when compared with those of the OGD/R group after 24 h of the reoxygenation process after OGD. * P < 0.05 vs. OGD/R group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Activation Assay, shRNA, Immunofluorescence

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of nuclear factor-kappa B (NF-κB) inhibition on oxygen-glucose deprivation/reoxygenation (OGD/R)-induced astrocytic swelling, NF-κB activation, and aquaporin-4 (AQP4) upregulation, as well as levels of interleukin-6 (IL-6) released into the surrounding medium. a NF-κB inhibition (using BAY 11-7082) significantly suppressed the increased nuclear levels of NF-κB and the upregulation of cytoplasmic p-IκBα in spinal cord astrocytes after 24 h of the reoxygenation phase after OGD. * P < 0.05 vs. OGD/R group (three replicates). b NF-κB and AQP4 immunofluorescence in spinal cord astrocytes after 24 h of the reoxygenation process after OGD showed significantly increased nuclear levels of NF-κB and membrane and cytoplasmic levels of AQP4 in the OGD/R group. Levels were markedly attenuated in the OGD/R + HMGB1 shRNA, OGD/R + BAY 11-7082, and OGD/R + EP groups (× 200, bar equal to 100 μm). * P < 0.05 vs. OGD/R group (three replicates). c NF-κB inhibition significantly reduced the increase in cellular volume of spinal cord astrocytes at 24 h during the reoxygenation process after OGD when compared with those of the OGD/R group. * P < 0.05 vs. OGD/R group (three replicates). d NF-κB inhibition significantly suppressed increased AQP4 levels in both the plasma membrane and cytoplasm of spinal cord astrocytes after 24 h of the reoxygenation process after OGD. * P < 0.05 vs. OGD/R group (three replicates). e NF-κB inhibition reduced increased levels of IL-6 in the surrounding medium when compared with those of the OGD/R group after 24 h of the reoxygenation process after OGD. * P < 0.05 vs. OGD/R group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Inhibition, Activation Assay, Immunofluorescence, shRNA

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of recombinant HMGB1 (rHMGB1) on aquaporin-4 (AQP4) expression in cultured spinal cord astrocytes. Incubation of cultured spinal cord astrocytes with rHMGB1 (0, 0.1, 1, 10, and 20 ng/ml) for 24 h did not induce dose-dependent increases in the membrane and cytoplasmic AQP4 expression (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Recombinant, Expressing, Cell Culture, Incubation

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of interleukin-6 (IL-6) on aquaporin-4 (AQP4) expression in cultured spinal cord astrocytes. a Spinal cord astrocytes were exposed to exogenous IL-6 at 0, 0.1, 1, or 10 ng/ml. After 24 h exposure, the membrane and cytoplasmic AQP4 expression in spinal cord astrocytes were markedly increased in the IL-6 0.1 ng/ml group, IL-6 1 ng/ml group, and IL-6 10 ng/ml group. * P < 0.05 vs. 0 ng/ml group (three replicates). b IL-6 levels increased in the surrounding medium of the OGD/R group after 24 h of the reoxygenation process after OGD. In comparison, this increase was significantly reduced in the OGD/R + HMGB1 shRNA group. * P < 0.05 vs. OGD/R group (three replicates). c The effects of astrocyte conditioned medium (ACM) on AQP4 expression in cultured spinal cord astrocytes. Twenty-four hours exposure of spinal cord astrocytes to the ACM obtained from the OGD/R group significantly increased the membrane and cytoplasmic AQP4 expression when compared with astrocytes incubated with the ACM obtained from the OGD/R + HMGB1 shRNA group. * P < 0.05 vs. astrocytes + OGD6h/R24h ACM group (three replicates). d Western blot analysis showed that the neutralizing anti-rat-IL-6 antibody could significantly reverse the upregulation effect of exogenous IL-6 or OGD/R ACM containing increased IL-6 on AQP4 expression in cultured spinal cord astrocytes. # P < 0.05 vs. astrocytes + IL-6 group; * P < 0.05 vs. astrocytes + OGD6h/R24h ACM group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Expressing, Cell Culture, shRNA, Incubation, Western Blot

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

Article Title: ?-defensins activate human mast cells via Mas-related Gene-X2 (MrgX2)

doi: 10.4049/jimmunol.1300023

Figure Lengend Snippet: LAD2 mast cells were stably transduced with scrambled shRNA control lentivirus or shRNA lentivirus targeted against MrgX2. (A) Western blotting was performed to determine MrgX2 expression in control and MrgX2 knockdown (KD) cells. (B) shRNA control and MrgX2 KD cells were stimulated with hBD2, hBD3, cortistatin (CST) or C3a and percent degranulation (β-hexosaminidase release) was determined. Data are mean ± SEM of three experiments. Statistical significance was determined by one-way ANOVA with Bonferroni's post test. * indicates p<0.01 and ** indicates p<0.001.

Article Snippet: MrgX2 antibody was purchased from Novus Biologicals (Littleton, CO).

Techniques: Stable Transfection, Transduction, shRNA, Control, Western Blot, Expressing, Knockdown

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

Article Title: ?-defensins activate human mast cells via Mas-related Gene-X2 (MrgX2)

doi: 10.4049/jimmunol.1300023

Figure Lengend Snippet: (A) RBL-2H3 cells stably expressing MrgX2 were stimulated with buffer, hBD2, hBD3 or cortistatin (CST) for 30 min and β-hexosaminidase release was measured. Data shown are representative of 3 similar experiments. Statistical significance was determined by one-way ANOVA with Bonferroni's post test. * indicates p<0.01 and ** indicates p<0.001. RBL-2H3 cells stably expressing MrgX2 were loaded with Indo-1AM and Ca2+ mobilization in response to (B) hBD2, (C) hBD3 or (D) CST was determined. HEK293 cells stably expressing MrgX2 were loaded with Indo-1AM and Ca2+ mobilization in response to (E) hBD3 or (F) CST was determined. Traces shown are representative of 3 individual experiments.

Article Snippet: MrgX2 antibody was purchased from Novus Biologicals (Littleton, CO).

Techniques: Stable Transfection, Expressing

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

Article Title: ?-defensins activate human mast cells via Mas-related Gene-X2 (MrgX2)

doi: 10.4049/jimmunol.1300023

Figure Lengend Snippet: (A) BMMCs were transiently transfected with HA tagged MrgX2 (solid line) or control plasmid vector (broken line) and MrgX2 receptor expression level was analyzed using flow cytometry. A representative histogram is shown. (B) Control and MrgX2 expressing BMMCs were incubated with DNP specific mouse IgE (1 μg/mL, 16 h). Cells were exposed to buffer (control), CST, hBD3, mCRAMP or DNP-BSA (10 ng/mL) for 30 minutes and β-hexosaminidase release was measured. LAD2 cells were stimulated with mCRAMP and (C) intracellular Ca2+ mobilization or (D) degranulation was determined. Traces are representative of 3 independent experiments. Bar graphs represent mean ± SEM of three experiments. Statistical significance was determined by one-way ANOVA with Bonferroni's post test. * indicates p<0.01.

Article Snippet: MrgX2 antibody was purchased from Novus Biologicals (Littleton, CO).

Techniques: Transfection, Control, Plasmid Preparation, Expressing, Flow Cytometry, Incubation

Journal: Nature Communications

Article Title: Non-canonical function of DGCR8 in DNA double-strand break repair signaling and tumor radioresistance

doi: 10.1038/s41467-021-24298-z

Figure Lengend Snippet: a Immunoblotting of Drosha, DGCR8, and β-actin in the LM2-DRR (expressing the pLCN DSB Repair Reporter) cell line transduced with DGCR8 shRNA. b Knockdown of DGCR8 decreased HR and NHEJ efficiency in LM2-DRR cells. Two days after co-transfection of I-SceI endonuclease and an exogenous donor for HR (pCAGGS DRR mCherry Donor EF1a BFP) into the DGCR8-knockdown LM2-DRR cells, the percentages of GFP-positive and mCherry-positive cells, gated on BFP-positive cells, were determined by flow cytometry. Repair by HR or NHEJ leads to mCherry or GFP expression. Data were normalized to the control cells. n = 3 biological replicates. c MYC-DGCR8-overexpressing LM2 cells were treated with IR (8 Gy) and cultured for 1 h, followed by pulldown with MYC beads and immunoblotting with the indicated antibodies. d Control and DGCR8-knockdown LM2 cells were treated with IR (8 Gy) and cultured for 1 h, followed by immunoprecipitation with an antibody against RNF168 or RNF8 and immunoblotting with the indicated antibodies. e Chromatin was extracted from LM2 cells that were treated with IR (8 Gy) and cultured for 1 h. The chromatin fractions, with or without MNase treatment, were immunoprecipitated with a DGCR8-specific antibody and immunoblotted with the indicated antibodies. f Quantification of MDC1, RNF8, RNF168, 53BP1, and BRCA1 foci in DGCR8-knockdown LM2 cells. Cells were incubated for 1 h after 2-Gy IR and immunostained with antibodies against γH2AX, MDC1, RNF8, RNF168, 53BP1, and BRCA1 (see representative images in Supplementary Fig. ). n = 3 biological replicates. g Control and DGCR8-knockdown LM2 cells with stable overexpression of FLAG-H2A and RNF8 or RNF168 were transfected with HA-ubiquitin (Ub), treated with IR (8 Gy), and cultured for 8 h, followed by immunoprecipitation with anti-FLAG beads and immunoblotting with antibodies against HA and FLAG. Before immunoprecipitation, lysates were heated at 95 °C for 5 min in the presence of 1% SDS (for denaturing), followed by a 10-fold dilution with lysis buffer and sonication. LE long exposure, SE short exposure. Statistical significance in b and f was determined by a two-tailed unpaired t -test. Error bars are mean ± SEM. n.s . not statistically significant. Source data are provided as a file.

Article Snippet: The cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, blocked with 3% bovine serum albumin in PBS, and incubated with antibodies against γH2AX (1:100, Cell Signaling Technology, #9718S), γH2AX (1:100, BD Biosciences, #560443), DGCR8 (1:100, Abcam, #ab191875), MDC1 (1:200, Bio-Rad, #AHP799), RNF8 (1:200, Proteintech, #14112-1-AP), RNF168 (1:100, Millipore, #ABE367), BRCA1 (1:20, Santa Cruz Biotechnology, #sc-6954), 53BP1 (1:100, Novus Biologicals, #NB100-304), FLAG (1:500, Sigma, #F7425), and MYC (1:500, Santa Cruz Biotechnology, #sc-40, clone 9E10) at 4 °C overnight, followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11008), Alexa Fluor 647 donkey anti-sheep IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-21448), Alexa Fluor 488 goat anti-mouse IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11001), Alexa Fluor 594 goat anti-rabbit IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11012), and/or Alexa Fluor 594 goat anti-mouse IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11005) at room temperature for 1 h. Coverslips were mounted on slides by using anti-fade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, #H-1200).

Techniques: Western Blot, Expressing, Transduction, shRNA, Knockdown, Cotransfection, Flow Cytometry, Control, Cell Culture, Immunoprecipitation, Incubation, Over Expression, Transfection, Ubiquitin Proteomics, Lysis, Sonication, Two Tailed Test

Journal: Nature Communications

Article Title: Non-canonical function of DGCR8 in DNA double-strand break repair signaling and tumor radioresistance

doi: 10.1038/s41467-021-24298-z

Figure Lengend Snippet: a Immunoblotting of USP36, USP51, and β-actin in parental and radioresistant LM2 cells with and without IR treatment (8 Gy followed by 24-h incubation). b Immunoblotting of DGCR8, USP36, USP51, and β-actin in USP36-knockdown and USP51-knockdown LM2 cells with or without IR treatment (8 Gy followed by 24-h incubation). c Co-IP of endogenous DGCR8 with endogenous USP51. LM2 and LM2-R cells were treated with 8-Gy IR. After 8 h, cells were lysed, immunoprecipitated with a DGCR8-specific antibody, and immunoblotted with antibodies against USP51 and DGCR8. SE short exposure, LE long exposure. d HEK293T cells with stable overexpression of MYC-DGCR8 were co-transfected with SFB-USP51 (wild-type or the C372S mutant) and HA-tagged ubiquitin or the lysine-specific mutant (K48 or K63), and then treated with IR (8 Gy). After 8 h, cells were lysed, denatured, and subjected to immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against HA and MYC. e Knockdown of USP51 decreased HR and NHEJ efficiency in LM2-DRR cells. Two days after co-transfection of I-SceI endonuclease and an exogenous donor for HR (pCAGGS DRR mCherry Donor EF1a BFP) into the USP51-knockdown LM2-DRR cells, the percentages of GFP-positive and mCherry-positive cells, gated on BFP-positive cells, were determined by flow cytometry. Repair by HR or NHEJ leads to mCherry or GFP expression. Data were normalized to the control cells. n = 3 biological replicates. f Quantification of γH2AX, DGCR8, MDC1, RNF8, RNF168, 53BP1, and BRCA1 foci in USP51-knockdown LM2 cells. Cells were incubated for 1 h after 2-Gy IR and immunostained with antibodies against γH2AX, DGCR8, MDC1, RNF8, RNF168, 53BP1, and BRCA1 (see representative images in Supplementary Fig. ). n = 3 biological replicates. g Control and USP51-knockdown LM2 cells with stable overexpression of FLAG-H2A and RNF8 or RNF168 were transfected with HA-ubiquitin (Ub), treated with IR (8 Gy), and cultured for 8 h, followed by immunoprecipitation with anti-FLAG beads and immunoblotting with antibodies against HA and FLAG. Before immunoprecipitation, lysates were heated at 95 °C for 5 min in the presence of 1% SDS (for denaturing), followed by a 10-fold dilution with lysis buffer and sonication. Statistical significance in e and f was determined by a two-tailed unpaired t -test. Error bars are mean ± SEM. Source data are provided as a file.

Article Snippet: The cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, blocked with 3% bovine serum albumin in PBS, and incubated with antibodies against γH2AX (1:100, Cell Signaling Technology, #9718S), γH2AX (1:100, BD Biosciences, #560443), DGCR8 (1:100, Abcam, #ab191875), MDC1 (1:200, Bio-Rad, #AHP799), RNF8 (1:200, Proteintech, #14112-1-AP), RNF168 (1:100, Millipore, #ABE367), BRCA1 (1:20, Santa Cruz Biotechnology, #sc-6954), 53BP1 (1:100, Novus Biologicals, #NB100-304), FLAG (1:500, Sigma, #F7425), and MYC (1:500, Santa Cruz Biotechnology, #sc-40, clone 9E10) at 4 °C overnight, followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11008), Alexa Fluor 647 donkey anti-sheep IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-21448), Alexa Fluor 488 goat anti-mouse IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11001), Alexa Fluor 594 goat anti-rabbit IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11012), and/or Alexa Fluor 594 goat anti-mouse IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11005) at room temperature for 1 h. Coverslips were mounted on slides by using anti-fade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, #H-1200).

Techniques: Western Blot, Incubation, Knockdown, Co-Immunoprecipitation Assay, Immunoprecipitation, Over Expression, Transfection, Mutagenesis, Ubiquitin Proteomics, Cotransfection, Flow Cytometry, Expressing, Control, Cell Culture, Lysis, Sonication, Two Tailed Test

Journal: Nature Communications

Article Title: Non-canonical function of DGCR8 in DNA double-strand break repair signaling and tumor radioresistance

doi: 10.1038/s41467-021-24298-z

Figure Lengend Snippet: a , b MYC-GFP-, WT DGCR8-, S677A-DGCR8-, and S677D-DGCR8-overexpressing LM2 cells with or without IR treatment ( a , 8 Gy followed by 1-h incubation; b , 8 Gy followed by 8-h incubation) were subjected to pulldown with MYC beads and immunoblotting with the indicated antibodies. c HEK293T cells with stable overexpression of MYC-tagged WT DGCR8, S677A-DGCR8, or S677D-DGCR8 were co-transfected with SFB-USP51 (WT or the C372S mutant) and HA-tagged ubiquitin, and then treated with IR (8 Gy). After 8 h, cells were lysed, denatured, and subjected to immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against HA and MYC. d Quantification of γH2AX, DGCR8, MDC1, RNF8, RNF168, 53BP1, and BRCA1 foci in DRCR8-knockdown LM2 cells with ectopic expression of WT DGCR8, S677A-DGCR8, or S677D-DGCR8. Cells were incubated for 1 h after 2-Gy IR and immunostained with antibodies against γH2AX, DGCR8, MDC1, RNF8, RNF168, 53BP1, and BRCA1 (see representative images in Supplementary Fig. ). n = 3 biological replicates. Statistical significance was determined by a two-tailed unpaired t -test. Error bars are mean ± SEM. e DRCR8-knockdown LM2 cells with ectopic expression of WT DGCR8 or the S677A mutant were transduced with FLAG-H2A and RNF8 or RNF168. The cells were then transfected with HA-ubiquitin (Ub), treated with IR (8 Gy), and cultured for 8 h, followed by immunoprecipitation with anti-FLAG beads and immunoblotting with antibodies against HA and FLAG. Before immunoprecipitation, lysates were heated at 95 °C for 5 min in the presence of 1% SDS (for denaturing), followed by a 10-fold dilution with lysis buffer and sonication. LE long exposure, SE short exposure. Source data are provided as a file.

Article Snippet: The cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, blocked with 3% bovine serum albumin in PBS, and incubated with antibodies against γH2AX (1:100, Cell Signaling Technology, #9718S), γH2AX (1:100, BD Biosciences, #560443), DGCR8 (1:100, Abcam, #ab191875), MDC1 (1:200, Bio-Rad, #AHP799), RNF8 (1:200, Proteintech, #14112-1-AP), RNF168 (1:100, Millipore, #ABE367), BRCA1 (1:20, Santa Cruz Biotechnology, #sc-6954), 53BP1 (1:100, Novus Biologicals, #NB100-304), FLAG (1:500, Sigma, #F7425), and MYC (1:500, Santa Cruz Biotechnology, #sc-40, clone 9E10) at 4 °C overnight, followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11008), Alexa Fluor 647 donkey anti-sheep IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-21448), Alexa Fluor 488 goat anti-mouse IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11001), Alexa Fluor 594 goat anti-rabbit IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11012), and/or Alexa Fluor 594 goat anti-mouse IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11005) at room temperature for 1 h. Coverslips were mounted on slides by using anti-fade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, #H-1200).

Techniques: Incubation, Western Blot, Over Expression, Transfection, Mutagenesis, Ubiquitin Proteomics, Immunoprecipitation, Knockdown, Expressing, Two Tailed Test, Transduction, Cell Culture, Lysis, Sonication