Journal: Cell & Bioscience

Article Title: KIF5B modulates central spindle organization in late-stage cytokinesis in chondrocytes

doi: 10.1186/s13578-019-0344-5

Figure Lengend Snippet: Cytokinetic phenotype in Kif5b knockdown ATDC5 cells. a Western blot of protein extracts from single cell clones with stable expression of sh-ctl or sh-kif5b constructs. b Immunofluorescence of KIF5B (green) in sh-Kif5b clone #4. Scale bar: 10 μm. c sh-ctl and sh-Kif5b cells stained with α-tubulin (red) and DAPI (blue). Yellow asterisks denote typical binucleated cells. Scale bar: 20 μm. d Quantification of the bi- and multi-nucleation rate in control cell clones ( n = 3, sh-ctl clone #1–3) and Kif5b-knockdown cell clones ( n = 5, sh-Kif5b clone #4–8). *** P < 0.0001; unpaired two-tailed t-test. Data are mean ± S.D. e Re-introduced GFP tagged full length KIF5B reduced the bi- and multi-nucleation rate in Kif5b knockdown ATDC5 cells clone #4 ( n = 10 independent experiments) and #5 ( n = 5 independent experiments). *** P < 0.0001; unpaired two-tailed t-test. Data are mean ± S.D. f Time-lapse images of sh-ctl and sh-Kif5b cells in mitosis. Scale bar: 10 μm. g Quantification of duration of cytokinesis in sh-ctl cells ( n = 84 cells from sh-ctl clone #1–3) and sh-Kif5b cells ( n = 68 cells from sh-Kif5b clone #4, #5 and #8). *** P < 0.0001; two-tailed Mann–Whitney U -test. The whisker plot shows median (lines), interquartile range (boxes) and 5% to 95% percentile (whiskers). Duration of cytokinesis was calculated from the furrow ingression to the final separation of the two daughter cells. Cells fusing back were not included in this analysis

Article Snippet: Other antibodies were as follow: anti-actin (Sigma, WB 1:2000), anti-α-tubulin (Sigma, WB: 1:10,000; immunofluorescences: 1:2000), anti-Aurora B (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-clathrin heavy chain (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-GM130 (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-PRC1 (Abcam, WB: 1:1000; Santa cruz, immunofluorescence: 1:100), phalloidin conjugated with Alexa Fluor 488 (Molecular probes) for F-actin labeling (1:300), CellLightTM Tubulin-GFP BacMam 2.0 (Molecular probes) for labeling tubulin in live cells, transferrin from human serum conjugated with rhodamine (Molecular probes).

Techniques: Knockdown, Western Blot, Clone Assay, Expressing, Construct, Immunofluorescence, Staining, Control, Two Tailed Test, MANN-WHITNEY, Whisker Assay

Journal: Cell & Bioscience

Article Title: KIF5B modulates central spindle organization in late-stage cytokinesis in chondrocytes

doi: 10.1186/s13578-019-0344-5

Figure Lengend Snippet: Midbody microtubule organization is impaired in Kif5b deficient cells. a Immunofluorescence of α-tubulin in primary chondrocytes in late cytokinesis. Scale bar: 10 μm. b Quantification of tubulin intensity in midbody in primary chondrocytes ( Kif5b fl/ + cells: n = 24; Col2cre ; Kif5b fl/ − cells: n = 27). *** P < 0.0001; two-tailed Mann–Whitney U -test. The whisker plot shows median (lines), interquartile range (boxes) and 10% to 90% percentile (whiskers). c Electron micrographs of midbody regions from sh-ctl and sh-Kif5b ATDC5 cells. Red lines denote the region of Flemming body. Yellow arrows and boxes denote the broken regions in Flemming body. Scale bar: 0.5 μm. d Quantification of the length of Flemming body in sh-ctl and sh-Kif5b cells (sh-ctl cells: n = 12; sh-Kif5b cells: n = 9). *** P < 0.0001; unpaired two-tailed t-test. Data are mean ± S.D. e Live imaging of sh-ctl and sh-Kif5b cells expressing GFP-tubulin in cytokinesis. Scale bar: 10 μm. f Quantification of the bi- and multi-nucleation rate in sh-Kif5b cells transiently expressing GFP, GFP-Kif5b and GFP-Kif5bΔMT ( n = 5 independent experiments). *** P < 0.0001; NS, P = 0.4039; unpaired two-tailed t-test. Data are mean ± S.D. g A model showing the function of KIF5B in cytokinesis. In late cytokinesis, KIF5B alone or together with other unknown molecules cross-links microtubules in the midbody, and therefore the structure of midbody can be stably maintained

Article Snippet: Other antibodies were as follow: anti-actin (Sigma, WB 1:2000), anti-α-tubulin (Sigma, WB: 1:10,000; immunofluorescences: 1:2000), anti-Aurora B (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-clathrin heavy chain (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-GM130 (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-PRC1 (Abcam, WB: 1:1000; Santa cruz, immunofluorescence: 1:100), phalloidin conjugated with Alexa Fluor 488 (Molecular probes) for F-actin labeling (1:300), CellLightTM Tubulin-GFP BacMam 2.0 (Molecular probes) for labeling tubulin in live cells, transferrin from human serum conjugated with rhodamine (Molecular probes).

Techniques: Immunofluorescence, Two Tailed Test, MANN-WHITNEY, Whisker Assay, Imaging, Expressing, Stable Transfection

Journal: Physiological Reports

Article Title: Circulating extracellular vesicle characteristics differ between men and women following 12 weeks of concurrent exercise training

doi: 10.14814/phy2.16016

Figure Lengend Snippet: EV surface marker expression is altered by AHRET in men and women. The proportion of EVs expressing SGCA (a), CD9 (b), VAMP3 (c), and THSD (d) were measured via Imaging Flow Cytometry and are shown as percentage of total gated EVs. Representative images of EV particles with bright‐field (BF) image of each particle and subsequent fluorescent channel images showing presence or absence of muscle‐derived EVs (SGCA+), microvesicles (VAMP3+), exosomes (CD9+) and apoptotic bodies (THSD+) vesicles are shown (e). N = 9. Statistical testing was done via three‐way ANOVA.

Article Snippet: Samples were then stained with the following antibodies and dilutions: anti‐human CD9 Alexa Fluor 700 (1:300 dilution; Novus Biologicals, CO), anti‐human VAMP3 Alexa Fluor 405 (1:300; Novus Biologicals), anti‐human thrombospondin (THSD‐1) Alexa Fluor 594 (1:100; Novus Biologicals), alpha sarcoglycan (SGCA) FITC (1:400; Biorbyt, St Louis, MO).

Techniques: Marker, Expressing, Imaging, Flow Cytometry, Derivative Assay

Journal: Applied Nanoscience

Article Title: Detection of heavy metals (Cu+2, Hg+2) by biosynthesized silver nanoparticles

doi: 10.1007/s13204-015-0452-4

Figure Lengend Snippet: Fig. 5 TEM micrographs of Cu-complex with AgNP (A, B, C) and SAED image (inset of C) from C

Article Snippet: The absorbance spectra of the AgNP were analyzed using a ‘SHIMADZU’ UV 1800 spectrophotometer and TEM images were taken using JEOL-JEM 2100 high-resolution transmission electron microscope (HRTEM).

Techniques:

Journal: Applied Nanoscience

Article Title: Detection of heavy metals (Cu+2, Hg+2) by biosynthesized silver nanoparticles

doi: 10.1007/s13204-015-0452-4

Figure Lengend Snippet: Fig. 4 TEM micrographs of AgNP (A, B) and SAED (C) image from (B)

Article Snippet: The absorbance spectra of the AgNP were analyzed using a ‘SHIMADZU’ UV 1800 spectrophotometer and TEM images were taken using JEOL-JEM 2100 high-resolution transmission electron microscope (HRTEM).

Techniques:

Journal: bioRxiv

Article Title: Spatio-temporal requirements of Aurora kinase A in mouse oocytes meiotic spindle building

doi: 10.1101/2024.04.01.587547

Figure Lengend Snippet: (A) Experimental design of WT oocytes treated with MLN during oocyte maturation. Red arrows indicate MLN addition time; blue arrows indicate fixation time. (B, F, J) Schematic of spindles at indicated stages, and the processes analyzed. (C, G, K) Representative confocal images of oocytes fixed at the indicated stages with and without MLN, immuno-stained with PCNT (gray), TACC3 (magenta), Tubulin (green) and DAPI (blue). Scale bars 10 µm. (D, H, L) Quantification of aMTOC parameters in (A), (G), (K) respectively. (E, I, M) Quantification of spindle and LISD parameters in (A), (G), (K) respectively. Black dots: DMSO-treated; gray dots: MLN-treated. See also Figure S1 which contains statistics.

Article Snippet: Primary antibodies and concentrations were used as follows: Pericentrin (Pcnt) (mouse, 1:100; BD Biosciences, #611814); TACC3 (Rabbit, 1:100; Novus Biologicals # NBP2-67671), Alpha Tubulin polyclonal (Sheep, 1:100; Cytoskeleton #ATN02); phosphorylated ABC (rabbit, 1:100; Cell Signaling Technology, #2914), phosphorylated CDC25B (1:100; Signalway Antibodies #11949); phosphorylated INCENP (Gift from Michael Lampson), Human anti-ACA (1:30, Antibodies Incorporated #15-234).

Techniques: Staining

Journal: bioRxiv

Article Title: Spatio-temporal requirements of Aurora kinase A in mouse oocytes meiotic spindle building

doi: 10.1101/2024.04.01.587547

Figure Lengend Snippet: (A) Experimental design of BC-KO oocytes treated with MLN; Red arrows indicate MLN addition time; blue arrows indicate fixation time. (B, F, J) Schematic of spindles at the indicated stages, and the processes analyzed. (C, G, K) Representative confocal images of oocytes fixed at the indicated stages with and without MLN, immuno-stained with PCNT (gray), TACC3 (magenta), Tubulin (green) and DAPI (blue). Scale bars 10 µm. (D, H, L) Quantification of aMTOC parameters in (A), (G), (K), respectively. (E, I, M) Quantification of spindle and LISD parameters in (A), (G), (K), respectively. Black dots: DMSO-treated; gray dots: MLN-treated. See also Figure S2 which contains statistics.

Article Snippet: Primary antibodies and concentrations were used as follows: Pericentrin (Pcnt) (mouse, 1:100; BD Biosciences, #611814); TACC3 (Rabbit, 1:100; Novus Biologicals # NBP2-67671), Alpha Tubulin polyclonal (Sheep, 1:100; Cytoskeleton #ATN02); phosphorylated ABC (rabbit, 1:100; Cell Signaling Technology, #2914), phosphorylated CDC25B (1:100; Signalway Antibodies #11949); phosphorylated INCENP (Gift from Michael Lampson), Human anti-ACA (1:30, Antibodies Incorporated #15-234).

Techniques: Staining

Journal: bioRxiv

Article Title: Spatio-temporal requirements of Aurora kinase A in mouse oocytes meiotic spindle building

doi: 10.1101/2024.04.01.587547

Figure Lengend Snippet: (A) Live cell light-sheet imaging of WT and BC-MLN-treated KO oocytes during Early pro-metaphase I; aMTOC (gray), SiR-tubulin (green), DNA (magenta). Time is indicated in h:min; Scale bar 10 μm (B, D) Quantification of spindle volume over time in WT (B) and BC-KO (D), respectively. (C, E) Quantification of area and aMTOC numbers over time in WT (C) and BC-KO (E), respectively. Yellow shadow: DMSO-treated; Purple shadow: MLN-treated. The red arrows indicate MLN addition time. Number of oocytes: WT DMSO: 11; WT MLN: 16; BC-KO DMSO:6; BC-KO MLN:8.

Article Snippet: Primary antibodies and concentrations were used as follows: Pericentrin (Pcnt) (mouse, 1:100; BD Biosciences, #611814); TACC3 (Rabbit, 1:100; Novus Biologicals # NBP2-67671), Alpha Tubulin polyclonal (Sheep, 1:100; Cytoskeleton #ATN02); phosphorylated ABC (rabbit, 1:100; Cell Signaling Technology, #2914), phosphorylated CDC25B (1:100; Signalway Antibodies #11949); phosphorylated INCENP (Gift from Michael Lampson), Human anti-ACA (1:30, Antibodies Incorporated #15-234).

Techniques: Imaging

Journal: bioRxiv

Article Title: Spatio-temporal requirements of Aurora kinase A in mouse oocytes meiotic spindle building

doi: 10.1101/2024.04.01.587547

Figure Lengend Snippet: (A) Schematic of the experimental design. ABC-KO oocytes microinjected with different AURKA fusions. Localization of AURKA is green. (B) Representative confocal images of Metaphase I ABC-KO oocytes expressing the indicated fusions. Non-injected ABC-KO oocytes were control. Oocytes were immuno-stained with TACC3 (magenta), tubulin (green), DAPI (blue). Localization of the targeted AURKA fusion is gray. (C) Quantification of spindle and LISD parameters in (B). Gray dots: Non-injected oocytes; pink dots: WT-AURKA; blue dots: MTOC-AURKA + chromatin-AURKA. (D, E) Representative confocal images of Metaphase I ABC-KO oocytes expressing the indicated fusions immuno-stained with tubulin (green), DAPI (DNA) in (D) and PCNT (green), TACC3 (magenta), DAPI (DNA) in (E). Localization of the targeted AURKA fusion is gray. Yellow arrow indicates MTOC-AURKA at kinetochores. Scale bar: 5µm. (F) Quantification of aMTOC parameters. (G) Quantification of spindle and LISD parameters. Gray dots: Non-injected oocytes; pink dots: WT-AURKA; green dots: MTOC-AURKA, and purple dots: chromatin-AURKA. (H) Live cell light-sheet imaging of WT and ABC-KO oocytes. ABC-KO oocytes expresses the indicated fusion, Sir-tubulin (green), DNA (magenta). (G) Time of MT nucleation and (H) time of spindle bipolarization (One-way ANOVA, **** p<0.0001). Data are represented as mean ± SEM. (K) Spindle volume during meiotic maturation. Time stamp (h:min) is relative to GVBD. In brackets are the number of oocytes analyzed in at least 3 independent experiments. See also Figure S3-S6.

Article Snippet: Primary antibodies and concentrations were used as follows: Pericentrin (Pcnt) (mouse, 1:100; BD Biosciences, #611814); TACC3 (Rabbit, 1:100; Novus Biologicals # NBP2-67671), Alpha Tubulin polyclonal (Sheep, 1:100; Cytoskeleton #ATN02); phosphorylated ABC (rabbit, 1:100; Cell Signaling Technology, #2914), phosphorylated CDC25B (1:100; Signalway Antibodies #11949); phosphorylated INCENP (Gift from Michael Lampson), Human anti-ACA (1:30, Antibodies Incorporated #15-234).

Techniques: Expressing, Injection, Control, Staining, Imaging

Journal:

Article Title: In Vivo Imaging of Bioluminescent Escherichia coli in a Cutaneous Wound Infection Model for Evaluation of an Antibiotic Therapy

doi: 10.1128/AAC.48.9.3436-3441.2004

Figure Lengend Snippet: Bacterial strain, plasmids, and primers used in the study

Article Snippet: Reactions were carried out in 50-μl volumes containing 5 μl of 10× PCR buffer (supplied with Taq DNA polymerase), 50 pmol of each oligonucleotide primer, each deoxynucleotide triphosphate (dATP, dCTP, dGTP, and dTTP) at a concentration of 0.2 mM, 1 U of Taq DNA polymerase, and 10 ng of plasmid DNA containing firefly luciferase (pSP luc ). table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain, plasmid, or primer Genotype or sequence Source or reference E. coli TOP10F′ Δ( ara - leu ) 7697 galU galK rpsL endA1 nupG Invitrogen (NO-C615-00) Plasmids pSP-luc Amp r Ori colE1 Promega (E1781) pRB474 Amp r Cm r Ori − Ori + 6 Primers LucF (5′-3′) AGG AA G GAT CC A GAG CAG ATT GTA CTG AGA G a This study LucR (5′-3′) AAT GCA GGT TAA CCT GGC TTA TCG AAA TT Open in a separate window a Underlined nucleotides show the position of the BamHI restriction site.

Techniques: Plasmid Preparation, Sequencing, Acetylene Reduction Assay

Journal:

Article Title: In Vivo Imaging of Bioluminescent Escherichia coli in a Cutaneous Wound Infection Model for Evaluation of an Antibiotic Therapy

doi: 10.1128/AAC.48.9.3436-3441.2004

Figure Lengend Snippet: Emission of light by E. coli cells as a function of storage time in Luria broth at two different temperatures (20 and 30°C). RLU, relative light units.

Article Snippet: Reactions were carried out in 50-μl volumes containing 5 μl of 10× PCR buffer (supplied with Taq DNA polymerase), 50 pmol of each oligonucleotide primer, each deoxynucleotide triphosphate (dATP, dCTP, dGTP, and dTTP) at a concentration of 0.2 mM, 1 U of Taq DNA polymerase, and 10 ng of plasmid DNA containing firefly luciferase (pSP luc ). table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain, plasmid, or primer Genotype or sequence Source or reference E. coli TOP10F′ Δ( ara - leu ) 7697 galU galK rpsL endA1 nupG Invitrogen (NO-C615-00) Plasmids pSP-luc Amp r Ori colE1 Promega (E1781) pRB474 Amp r Cm r Ori − Ori + 6 Primers LucF (5′-3′) AGG AA G GAT CC A GAG CAG ATT GTA CTG AGA G a This study LucR (5′-3′) AAT GCA GGT TAA CCT GGC TTA TCG AAA TT Open in a separate window a Underlined nucleotides show the position of the BamHI restriction site.

Techniques:

Journal:

Article Title: In Vivo Imaging of Bioluminescent Escherichia coli in a Cutaneous Wound Infection Model for Evaluation of an Antibiotic Therapy

doi: 10.1128/AAC.48.9.3436-3441.2004

Figure Lengend Snippet: Effects of increasing concentrations of SMX-TMP on the light emission (relative light units) of E. coli detected with the bioluminescence imaging system. Each concentration of SMX-TMP (0.3, 0.6, 0.9, and 1.2 mg/liter) was incubated with the bioluminescent E. coli for 30 min (▪) and 4 h (□) at 35°C in Luria broth. The sample labeled “Control” is the untreated strain.

Article Snippet: Reactions were carried out in 50-μl volumes containing 5 μl of 10× PCR buffer (supplied with Taq DNA polymerase), 50 pmol of each oligonucleotide primer, each deoxynucleotide triphosphate (dATP, dCTP, dGTP, and dTTP) at a concentration of 0.2 mM, 1 U of Taq DNA polymerase, and 10 ng of plasmid DNA containing firefly luciferase (pSP luc ). table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain, plasmid, or primer Genotype or sequence Source or reference E. coli TOP10F′ Δ( ara - leu ) 7697 galU galK rpsL endA1 nupG Invitrogen (NO-C615-00) Plasmids pSP-luc Amp r Ori colE1 Promega (E1781) pRB474 Amp r Cm r Ori − Ori + 6 Primers LucF (5′-3′) AGG AA G GAT CC A GAG CAG ATT GTA CTG AGA G a This study LucR (5′-3′) AAT GCA GGT TAA CCT GGC TTA TCG AAA TT Open in a separate window a Underlined nucleotides show the position of the BamHI restriction site.

Techniques: Imaging, Concentration Assay, Incubation, Labeling, Control

Journal:

Article Title: In Vivo Imaging of Bioluminescent Escherichia coli in a Cutaneous Wound Infection Model for Evaluation of an Antibiotic Therapy

doi: 10.1128/AAC.48.9.3436-3441.2004

Figure Lengend Snippet: In vivo monitoring of E. coli(pRB474) bioluminescence in the cutaneous wound by use of the bioluminescence imaging system. Each set of data represents the mean (±standard deviation |) number of relative light units (RLU) for three hairless rats either untreated (♦) or treated with SMX-TMP (▪; 30 mg/kg) and imaged for 5 min at 0, 4, 8, 24, 36, and 48 h postinfection. *, statistically different by the Student t test.

Article Snippet: Reactions were carried out in 50-μl volumes containing 5 μl of 10× PCR buffer (supplied with Taq DNA polymerase), 50 pmol of each oligonucleotide primer, each deoxynucleotide triphosphate (dATP, dCTP, dGTP, and dTTP) at a concentration of 0.2 mM, 1 U of Taq DNA polymerase, and 10 ng of plasmid DNA containing firefly luciferase (pSP luc ). table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain, plasmid, or primer Genotype or sequence Source or reference E. coli TOP10F′ Δ( ara - leu ) 7697 galU galK rpsL endA1 nupG Invitrogen (NO-C615-00) Plasmids pSP-luc Amp r Ori colE1 Promega (E1781) pRB474 Amp r Cm r Ori − Ori + 6 Primers LucF (5′-3′) AGG AA G GAT CC A GAG CAG ATT GTA CTG AGA G a This study LucR (5′-3′) AAT GCA GGT TAA CCT GGC TTA TCG AAA TT Open in a separate window a Underlined nucleotides show the position of the BamHI restriction site.

Techniques: In Vivo, Imaging, Standard Deviation

Journal:

Article Title: In Vivo Imaging of Bioluminescent Escherichia coli in a Cutaneous Wound Infection Model for Evaluation of an Antibiotic Therapy

doi: 10.1128/AAC.48.9.3436-3441.2004

Figure Lengend Snippet: Monitoring of the effects of SMX-TMP on bioluminescent E. coli(pRB474) in a hairless rat. Bioluminescent bacteria were injected into the wound (5 × 107 CFU). Shown is one treated (SMX-TMP) E. coli(pRB474)-infected rat (right set of panels) and one untreated E. coli(pRB474)-infected rat (left set of panels) imaged dorsally for 5 min at 4, 24, and 48 h postinfection.

Article Snippet: Reactions were carried out in 50-μl volumes containing 5 μl of 10× PCR buffer (supplied with Taq DNA polymerase), 50 pmol of each oligonucleotide primer, each deoxynucleotide triphosphate (dATP, dCTP, dGTP, and dTTP) at a concentration of 0.2 mM, 1 U of Taq DNA polymerase, and 10 ng of plasmid DNA containing firefly luciferase (pSP luc ). table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain, plasmid, or primer Genotype or sequence Source or reference E. coli TOP10F′ Δ( ara - leu ) 7697 galU galK rpsL endA1 nupG Invitrogen (NO-C615-00) Plasmids pSP-luc Amp r Ori colE1 Promega (E1781) pRB474 Amp r Cm r Ori − Ori + 6 Primers LucF (5′-3′) AGG AA G GAT CC A GAG CAG ATT GTA CTG AGA G a This study LucR (5′-3′) AAT GCA GGT TAA CCT GGC TTA TCG AAA TT Open in a separate window a Underlined nucleotides show the position of the BamHI restriction site.

Techniques: Bacteria, Injection, Infection

Journal: Scientific Reports

Article Title: Changes in EGFR activity following CRISPR/Cas9-editing of the EGF binding domain

doi: 10.1038/s41598-026-37579-8

Figure Lengend Snippet: Distribution of EGF and EGFR and phosphorylation status of EGFR in EGF treated cells. ( A ) ICC for EGF and EGFR in untreated cells and cells treated with EGF for 30 min Confocal imaging was done on laser scan confocal Nikon Ti microscope at 100x magnification; staining was done for nuclei (Hoechst 33342), EGF (ab9595 Abcam, Cambridge, United Kingdom) and EGFR (antibody recognizing extracellular domain of human EGFR: epitope 6-273 aa, antibody clone sc-101; Santa Cruz Biotechnology, TX, United States). The scale bars are 50 μm. ( B ) Phosphorylation of EGFR upon treatment with EGF was determined by Western blot. Whole cell lysates were obtained from cells without treatment or 30 min after treatment with recombinant EGF. Three independent experiments were done and imaged using Li-Cor system; representative Western blots are shown. Normalization was done using α-Tubulin as a loading control (Catalog number 926-42213 Li-Cor, NE, United States). Antibodies for phosphorylated EGFR included Y1068 (Catalog number #3777, Cell Signaling, MA, United States), Y1086 (Catalog number #2220S, Cell Signaling, MA, United States) and Y1173 (Catalog number ab32578 Abcam, Cambridge, United Kingdom). ( C ) Western blot quantification was done using software provided by Li-Cor and plotted using Graph Pad Prizm. This bar graph represents relative quantification of phosphorylation of EGFR (for tyrosine positions Y1068, Y1086 and Y1173) for wild type ME180 cells and mutant clone VII11 at 30 min after EGF treatment. To show relative phosphorylation values, the highest phosphorylation signal (that for Tyrosine 1068 in wild type cells) was set as 1. Results are presented as the means ± SD of at least three independent experiments.

Article Snippet: Normalization was done using anti-Beta-Actin monoclonal primary antibody (Catalog number 926-42210 Li-Cor, NE, United States) or anti-Alpha-Tubulin Mouse monoclonal antibody (Catalog number 926-42213 Li-Cor, NE, United States).

Techniques: Phospho-proteomics, Imaging, Microscopy, Staining, Western Blot, Recombinant, Control, Software, Quantitative Proteomics, Mutagenesis

Journal: Cells

Article Title: Human Pericardial Fluid-Derived Cells Exhibit Mesothelial-like Properties and Exert Proangiogenic Effects on Endothelial Cells

doi: 10.3390/cells14231855

Figure Lengend Snippet: ( a – c ) Characterization of pericardial fluid-derived MCs ( a ) Representative images showing MC culture stages: cell layers; ( b ) 2-day explant culture; ( c ) 10-day MC culture; ( d ) Growth kinetics of cardiac MCs; ( e – h ) Flow cytometry analysis of immunophenotypic markers. Blue histograms represent cells stained with antibodies against ( e ) E-cadherin, ( f ) Calretinin, ( g ) CD34, and ( h ) CD45; green histograms show IgG isotype controls; ( i ) Immunoblotting data show the expression of Notch1 FL (full length), N1ICD, Jagged1, E-cadherin, and tubulin proteins in MCs obtained from pericardial fluid. All the data were obtained from cells isolated from four patients (pat).

Article Snippet: Membranes were blocked with 5% non-fat milk in TBST and subsequently incubated overnight at 4 °C with primary antibodies anti-Tubulin (Cell Signaling, Boston, MA, USA), anti-Notch1 (Cell Signaling, Boston, MA, USA), anti-E-cadherin (Abcam, Cambridge, UK) and anti-Jagged1 (ABclonal Biotechnology Co., Ltd., China).

Techniques: Derivative Assay, Flow Cytometry, Staining, Western Blot, Expressing, Isolation