Journal: eLife

Article Title: Genome-wide CRISPR screens of oral squamous cell carcinoma reveal fitness genes in the Hippo pathway

doi: 10.7554/eLife.57761

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-human actin (clone C4), Mouse monoclonal , Merck Milipore , MAB1501 , WB – (1:5000).

Techniques: Recombinant, Genome Wide, Knock-Out, CRISPR, Plasmid Preparation, Cloning, Fluorescence, Transfection, Derivative Assay, Western Blot, Purification, Reverse Transcription, SYBR Green Assay, Software

Journal: International Journal of Environmental Research and Public Health

Article Title: Liquid-Diet with Alcohol Alters Maternal, Fetal and Placental Weights and the Expression of Molecules Involved in Integrin Signaling in the Fetal Cerebral Cortex

doi: 10.3390/ijerph7114023

Figure Lengend Snippet: Maternal alcohol changed expression levels of molecules involved in integrin signaling in the fetal cerebral cortices. A. Brain from a GD18 fetal head (top) and dissected cerebral cortices (bottom). B. Bands representing expression levels of integrin subunits (β 1 , α 3 and α 6 ), phospholipase Cγ isoforms (PLC-γ 1 and PLC-γ 2 ), N-cadherin (CADH), β-Actin and GPDH from control and alcohol exposed fetal cerebral cortices. Each band represents expression of molecule in a pooled sample of cortices from two fetuses per mother. C. Mean + standard errors of mean values of the optical densities of bands in arbitrary units (au) from control and alcohol exposed cerebral cortices. * Significantly different from the respective controls at p < 0.05.

Article Snippet: β-Actin , Mouse monoclonal (Clone C4) , BD Biosciences, San Jose, CA, USA.

Techniques: Expressing

Journal:

Article Title: Skin-Homing Receptors on Effector Leukocytes Are Differentially Sensitive to Glyco-Metabolic Antagonism in Allergic Contact Dermatitis 1

doi:

Figure Lengend Snippet: E-selectin-binding PSGL-1 is expressed on NK cells. A, Western blot analysis of E-selectin ligand expression was performed on DX5+ CD90− NK cell lysates (40 μg/lane) from both wt and PSGL-1−/− mice. Compared with total LN leukocyte, CD90+ T cell or DX5− CD90− cell lysates, a demonstrable E-selectin-Ig-stained band was evident in DX5+ CD90− NK cell lysate at 260 kDa, which was absent in DX5+ CD90− NK cell lysate from PSGL-1−/− mice. Protein loading was controlled for by staining lysates with anti-β-actin. B, Western blot analysis of E-selectin ligand expression was performed on control KG1a cell lysate and on total PBMC, CD3+ T cell, CD56+ CD3− NK cell, and CD56− CD3− cell lysates (30 μg/lane). Compared with E-selectin-Ig-stained bands principally represented by CD44 (95kDa) and PSGL-1 (130 and 260kDa) in KG1a lysate, there were also E-selectin-Ig-stained bands at 130 and 260 kDa in CD56+ CD3− NK cell lysates that comigrated with KG1a PSGL-1, in addition to another band at 155 kDa. Protein loading was controlled by staining lysates with anti-PSGL-1 moAb KPL-1. C, To confirm the E-selectin-binding function of PSGL-1, PSGL-1 was immunoprecipitated with anti-PSGL-1 moAb KPL-1 or isotype Ab from component cell lysates and Western blotted with E-selectin-Ig or KPL-1. As shown, PSGL-1 bound E-selectin-Ig in all immunoprecipitates (C). Experiments were performed a minimum of three times.

Article Snippet: To control for protein loading, blots were performed in parallel using anti-PSGL-1 moAb KPL-1 or anti- β -actin moAb C4 (BD Biosciences).

Techniques: Binding Assay, Western Blot, Expressing, Staining, Immunoprecipitation

Journal:

Article Title: Skin-Homing Receptors on Effector Leukocytes Are Differentially Sensitive to Glyco-Metabolic Antagonism in Allergic Contact Dermatitis 1

doi:

Figure Lengend Snippet: PSGL-1 is the principal E-selectin ligand on the NK cell surface. Lysates (30 μg/lane) were prepared of DX5+ CD90− NK cells from wt, PSGL-1−/−, or FT4/7−/− mice. Where indicated, DX5+ CD90− NK cells were treated with protease (0.1% bromelain) before lysate preparation. Western blot analysis of E-selectin ligand showed that protease treatment removed E-selectin-binding PSGL-1 (260kDa) and that PSGL-1 E-selectin-binding determinants were absent in FT4/7-deficient NK cells. Western blotting with anti-PSGL-1 moAb KPL-1 confirmed that PSGL-1 was eliminated on protease-treated cells and absent in PSGL-1−/− mice and that PSGL-1 was present in all other groups. Equivalent protein loading was validated by staining lysates with anti-β-actin moAb. Experiments were performed a minimum of three times.

Article Snippet: To control for protein loading, blots were performed in parallel using anti-PSGL-1 moAb KPL-1 or anti- β -actin moAb C4 (BD Biosciences).

Techniques: Western Blot, Binding Assay, Staining

Journal:

Article Title: Skin-Homing Receptors on Effector Leukocytes Are Differentially Sensitive to Glyco-Metabolic Antagonism in Allergic Contact Dermatitis 1

doi:

Figure Lengend Snippet: Oral efficacy of 4-F-GlcNAc on allergic CHS. CHS responses were generated by sensitizing wt or Rag1−/− mice on days 0 and 1 and challenged on day 5 with DNFB or vehicle control. Mice (10 mice/group) were administered i.p. or p.o. with 0.9% saline or 100 mg/kg 4-F-GlcNAc from days 1–5. Ear thickness measurements before and 24 h after DNFB-challenge were monitored to assess anti-inflammatory efficacy of 4-F-GlcNAc. DX5+ NK1.1+ NK cells from ILN of saline or treated wt and Rag1−/− mice and L-selectin− TCR-β + T cells from ILN of saline or treated wt mice were analyzed for E-selectin ligand expression by flow cytometry. Fresh DX5+ CD90− NK cells lysates were also prepared from saline or treated wt mice and analyzed for E-selectin ligand (PSGL-1) and β-actin expression by Western blotting. Both i.p. and p.o. 4-F-GlcNAc treatment caused significant reductions in mean ear swelling (statistically significant difference compared with saline treatment control; Student’s paired t test, **, p < 0.001 and *, p < 0.01, respectively), while CHS responses in Rag1-deficient mice were not affected (A). E-selectin ligand expression on DX5+ NK1.1+ NK cells from all groups was analyzed by flow cytometry and showed that ligand expression was relatively insensitive to 4-F-GlcNAc (B). Western blot analysis of E-selectin ligand and PSGL-1 expression on DX5+ CD90− NK cells (30 μg/lane) revealed that 4-F-GlcNAc did not ablate E-selectin-binding PSGL-1 (C). Anti-PSGL-1 and anti-B-actin immunoblots showed that loaded protein levels were similar (C). There was no staining evident in isotype control blots or in E-selectin-Ig blots performed in the presence of 5 mM EDTA. Flow cytometric analysis of E-selectin ligand expression on L-selectin− and TCR-β+ cells revealed that both i.p. and p.o. 4-F-GlcNAc treatments significantly reduced E-selectin ligand expression compared with saline treatment controls (statistically significant difference compared with corresponding saline treatment control; Student’s paired t test, **, p < 0.004; *, p < 0.04) (D). E-selectin ligand expression on L-selectin+ TCR-β+ T cells was unchanged following 4-F-GlcNAc treatment (D). Experiments were performed a minimum of three times.

Article Snippet: To control for protein loading, blots were performed in parallel using anti-PSGL-1 moAb KPL-1 or anti- β -actin moAb C4 (BD Biosciences).

Techniques: Generated, Expressing, Flow Cytometry, Western Blot, Binding Assay, Staining