Journal: Frontiers in Cardiovascular Medicine

Article Title: Deficiency of Myeloid Pfkfb3 Protects Mice From Lung Edema and Cardiac Dysfunction in LPS-Induced Endotoxemia

doi: 10.3389/fcvm.2021.745810

Figure Lengend Snippet: Myeloid-specific Pfkfb3 deficiency attenuates LPS-induced inflammatory responses. (A) Representative images (left) and quantification (right) for immunohistochemical staining of the neutrophil marker Ly6G in lung sections of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 5). (B) Representative images (left) and quantification (right) for immunohistochemical staining of the macrophage marker Mac2 in lung sections of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 5). (C–E) qPCR analysis of the mRNA levels of Il1b (C) , Il6 (D) and Nos2 (E) in the lung of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 6). (F,G) ELISA analysis of Il1b (F) and Il6 (G) in serum of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 4). (H) NO levels in serum of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 3–4). All data are represented as mean ± SEM, ** P < 0.01 and *** P < 0.001 for Pfkfb3 WT vs. Pfkfb3 ΔMϕ (unpaired two-tailed Student's t test).

Article Snippet: After antigen retrieval with Antigen Unmasking Solution (H-3301, Vector Laboratories, Burlingame, CA, USA) at 98°C for 10 min, sections were blocked with avidin solution with 10% normal rabbit serum for 1 h at room temperature, and incubated in biotin blocking solution with primary antibodies against Mac2 (3 μg/mL, CL8942F, Cedarlane, Burlington, NC, USA), or Ly6G (3 μg/mL, 551459, BD biosciences, San Jose, CA, USA) at 4°C overnight.

Techniques: Immunohistochemical staining, Staining, Marker, Injection, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Proteomic Profiling of Two Distinct Populations of Extracellular Vesicles Isolated from Human Seminal Plasma

doi: 10.3390/ijms21217957

Figure Lengend Snippet: Isolation and characterization of LEV and SEV. Total EV in seminal plasma of vasectomized men were collected by UC at the interface of a sucrose block gradient. LEV and SEV were separated by their distinct velocities during upward displacement into a continuous sucrose density gradient. ( A ) Gradient fractions were analyzed by SDS-PAGE followed by Sypro ruby staining for total protein. ( B ) Gradient fractions were analyzed by immunoblotting for the presence of EV associated proteins, including CD9, CD81, PSCA, Galectin-3, CD63, HSP70, Annexin A1, and GLIPR2. Molecular weight markers are indicated on the left in kDa. ( C ) Particles in the LEV and SEV containing fractions were analyzed by TEM. Scale bar, 500 nm. Arrows exemplify incidental SEV in the LEV isolate. ( D ) Size distribution of the particles in LEV and SEV isolates as determined by NTA.

Article Snippet: The primary antibodies used were mouse anti human CD9 (HI9a, 312102, Biolegend, San Diego, California, USA, 1:2000); mouse anti human CD81 (B11, sc-166029, Santa Cruz Biotechnology, Dallas, Texas, USA, 1:500); mouse anti human HSP70 (N27F3-4, ADI-SPA-820-D, Enzo, Bruxelles, Belgium, 1:1000); mouse anti human Flotillin-1 (clone 18, 610821, BD Biosciences, San Jose, California, USA, 1:1000); mouse anti-human PSCA (clone 7F5, sc-80654, Santa Cruz Biotechnology, Dallas, Texas, USA, 1:1000); mouse anti human Annexin A1 (clone 29, 610066, BD Biosciences, San Jose, California, USA, 1:1000); mouse anti human CD47 (B6H12, sc-12730, Santa Cruz Biotechnology, Dallas, Texas, USA, 1:500); rat anti human galectin-3 (M3/38, CL8942B, CEDARLANE, Burlington, Vermont, USA, 1:500) and rabbit anti human GLIPR2/GAPR-1 (Eberle et al., 2002, 1:5000, kindly provided by J.B. Helms).

Techniques: Isolation, Blocking Assay, SDS Page, Staining, Western Blot, Molecular Weight

Journal: Oncogene

Article Title: A role for collagen XXIII in cancer cell adhesion, anchorage-independence, and metastasis

doi: 10.1038/onc.2011.406

Figure Lengend Snippet: Whole cell lysates were collected from H460 clonal cell lines and probed for various proteins involved in cell adhesion. In the knockdown cell lines, a reduction in OB-cadherin, gamma-, beta-, and alpha-catenin, vimentin and galectin-3 was observed. Analysis of the canonical cadherins revealed no changes in P-cadherin. E- and N-cadherin are not expressed in these cells. Actin was used as a loading control.

Article Snippet: The antibodies used were mouse monoclonal antibodies to collagen XXIII, galectin-3 (R&D Systems), integrins α2, α3, α5, β3, β-catenin, p120-catenin, E, N, and P-Cadherin, FAK, pFAK397, (BD Biosciences), actin (Chemicon), vimentin (DAKO) and rabbit antibodies to ILK1, α-catenin, γ-catenin, OB-Cadherin, AKT, Src, pSrcY416, and integrins α4, αv, α6, β1, β4, β5 (Cell Signal).

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: Transmembrane Mucin 1 Blocks Fluorescein Ingress to Corneal Epithelium

doi: 10.1167/iovs.63.2.31

Figure Lengend Snippet: (A) The expression of MUC16 protein in HCECs separately transduced with two lentiviral-based shRNA vectors (shMUC16-1 or shMUC16-2) were examined through a Western blot analysis. The a-tubulin was used as a loading control. (B) Representative flow cytometric analyses of fluorescence intensity and MUC16 expressions in transduced HCECs followed by fluorescein staining were shown in contour plots. (C) Representative flow cytometric analyses of fluorescence intensity and MUC16 expression in HCECs cultured in KSFM ( left ) or MPM ( right ) followed by fluorescein staining were presented in contour plots. (D) The frozen tissue sections prepared from fluorescein-stained corneal epithelia were stained with GAL3 ( red , immunofluorescence), and the cell nuclei were counterstained with DAPI ( blue , immunofluorescence).

Article Snippet: The cryosections were then stained with a biotin-conjugated anti-rabbit MUC1 rabbit antibody (no. NBP1-60046B; Novus Biologicals, Littleton, CO, USA), anti-ZO-1 (no. 33-9100; ThermoFisher Scientific, Waltham, MA, USA), or anti-galectin-3 (GAL3) mouse antibody (no. A3A12, Novus Biologicals) for one hour followed by incubation in a Texas Red conjugate of NeutrAvidin biotin-binding protein (no. A2665; Invitrogen, Carlsbad, CA, USA), Alexa Fluor 594 goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (no. A-11012; Invitrogen), or Alexa Fluor 564-conjugated goat anti-mouse IgG antibody (no. A-11003) for one hour in the darkness.

Techniques: Expressing, Transduction, shRNA, Western Blot, Control, Fluorescence, Staining, Cell Culture, Immunofluorescence