human β 1 integrin subunits  (Proteintech)

Journal: bioRxiv

Article Title: THE ASSOCIATION OF CD47 WITH INTEGRIN Mac-1 REGULATES MACROPHAGE RESPONSES BY STABILIZING THE EXTENDED INTEGRIN CONFORMATION

doi: 10.1101/2022.09.30.510402

Figure Lengend Snippet: (A) Purified inflammatory peritoneal mouse macrophages were immunoprecipitated with anti-α M rabbit polyclonal antibody or rabbit polyclonal anti-CD47 antibody, and blots were analyzed with rabbit polyclonal antibody against the α M or CD47. Blots of the total cell lysates of WT and CD47-deficient macrophages using rabbit polyclonal antibody against the α M or CD47are shown in the left panel. M, molecular weight markers. The molecular weight of the α M (165 kDa) and β 2 (95 kDa) integrin subunits and CD47 (47 kDa) are indicated on the right of the panel. (B) Murine IC-21 macrophages were immunoprecipitated with anti-α M rabbit polyclonal antibody or rabbit polyclonal anti-CD47 antibody, and blots were analyzed with rabbit polyclonal antibody against the α M or CD47. (C) Biotinylated Mac-1-HEK293 cells were lysed and immunoprecipitated with mAb 44a against the α M subunit or isotype control IgG1. Blots were disclosed with streptavidin-alkaline phosphatase (AP). (D) Suspended (denoted “s”) or adherent (denoted “a”) Mac-1-HEK293 cells were lysed and immunoprecipitated with anti-α M mAb 44a or anti-β 2 mAb IB4. Blots were analyzed with anti-α M , anti-β 2 , and anti-CD47 antibodies. (E) The ratios of CD47 to the α M and β 2 integrin subunits in the immunoprecipitates from suspended and adherent cells were determined from the densitometry analyses of blots. The ratio of CD47 to each integrin subunit in suspended cells was taken as 1.0. (F) Lysates of biotinylated Mac-1-HEK293 cells were immunoprecipitated with anti-CD47 mAb B6H12; then immunoprecipitates were subjected to Western blotting probed with streptavidin-AP ( left panel; 1 IP). After the first round of immunoprecipitation, the supernatant was immunoprecipitated with anti-α M mAb 44a ( middle panel; 2 IP). The third round of immunoprecipitation (3 IP) was performed using anti-β 1 mAb ( right panel ).

Article Snippet: The mouse anti-human α M mAb (catalog #66519-1-Ig) and the rabbit polyclonal antibody, which recognizes both mouse and human β 1 integrin subunits (catalog #12594-1-AP), were from Proteintech (Rosemont, IL).

Techniques: Purification, Immunoprecipitation, Molecular Weight, Western Blot

Journal: Biomaterials

Article Title: Modular GAG-matrices to promote mammary epithelial morphogenesis in vitro

doi: 10.1016/j.biomaterials.2016.10.007

Figure Lengend Snippet: (A) Storage modulus (Pa) of BME and soft and stiffPEG-HEP matrices (crosslinking degree of γ=0.63 and γ=1.25, respectively). (B) Top row: Bright field images of MEC colony morphology after culture in reference BME and soft and stiff PEG-HEP matrices (scale bar = 100 μm). Middle/bottom row: Confocal immunofluorescence of polarization markers in MEC colony cross sections from BME and soft and stiff PEG-HEP matrices (scale bar = 20 μm). Middle row: Immunofluorescence images of MECs stained for polarity markers β4-integrin (arrows; red) and β-catenin (arrowhead; green), and nuclei (DAPI; blue). Bottom row: Immunofluorescence images of MECs stained for laminin-332 (arrow; green), actin filaments (red), and nuclei (DAPI; blue). (C) Left: Percentage of acini containing cleared lumens. Right: Representative images of DAPI-stained acini showing luminal clearance. (D) Left: Percentage of acini containing apically oriented Golgi apparatus. Right: Representative images of GM-130- (Golgi apparatus) and DAPI-stained (nucleus) acini showing orientation of the Golgi relative to the nuclei. (E) Percentage of acini showing an invasive (non-spherical) phenotype. (F and G) Ultra-structural analysis of MECs cultured in soft PEG-HEP matrices. (F) Left image shows several desmosomes (red arrows) at the epithelial cell-cell interface (scale bar = 500 nm). Right image is a magnified inset of the left image showing the desmosomes (red arrows) with connected cytoplasmic filaments (red arrowheads, scale bar = 500 nm). (G) The top left overview image is a representative MEC colony with cleared lumen (L). Note that the hydrogel (G) polymeric network appears degraded in the periphery of the basal (B) acini surface (scale bar = 20 μm). The left bottom image is a magnified inset of the left top image (scale bar = 2 μm). The right image is further magnified at the cell-matrix interface, showing the typical dense dark structures of hemidesmosomes (arrows), which are connected to cytoplasmic filaments (arrowheads, scale bar = 500 nm). G = gel, L = lumen, B = basal side of acini. A, C, D and E are mean ± S.D. (A) ANOVA with Tukey's multiple comparison, n = 3. (C) and (D) n = 50 acini counted from three independent experiments; un-paired t-test. (E) n = 100-150 from one representative experiment; Kruskal-Wallis test with Dunn's multiple comparisons. All images and measurements in this figure are of cells after 14 days in culture. n.s. = not significant; p ≥ 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: For functional integrin blocking studies antibodies directed against human integrin subunit β 1 (BD Biosciences 552828) and integrin heterodimer α 6 β 4 (α 6 (BD Biosciences 555734), β 4 (Millipore MAB2059)) were added to the PEG-MCP precursor solution and to the cell culture media at a final concentration of 20 μg/ml.

Techniques: Immunofluorescence, Staining, Cell Culture

Journal: Biomaterials

Article Title: Modular GAG-matrices to promote mammary epithelial morphogenesis in vitro

doi: 10.1016/j.biomaterials.2016.10.007

Figure Lengend Snippet: (A) Storage modulus (Pa) of matrices with decreasing biomolecular polymer network functionalization. Left to right: MMP-cleavable PEG-HEP, MMP-insensitive PEG-scr-HEP, and MMP-cleavable PEG-PEG matrices. (B) Bright field images showing the colony morphology of MECs grown in soft synthetic matrices with different degrees of biofunctionality: (I) PEG-HEP, (II) PEG-scr-HEP, and (III) PEG-PEG matrices (scale bar = 200 μm). Bottom row: magnified image of the white box in top row (scale bar = 50 μm). (C) Confocal immunofluorescence images of MEC colony cross sections grown in the different matrices (as in B, scale bar = 20 μm). Top row: Immunfluorescence of β4-integrin (arrows; red), β-catenin (arrowhead; green), and nuclei (DAPI; blue). Bottom row: Immunfluorescence of the basement membrane protein laminin-332 (arrows; green), actin filaments (red), and nuclei (blue) (D) MECs were grown in PEG-MCP-HEP, PEG-scr-HEP, or PEG-MCP-PEG matrices, brightfield images were taken, and the colony diameters measured. (E) Percentage of acini containing cleared lumens. (F) Percentage of acini containing apically oriented Golgi apparatus. A, D, E and F are mean ± S.D. (A) ANOVA with Tukey's multiple comparison, n = 3; (E) and (F) unpaired t-test, n = 50 from three independent experiments. (D) Kruskal-Wallis test with Dunn's multiple comparisons; n= 100-150 from one representative experiment. All images and measurements in this figure are of cells after 14 days in culture. n.s. = not significant (p ≥ 0.05), ** p< 0.01, *** p< 0.001.

Article Snippet: For functional integrin blocking studies antibodies directed against human integrin subunit β 1 (BD Biosciences 552828) and integrin heterodimer α 6 β 4 (α 6 (BD Biosciences 555734), β 4 (Millipore MAB2059)) were added to the PEG-MCP precursor solution and to the cell culture media at a final concentration of 20 μg/ml.

Techniques: Immunofluorescence

Journal: Biomaterials

Article Title: Modular GAG-matrices to promote mammary epithelial morphogenesis in vitro

doi: 10.1016/j.biomaterials.2016.10.007

Figure Lengend Snippet: (A) Top row: Confocal immunofluorescence images of laminin-332 (top, green) or laminin-111 (bottom, green) expression in MEC colonies grown in soft, degradable PEG-HEP hydrogels for the number of days indicated (scale bar = 20 μm). Top row: After two days laminin-332 was deposited partially around the outer colony surface. By day 4 of culture, laminin-332 appears fully distributed around the outer acini surface (actin filaments, red; nuclei, blue). Bottom row: No extracellular laminin-111 deposition was observed in the cultures. (B) MECs were grown in soft, degradable PEG-HEP matrices in the presence or absence of β1 or α6β4 integrin blocking antibodies. Brightfield images were taken, and the colony diameters measured. MEC colony growth, as measured by brightfield microscopy, is impaired by blocking β1 or α6β4 integrin function. (C) Colony diameter after 14 days. Kruskal-Wallis test with Dunn's multiple comparisons, n = 130-150 from one representative experiment. n.s. = not significant (P ≥ 0.05); *** P < 0.001. (D) Bright field images of MECs grown in soft, degradable PEG-HEP matrices after treatment with integrin blocking antibodies (β1 or α6β4; scale bar = 20 μm). (E) Diagram of the hypothesized mechanism of polarized acini development in soft, degradable PEG-HEP matrices. MECs embedded within soft, enzymatically degradable PEG-HEP hydrogels constitutively secrete LN-332, which binds to heparin and through binding to integrins acts as a signaling molecule to promote MEC morphogenesis into polarized acini.

Article Snippet: For functional integrin blocking studies antibodies directed against human integrin subunit β 1 (BD Biosciences 552828) and integrin heterodimer α 6 β 4 (α 6 (BD Biosciences 555734), β 4 (Millipore MAB2059)) were added to the PEG-MCP precursor solution and to the cell culture media at a final concentration of 20 μg/ml.

Techniques: Immunofluorescence, Expressing, Blocking Assay, Microscopy, Binding Assay