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

Article Title: Rheumatoid arthritis synovial stromal cells inhibit apoptosis and up-regulate Bcl-xL expression by B cells in a CD49/CD29-CD106-dependent mechanism.

doi: 10.4049/jimmunol.164.2.1110

Figure Lengend Snippet: FIGURE 7. Stimulation by VLA-4 can induce Bcl-xL protein and mRNA in B cells. B cells (1 3 106) were cultured for 3 days with mAbs to CD11a/CD18 and/or CD29/CD49d in 24-well culture plates coated with rabbit anti-mouse IgG. Afterward, the expression of Bcl-2 and Bcl-xL pro- tein was assessed by Western blotting, and the percentages of viable and apoptotic cells were measured as described in Materials and Methods, and mRNA for Bcl-2, Bcl-xL, and G6PD were assessed by RT-PCR as de- scribed in Materials and Methods. The results of Southern blotting are shown in Expt. 1, and the expression of PCR products by ethidium bromide staining is shown in Expt. 2. One to three micrograms of cDNA was used for each amplification. The number of PCR cycles was modified to ensure that the PCR products obtained were from the linear phase of amplification. The cycle number of each PCR is shown in the figure. Representative data from two of five experiments with similar results are shown.

Article Snippet: Mouse IgG1 (MOPC) mAb, mouse anti-human IgM heavy chain (DA4.4) conjugated with biotin, mouse anti-human CD11a mAb (TS1/22), and mouse antihuman CD18 mAb (TS1/18) were prepared from hybridoma cell lines purchased from American Type Culture Collection (Manassas, VA).

Techniques: Cell Culture, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Southern Blot, Staining

Journal: Cancer immunology research

Article Title: C3aR Signaling Inhibits NK-cell Infiltration into the Tumor Microenvironment in Mouse Models

doi: 10.1158/2326-6066.CIR-21-0435

Figure Lengend Snippet: a. Immunoprecipitation to assess interaction between C3aR and CD11a (αL subunit that is specific to LFA-1)(n=2). b, c. Representative CD11a staining in activated human NK cells with or without C3a stimulation (n=3). d, e. Representative LFA-1 epitope 24 staining in activated human NK cells with or without C3a stimulation (n=3). f, g. Phospo-CD18 (T758) staining with or without C3a stimulation (n=3). Scale bar: 50 µm. Boxed areas indicate area shown in zoomed images.

Article Snippet: Primary rabbit anti-human CD11a (Cat#ab52895, Abcam, USA), primary rabbit anti-human CD18 (phospo-T758; Cat#ab63388, Abcam, USA), primary mouse anti-human CD11a+CD18 antibody [24] (Cat# ab13219, Abcam, USA), or respective control antibody (Rabbit IgG and Mouse IgG1) was diluted as suggested by the provider, infused into the microfluidic devices, and incubated for overnight at 4 o C. Samples were then washed three times and incubated with diluted secondary antibody (1:200 dilution; anti-mouse goat IgG, Cat# A32723 or anti-rabbit goat IgG, Cat# A32721, Thermofisher Scientific, USA) in the dark at room temperature for one hour.

Techniques: Immunoprecipitation, Staining

Journal: International Journal of Molecular Sciences

Article Title: Ganglioside GD2 Enhances the Malignant Phenotypes of Melanoma Cells by Cooperating with Integrins

doi: 10.3390/ijms23010423

Figure Lengend Snippet: Physical association of GD2 and integrin β1. ( A ) Cell surface expressions of integrin β1 in GD2+ and GD2− cells were analyzed by flow cytometry using anti-integrin β1 mAb. ( B ) The mRNA expression of integrin β1 was analyzed by qRT-PCR using GD2+ and GD2− cells. The experiment was performed in triplicates and the mean ± SD are presented. ( Ca ) Expressions of integrin β1 as well as GD2 were analyzed by Western immunoblotting. GD2 and integrin β1 were detected separately with anti-GD2 mAb and anti-integrin β1 mAb. ( Cb ) The binding of ganglioside GD2 and integrin β1 was analyzed by immunoprecipitation with rabbit anti-integrin β1 antibodies, and subsequent immunoblotting with anti-integrin β1 mAb or anti-GD2 mAb.

Article Snippet: Rabbit anti-integrin β1 polyclonal Ab was from Bioss, Woburn, MA, USA.

Techniques: Flow Cytometry, Expressing, Quantitative RT-PCR, Western Blot, Binding Assay, Immunoprecipitation

Journal: International Journal of Molecular Sciences

Article Title: Ganglioside GD2 Enhances the Malignant Phenotypes of Melanoma Cells by Cooperating with Integrins

doi: 10.3390/ijms23010423

Figure Lengend Snippet: Colocalization of GD2 and integrin β1. ( A ) Cell surface localization of GD2 and integrin β1 was examined by immunocytochemistry using GD2+ cells. After fixation and permeabilization, cells were stained with mouse anti-GD2 mAb and rabbit anti-integrin β1 mAb. Then, cells were incubated with an Alexa 568-conjugated goat anti-mouse IgG antibody and an Alexa 488-conjugated donkey anti-rabbit IgG antibody. Microscopic visualization was performed using a confocal microscope. The green color indicates integrin β1 and the red color indicates GD2. Scale bar = 15 μm. ( B ) Association between GD2 and integrin β1 was analyzed by PLA. GD2+ and GD2− cells were incubated with anti-GD2 and anti-integrin β1 mAb. Duolink TM in situ PLA probes anti-mouse PLUS and anti-rabbit MINUS were added, then a ligation–ligase solution was added. Finally, an amplification reaction was carried out. Cells were visualized under a confocal microscope. Scale bar = 20 μm.

Article Snippet: Rabbit anti-integrin β1 polyclonal Ab was from Bioss, Woburn, MA, USA.

Techniques: Immunocytochemistry, Staining, Incubation, Microscopy, In Situ, Ligation, Amplification

Journal: International Journal of Molecular Sciences

Article Title: Ganglioside GD2 Enhances the Malignant Phenotypes of Melanoma Cells by Cooperating with Integrins

doi: 10.3390/ijms23010423

Figure Lengend Snippet: Knockdown of integrin β1 and its effects on cell phenotypes. ( A , B ) Knockdown efficiency of integrin β1 was examined with 4 types of siRNA (37, 75, 74, and ITG1). Using cell lysates and RNAs from GD2+ and GD2− cells, Western immunoblotting ( A ) and qRT-PCR ( B ) were performed, respectively. Gene expression levels were analyzed using the Student’s t -test. ** p < 0.01. ( C ) Cell proliferation was analyzed by the MTT assay, using GD2+ and GD2− cells treated with anti-integrin β1 si-RNA ITG1. Cells (3 × 10 3 ) were seeded in 96-well plates. MTT assay was performed, as described in . The analysis was performed in triplicates (and the mean ± SD are presented) and analyzed by two-way ANOVA with the Tukey post-hoc test. * p < 0.05, ** p < 0.01. ( D ) Cell adhesion was analyzed by the RT-CES system. GD2+ and GD2− cells were transfected with integrin β1 si-RNA, ITG1, and used for RT-CES, as described in . ( E , F ) Invasion activity was analyzed using GD2+ and GD2− cells treated by integrin β1 si-RNA ITG1 with cell culture inserts. ( F ) A summary of the invasion assay. The invasion assay was performed in triplicates (and the mean ± SD are presented) were analyzed by Student’s t -test. * p < 0.05. Scale bar = 20 μm.

Article Snippet: Rabbit anti-integrin β1 polyclonal Ab was from Bioss, Woburn, MA, USA.

Techniques: Western Blot, Quantitative RT-PCR, Expressing, MTT Assay, Transfection, Activity Assay, Cell Culture, Invasion Assay

Journal: International Journal of Molecular Sciences

Article Title: Ganglioside GD2 Enhances the Malignant Phenotypes of Melanoma Cells by Cooperating with Integrins

doi: 10.3390/ijms23010423

Figure Lengend Snippet: Multiple phospho-tyrosine bands were detected during the cell adhesion of GD2+ cells. ( A ) Immunoblotting with anti-phosphotyrosine mAb PY20. GD2+ S1 and GD2− V4 cells were transfected with anti-integrin β1 si-RNA ITG1. After 36 h of culture in regular medium, the cells were prepared, as described in . Then, the cells were lysed and used for immunoblotting. ( B ) Band intensities in A were scanned by Images J TM and plotted for S1 bands ( a ) and V4 bands ( b ).

Article Snippet: Rabbit anti-integrin β1 polyclonal Ab was from Bioss, Woburn, MA, USA.

Techniques: Western Blot, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Ganglioside GD2 Enhances the Malignant Phenotypes of Melanoma Cells by Cooperating with Integrins

doi: 10.3390/ijms23010423

Figure Lengend Snippet: Intracellular distribution of integrin β1 before and during adhesion to CL type I. ( A ) GD2+ (S1) and control GD2− (V4) cells were detached using 0.5 mM EDTA/PBS. Cell suspension (5 × 10 5 cells) was placed in collagen I pre-coated plates in DMEM, and incubated for 0~30 min at 37 °C. Then, cell lysates were prepared using 1% Brij 020 in a TNE buffer and separated by Optiprep gradient ultracentrifugation at 42,000 rpm and 4 °C for 5 h, and fractionated (500 μL). Each fraction (13.5 μL) was used for immunoblotting with anti-integrin β1 mAb, anti-GD2 mAb, anti-flotillin, or anti-caveolin-1 antibodies. Flotillin and caveolin-1 were used as GEM/raft markers. ( B ) Band intensities of integrin β1 were measured by ImageJ TM software and the relative intensity of bands are presented.

Article Snippet: Rabbit anti-integrin β1 polyclonal Ab was from Bioss, Woburn, MA, USA.

Techniques: Incubation, Western Blot, Software