integrin αv integrin β1 polyclonal rabbit anti human  (Bioss)

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