Monoclonal anti talin 8d4

Cells were lysed with ice-cold cell lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10 mM MgCl₂, 1% NP-40, and 10% glycerol) plus protease inhibitor cocktails (Roche) on ice for 30 min and immunoprecipitated with the α4β7 mAb, Act-1 (1 µg). Mouse IgG was used as a control. After overnight incubation with antibodies at 4°C, Protein G Sepharose (Invitrogen) was added to the reaction mixture and further incubated for 2–4 h at 4°C. After three washes with ice-cold lysis buffer, beads were mixed with sample buffer before separation on a 4–20% SDS polyacrylamide gel (Novex; Invitrogen). Western blots were stained with monoclonal anti–human β7 (EPR1357, 1:3,000; Abcam), monoclonal antitalin (8d4; 1:1,000; Sigma-Aldrich), and monoclonal anti–β-actin (1:5,000; Sigma-Aldrich). Signal was detected and results were quantified using an Odyssey imaging system (LI-COR Biosciences).

The following antibodies were purchased from BioLegend: CD4 (GK1.5), CD8 (53–6.7), CD44 (1M7), CD62L (MEL-14), B220 (RA3-6B2), CD11c (N418), and F4/80 (BM8). Monoclonal anti-GFP (1:5,000; Takara Bio Inc.), monoclonal anti–β-actin (1:5,000; Sigma-Aldrich), monoclonal anti–human β7 (EPR1357, 1:3,000; Abcam), monoclonal antitalin (8d4, 1:1,000; Sigma-Aldrich), and polyclonal anti–kindlin 3 antibody (1:500; Abgent; Ye et al., 2013 (link)) were used for immunoblotting. FIB504 against human/mouse β7 prepared by using hybridoma cells was used for immunofluorescence at 2 µg/ml. Act-1 against human α4β7 was used for immunoprecipitation as previously described (Sun et al., 2011 (link)). Secondary Alexa Fluor–labeled antibodies for Western blot analysis were from Rockland or Thermo Fisher Scientific. CFSE and eFluor 670 were purchased from Invitrogen and BioLegend, respectively. Human and mouse CXCL12 were purchased from R&D Systems. PMA was purchased from Sigma-Aldrich.