Anti ilk 4g9

β1^WT and β1^KO Fo and transitional B cells were lysed with buffer containing 20 mM Hepes (pH 7.6), 2 mM MgCl₂, 150 mM NaCl, 10% glycerol, 0.1% NP40, 1 mM PMSF, and protease inhibitor mix (Sigma-Aldrich). Following sonication, the cell lysate was centrifuged at 16,100 g for 10 min at 4°C. The protein concentration of the supernatant was measured by Bradford assay, and 1 mg of total protein was then mixed with anti-ILK (4G9; rabbit monoclonal; Cell Signaling Technology) or anti-rabbit IgG isotype control antibody (Biomol) and rotated ON at 4°C. Subsequently, samples were incubated with Dynabeads Protein G (Thermos Fischer Scientific) for 2 h at 4°C, beads were washed, and associated proteins were eluted by addition of 2× sample buffer and a boiling step of 10 min at 95°C. Following separation of the proteins by SDS-PAGE, proteins were detected with anti-ILK (4G9; rabbit monoclonal; Cell Signaling Technology), anti-Grb2 (Y237; rabbit monoclonal; Abcam), and anti-FAK antibody (rabbit polyclonal; Cell Signaling Technology).

The following monoclonal antibodies were used: anti-α-tubulin (Clone DM 1A; Sigma-Aldrich), anti-acetylated α-tubulin (Clone 6-11B-1; Sigma-Aldrich), anti-EB1 (Santa Cruz), anti-EB1 (Sigma Aldrich), anti-tyrosinated α-tubulin (YL1/2, Santa Cruz) and anti-vinculin (Sigma-Aldrich). The following polyclonal antibodies were used: anti-β-catenin (Sigma-Aldrich), anti-β1-integrin (GTX128839; GeneTex), anti-detyrosinated α-tubulin (Millipore), anti-ILK (4G9, Cell Signaling), anti-MCAK (Thermo Fisher Scientific) and anti-GAPDH (HyTest). Control rabbit and mouse IgG was purchased from Santa Cruz. Cruz. Horseradish peroxidase-conjugated secondary antibodies against mouse and rabbit were obtained from Biorad (mouse and rabbit) and Bethyl (rat). Alexa-labeled secondary antibodies were purchased from Thermo Fisher. The plasmid mCherry-Vinculin-N-21 was a gift from Michael Davidson (Addgene plasmid #55160; RRID; Addgene_55160). The plasmid EB1-GFP (JB131) was a gift from Jennifer Tirnauer and Tim Mitchison (Addgene plasmid #39299; RRID:Addgene_39299). The plasmid mCherry-EB1 was a gift from Torsten Wittmann (Addgene plasmid #80603; RRID; Addgene_80603).