Anti phospho src family y416 antibody

CD4⁺ CD45RA⁺ CD25^lo naive T reg cells, CD4⁺ CD45RA⁻ CD25^hi eT reg cells, CD4⁺ CD45RA⁺ CD25⁻ T cells, and CD8⁺ CD45RA⁺ CD25⁻ T cells from PBMCs were sorted by FACSAria II (BD) and cultured with or without 10 µM imatinib for 1 h. Cells were treated with NP-40 buffer (1% NonidetP-40, 20 mM Tris pH 7.8, 150 mM NaCl, 2 mM MgCl₂), supplemented with the protease inhibitors PMSF (1 mM) and CLAP (5 µg/ml each of chymostatin, pepstatin A, antipain hydrochloride, and 10 µg/ml leupeptin hemisulphate) and a phosphatase inhibitor cocktail (Roche), and subsequently with Laemmli buffer. Lysates from 2 × 10⁵ cells from each sample were run on 7.5% polyacrylamide gel. Anti-Lck (3A5) antibody (Santa Cruz Biotechnology) and anti-Phospho-Src family (Y416) antibody (Cell Signaling Technology) were used for detection, and analysis was performed by LAS-4000 with ImageReader program (Fujifilm). Quantification of band signal intensity with background subtraction was calculated by MultiGauge software (Fujifilm).

Sorted cells were treated with NP-40 buffer (1% Nonidet P-40, 20 mM Tris, pH 7.8, 150 mM NaCl, 2 mM MgCl₂), supplemented with the protease inhibitors PMSF (1 mM) and CLAP (5 μg/ml each of chymostatin, pepstatin A, antipain hydrochloride, and 10 μg/ml leupeptin hemisulfate), and a phosphatase inhibitor cocktail (Roche), and subsequently with Laemmli buffer. Lysates from 1 × 10⁵ cells from each sample were run on 7.5% polyacrylamide gel or 12–230 kD capillary with total protein normalization on Jess (Proteinsimple) with anti-phospho-Src family (Y416) antibody (Cell Signaling Technology) for detection. Analysis was performed by LAS-4000 (Fujifilm) or Jess (Proteinsimple).