SYK is a substrate of PTPROt in vivo. (A) Coimmunoprecipitation of SYK and WT or mutant PTPROt in tet-inducible clones. Tet-inducible FLAG-tagged PTPROt WT (WT₁,WT₂) or mutant (DA₁,DA₂) clones were cultured with doxycycline (Dox) overnight. Thereafter, cells were serum starved and stimulated with goat anti–human IgG (10 μg/mL) for 8 minutes. Cells were then lysed and samples were immunoprecipitated with anti-FLAG (PTPROt) antibody. The tyrosyl-phosphorylated proteins pulled down by anti-FLAG (PTPROt) were detected by immunoblotting with the antiphosphotyrosine antibody 4G10 (top panel). The blot was then stripped and reprobed with anti-SYK (middle panel) and anti-FLAG (bottom panel). (B) PTPROt expression in lymphoblastoid B cells and tet-inducible PTPROt clones. Lanes 1, 2, and and 4 show total cell lysates from PTPROt WT cells without or with Dox induction (lanes 1 and 2, respectively) and CRL-8062 lymphoblastoid cells (lane 4). Lane 3 shows control anti-FLAG (PTPROt) immunoprecipitate from Dox-induced PTPROt WT cells. Samples were size-fractionated, blotted, and analyzed with anti PTPROt (top panel) or β-actin antibody (bottom panel). (C) Coimmunoprecipitation of SYK and PTPROt in lymphoblastoid cells. CRL-8062 cells were serum starved and subsequently stimulated with anti-IgG. Thereafter, cells were lysed and size-fractionated directly (lane 1) or following immunoprecipitation with anti-SYK antibody (lane 2). Anti-FLAG (PTPROt) immunoprecipitate from Dox-induced PTPROt-WT cells was analyzed simultaneously to serve as a positive control (lane 3). After size fractionation, the samples were immunoblotted and analyzed with PTPROt antiserum (top panel). Thereafter, the blot was stripped and reprobed with anti-SYK (bottom panel).