The rabbit polyclonal antibody that recognizes both PTPROt and GLEPP-1 has been described (32). Also used were mouse monoclonal antibodies against phosphotyrosine (PY99, Santa Cruz Biotechnology and PY20, BD Transduction Laboratories); HA [clone HA-7, coupled to beads (immunoprecipitation), Sigma-Aldrich and clone HA.11 (blotting and PLA), BioLegend]; actin (clone AC-40, Sigma-Aldrich); tubulin (clone DMIA, Sigma-Aldrich); and FLAG (clone M2, Sigma-Aldrich). Rabbit polyclonal antibodies were used against Src, pTyr416-Src, and pTyr527-Src (Cell Signaling Technology) and Grb2 (S-255, Santa Cruz Biotechnology). A rabbit monoclonal antibody that recognizes Src (Clone 32G6, Cell Signaling Technology) was used for PLA.

Wild-type PTPROt was cloned into the pcDNA3 expression vector (Invitrogen/Thermo Fisher Scientific). C-terminal HA or FLAG tags, as well as the Y399F, C325S, and/or R331M mutations, were introduced into the mouse Ptprot complementary DNA using PCR-directed mutagenesis followed by sequencing.

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