Figure 1
Induced expression of LMP2A in DT40 B cells causes PTK substrate phosphorylation. (A) LMP2A expression strategy. Treatment of DT40 transfectants with 4-HT induces Cre-mediated excision of a loxP-flanked puromycin resistance cassette which otherwise prevents the promotor (P) from activating transcription of a cDNA encoding triple-FLAG-tagged LMP2A (LMP2A3xF). (B), Following 4-HT treatment for the indicated time points, expression of LMP2A3xF was monitored on the protein level by anti-FLAG immunoblotting of cellular lysates. (C) LMP2A transfectants of DT40 B cells were treated with 4-HT for the indicated times (lanes 1-9), left untreated (lane 10) or stimulated for 3 min with anti-IgM antibodies (lane 11). Upon lysis, tyrosine-phosphorylated proteins were purified using anti-phosphotyrosine antibodies (covalently coupled to NHS-Sepharose) and subjected to anti-phosphotyrosine (anti-pTyr) immunoblotting. Relative molecular masses of marker proteins are indicated on the left in kDa. (D) The same cells as before were treated with 4-HT for the indicated times (lanes 1-8), left untreated (lane 9) or stimulated for 3 min with anti-IgM antibodies (lane 10). After lysis in 1% NP40-containing lysis buffer, tyrosine-phosphorylated proteins were affinity purified using a fusion protein consisting of GST and the tandem SH2 domains of Syk (GST-Syk[SH2]2). The GST-Syk[SH2]2 fusion protein specifically binds to doubly phosphorylated ITAM motifs. Purified proteins were subjected to anti-phosphotyrosine immunoblotting. The positions of phosphorylated LMP2A and the (non-phosphorylated) GST fusion protein are indicated on the right.