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. 2001 Aug 21;98(18):10172–10177. doi: 10.1073/pnas.191130798

Figure 3.

Figure 3

Lyn-mediated phosphorylation of GADD34. (A) Antiphosphotyrosine (PY) and anti-GADD34 (GADD) immunoblots of anti-FLAG immunoprecipitates of HEK293 cells transfected with empty vectors, pFLAG-CMV2-GADD34 (5 μg), and pcDNA3-Lyn constructs (5 μg) as indicated. Vector DNA was added to make 10 μg total whenever required. Equal amounts of GADD34 protein were recovered in the immunoprecipitates of GADD34-transfected cells. (B) Antiphosphotyrosine (PY) and anti-GADD34 (GADD) immunoblots of anti-GADD34 immunoprecipitates of BaF-3 cells untreated or treated with 50 μg/ml MMS for 1 h. (C) Antiphosphotyrosine (PY) and anti-FLAG (FLAG) immunoblots of anti-FLAG immunoprecipitates of DT40 and D33 cells transfected with pFLAG-CMV2-GADD34 (5 μg) and pcDNA3-Lyn (5 μg) and treated with 50 μg/ml MMS as indicated. (D) Anti-Lyn immune kinase assays with the exogenous substrates BSA, FLAG-GADD34 (GADD), and acid-denatured enolase. Lysates were prepared from DT40 wild-type or D33 Lyn-deficient cells and immunoprecipitated with the polyclonal anti-Lyn antibody. Equal amounts of protein from immunoprecipitates were used. Asterisks indicate positions of phosphorylated substrates. Lyn is a prominent doublet of 53 and 56 kDa. BSA is expected to appear as a 68-kDa band. Size markers on the left are in kilodaltons.