crosstide (akt substrate peptide  (GenScript corporation)

Journal:

Article Title: Protein Kinase B/Akt Participates in GLUT4 Translocation by Insulin in L6 Myoblasts

doi:

Figure Lengend Snippet: AAA-PKB acts as a dominant-negative mutant of PKB. (A) L6-GLUT4myc myoblasts grown in six-well plates were transiently transfected with HA-tagged wild-type PKBα/Akt1 (HA-PKB; 0.4 μg per well) or with HA-tagged AAA-PKBα/Akt1 (HA-AAA-PKB; 0.4 μg per well) and incubated for 48 h in culture. Cells were serum deprived for 5 h and were left untreated (basal) or treated with 100 nM insulin for 10 min (insulin). Cell lysates were prepared, HA-tagged proteins were immunoprecipitated with anti-HA antibodies (3 μg), and PKB kinase activity in the immune complexes was measured as described in Materials and Methods. The basal activity of HA-PKB was assigned a value of 1.0; all other activities were expressed relative to this value. The results represent the means ± SE of data from three independent experiments. (B) Cells were transfected with HA-PKB (0.4 μg per well) in combination either with pcDNA3 vector alone (4 μg per well) or with untagged AAA-PKB in pcDNA3 (4 μg per well) as indicated. Cells were treated with insulin as indicated, and lysates were prepared and processed for the PKB/Akt kinase assay as described for panel A. The basal activity of HA-PKB cotransfected with pcDNA3 was assigned a value of 1.0; all other activities were expressed relative to this value. The results represent the means ± SE of data from five independent experiments.

Article Snippet: PKB/Akt substrate peptide (Crosstide) was from Upstate Biotechnology (Lake Placid, N.Y.).

Techniques: Dominant Negative Mutation, Transfection, Incubation, Immunoprecipitation, Activity Assay, Plasmid Preparation, Kinase Assay