Journal:

Article Title: Ras-Dependent Regulation of c-Jun Phosphorylation Is Mediated by the Ral Guanine Nucleotide Exchange Factor-Ral Pathway

doi:

Figure Lengend Snippet: Insulin and Ral guanine nucleotide exchange activity induce activation of JNK. (A) Activation of JNK activity by insulin and Rlf-CAAX. A14 cells were either transfected with pCD20 and treated with insulin (1 μM) for 30 min (left two lanes) or cotransfected with pCD20 and either pMT2HA (v), pMT2HA-RlfCAAX (wt), or pMT2HA-Rlf-R328E-CAAX (R328E) as indicated (right three lanes). Transfected cells were isolated by MACS. Endogenous JNK was isolated by using GST–c-Jun1–79 precoupled to glutathione beads. JNK activity using GST–c-Jun1–79 as a substrate was assayed directly by adding ATP. Phosphorylation of GST–c-Jun1–79 was monitored by Western blotting using anti-c-Jun phosphoserine 73. (B) Rlf-CAAX does not induce ERK phosphorylation. A14 cells were cotransfected with pCD20 and either pMT2HA (v), pMT2HA-Rlf-CAAX (wt), pMT2HA-Rlf-R328E-CAAX (R328E), or pMT2HA-RasV12 (Ras) as indicated and transfected cells were isolated by magnetic cell sorting. Phosphorylation of ERK was detected by Western blotting using anti-ERK phosphothreonine-202 phosphotyrosine-204 polyclonal antibody. The positions of phosphorylated ERK1 and ERK2 are indicated.

Article Snippet: The c-Jun antibody used was rabbit polyclonal H79 (Santa Cruz) or rabbit polyclonal anti-c-Jun Ab1 (Oncogene Science).

Techniques: Activity Assay, Activation Assay, Transfection, Isolation, Western Blot, FACS

Journal:

Article Title: Ras-Dependent Regulation of c-Jun Phosphorylation Is Mediated by the Ral Guanine Nucleotide Exchange Factor-Ral Pathway

doi:

Figure Lengend Snippet: Growth factor-induced phosphorylation of endogenous c-Jun requires Ral signaling. A14 or HEK-293 cells were transiently transfected with either empty vector or a construct expressing either RalB-N28, RalBP-ΔGAP, or RalGDS-RBD as indicated. After 24 h, the cells were serum starved for 16 h and then stimulated with insulin (1 μM) or EGF (20 ng/ml) as indicated, and transfected cells were isolated by MACS. (A) RalB-N28 blocks insulin-induced c-Jun phosphorylation. A14 cells transiently expressing empty pSG5 or pSG5-RalB-N28 as indicated were treated with insulin for the times indicated, and transfected cells were isolated. Samples were analyzed for c-Jun phosphorylation on serine 73 (top panel), c-Jun protein expression (middle panel) or RalB-protein expression (bottom panel). The numbers below the panels indicate fold induction compared to the unstimulated cells. (B) RalB-N28 does not block insulin-induced ATF2 or ERK phosphorylation. Equal amounts of whole-cell extracts isolated in panel A were analyzed for increases in ATF2 phosphorylation and for phosphorylation of ERK. ATF2 phosphorylation was detected by Western blotting using an anti-ATF2 phosphothreonine-71 polyclonal antibody (upper panel). Phosphorylation of ERK was monitored by Western blotting using anti-ERK phosphothreonine-202 phosphotyrosine-204 polyclonal antibody (lower panel). Fold induction is indicated below the panels. (C) RalB-N28 blocks EGF-induced c-Jun phosphorylation in HEK-293 cells. HEK-293 cells transiently expressing empty pSG5 or pSG5-RalB-N28 as indicated were treated with EGF for the times indicated, and transfected cells were isolated. Samples were analyzed for c-Jun phosphorylation on serine 73 (top panel), c-Jun protein expression (middle panel), or RalB-protein expression (bottom panel). (D) RalBP-ΔGAP and RalGDS-RBD block insulin-induced phosphorylation of c-Jun. A14 cells transiently expressing empty pMT2HA, pRK5-MYC-RalBP-ΔGAP, or pMT2HA-RalGDS-RBD as indicated were treated with insulin for the times indicated, and transfected cells were isolated. Isolated samples were analyzed for c-Jun phosphorylation on serine 73 (top panel), c-Jun protein expression (second panel), Myc-RalBP-ΔGAP expression (third panel), or HA-RalGDS-RBD expression (bottom panel).

Article Snippet: The c-Jun antibody used was rabbit polyclonal H79 (Santa Cruz) or rabbit polyclonal anti-c-Jun Ab1 (Oncogene Science).

Techniques: Transfection, Plasmid Preparation, Construct, Expressing, Isolation, Blocking Assay, Western Blot