polyclonal antibodies against raf 1  (Santa Cruz Biotechnology)

Journal: ASN NEURO

Article Title: Crosstalk Between MAPK/ERK and PI3K/AKT Signal Pathways During Brain Ischemia/Reperfusion

doi: 10.1177/1759091415602463

Figure Lengend Snippet: Ischemia increases interactions between SOS1 and EGFR or between p-AKT and Raf-1 and stimulates Raf-1 Ser259 phosphorylation, but not Ser338 phosphorylation. Two hr of focal ischemia was induced by MCAO. (a) Coimmunoprecipitation was performed with EGFR antibody, and blots were stained with either SOS1 antibody or EGFR antibody. Bands of 170 kDa represent SOS1 (upper rows) or EGFR (lower rows), respectively. (b) Bands of 74 kDa represent p-Raf-1 Ser259 (first rows), Ser338 (seconnd rows), or total Raf-1 (bottom rows). (c) Coimmunoprecipitation was performed with Raf-1 antibody, and blots were stained with either p-AKT antibody or Raf-1 antibody. Bands of 60 and 74 kDa represent phosphorylated AKT (p-AKT; upper rows) or Raf-1 (lower rows), respectively. Immunoblots from a representative experiment. Similar results are obtained from three independent experiments. (a) Average binding of SOS1 and EGFR is quantified as ratios between SOS1 and EGFR. (b) Average phosphorylation of Ser259 or Ser338 is quantified as ratios between Ser259 and Raf-1 or between Ser338 and Raf-1. Average binding of p-AKT and Raf-1 is quantified as ratios between p-AKT and Raf-1. SEM values are indicated by vertical bars. *Statistically significant ( p < .05) difference from the corresponding core or penumbra from contralateral hemisphere, but not from each other. SOS1 = Son of sevenless 1; EGFR = epidermal growth factor receptor; AKT = protein kinase B; MCAO = middle cerebral artery occlusion.

Article Snippet: The rabbit polyclonal antibody against EGFR (2232), rabbit polyclonal antibody against c-Raf (9422), rabbit polyclonal antibody against p-c-Raf (Ser338) (9427), rabbit polyclonal antibody against p-c-Raf (Ser259) (9421), and rabbit polyclonal antibody against Akt (9272) used for western blotting were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Phospho-proteomics, Staining, Western Blot, Binding Assay

Journal: ASN NEURO

Article Title: Crosstalk Between MAPK/ERK and PI3K/AKT Signal Pathways During Brain Ischemia/Reperfusion

doi: 10.1177/1759091415602463

Figure Lengend Snippet: Diagram of crosstalk between Raf/MAPK/ERK and PI3K/AKT signal pathways during brain ischemia and reperfusion. Ischemia induces EGFR transactivation and phosphorylation at Y1173, Y845, and Y1045. The activation of EGFR, in turn, significantly stimulates PI3K/AKT signal pathway. Subsequently, AKT phosphorylates Raf-1 at its inhibitory phosphorylation site Ser259 and inhibits Raf-1 activity. The inhibition of Raf-1 leads to inactivation of its downstream signal MAPK/ERK. During reperfusion, ROS stimulates both EGFR and PTEN. The latter, in turn, inhibits PI3K/AKT signal pathway and restores the activity of Raf-1/MAPK/ERK 1/2 signal pathway, which is responsible for reperfusion-induced cell damage. EGFR = epidermal growth factor receptor; ROS = reactive oxygen species; PTEN = phosphatase and tensin homolog; SOS1 = Son of sevenless 1; AKT = protein kinase B; MAPK = mitogen-activated protein kinase; ERK = extracellular signal-regulated kinase.

Article Snippet: The rabbit polyclonal antibody against EGFR (2232), rabbit polyclonal antibody against c-Raf (9422), rabbit polyclonal antibody against p-c-Raf (Ser338) (9427), rabbit polyclonal antibody against p-c-Raf (Ser259) (9421), and rabbit polyclonal antibody against Akt (9272) used for western blotting were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Phospho-proteomics, Activation Assay, Activity Assay, Inhibition