Western blot analysis of Smad2/3 Control Cell Extracts from HT-1080 cells, untreated (-) or treated with hTGF-β3 #8425 (10 ng/ml, 30 min; +), using Phospho-Smad2 (Ser465/467) (138D4) Rabbit mAb #3108 (upper) or Smad2 (86F7) Rabbit mAb #3122 (lower).
Boil for 3 minutes prior to use. Load 10 μl of phosphorylated and nonphosphorylated Smad2/3 Control Cell Extracts per lane.
Supplied in SDS Sample Buffer: 62.5 mM Tris- HCl (pH 6.8 at 25°C), 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue or phenol red.Store at –20°C, or at –80°C for long-term storage.
Nonphosphorylated Smad2/3 Control Cell Extracts: Total cell extracts from HT-1080 cells, serum-starved overnight to serve as a negative control. Supplied in SDS Sample Buffer.
Phosphorylated Smad2/3 Control Cell Extracts: Total cell extracts from HT-1080 cells, serum-starved overnight and treated with 10 ng/ml hTGF-β3 #8425 for 30 min to serve as a positive control. Supplied in SDS Sample Buffer.
Members of the Smad family of signal transduction molecules are components of a critical intracellular pathway that transmit TGF-β signals from the cell surface into the nucleus. Three distinct classes of Smads have been defined: the receptor-regulated Smads (R-Smads), which include Smad1, 2, 3, 5, and 8; the common-mediator Smad (co-Smad), Smad4; and the antagonistic or inhibitory Smads (I-Smads), Smad6 and 7 (1-5). Activated type I receptors associate with specific R-Smads and phosphorylate them on a conserved carboxy terminal SSXS motif. The phosphorylated R-Smad dissociates from the receptor and forms a heteromeric complex with the co-Smad (Smad4), allowing translocation of the complex to the nucleus. Once in the nucleus, Smads can target a variety of DNA binding proteins to regulate transcriptional responses (6-8).
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