• Western blot analysis of extracts from PC-3 cells, untreated or LY294002/wortmannin-treated, and NIH/3T3 cells, serum-starved or PDGF-treated, using Phospho-Akt (Ser473) (D9E) XP ® Rabbit mAb (upper) or Akt (pan) (C67E7) Rabbit mAb #4691 (lower). • Immunohistochemical analysis of paraffin-embedded MDA-MB-468 xenograft using Phospho-Akt (Ser473) (D9E) XP ® Rabbit mAb (left) or PTEN (138G6) Rabbit mAb #9559 (right). Note the presence of P-Akt staining in the PTEN deficient MDA-MB-468 cells. • Immunohistochemical analysis of paraffin-embedded human breast carcinoma comparing SignalStain ® Antibody Diluent #8112 (left) to TBST/5% normal goat serum (right) using Phospho-Akt (Ser473) (D9E) XP ® Rabbit mAb #4060.

• Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Phospho-Akt (Ser473) (D9E) XP ® Rabbit mAb. Grabcevich sbornik zadachi po fizike dinamika reshebnik. • Immunohistochemical analysis using Phospho-Akt (Ser473) (D9E) XP ® Rabbit mAb on SignalSlide® Phospho-Akt (Ser473) IHC Controls #8101 (paraffin-embedded LNCaP cells, untreated (left) or LY294002-treated (right)). • Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Phospho-Akt (Ser473) (D9E) XP ® Rabbit mAb. • Immunohistochemical analysis of paraffin-embedded PTEN heterozygous mutant mouse endometrium using Phospho-Akt (Ser473) (D9E) XP ® Rabbit mAb. (Tissue section courtesy of Dr. Sabina Signoretti, Brigham and Women's Hospital, Harvard Medical School, Boston, MA.) • Immunohistochemical analysis of paraffin-embedded U-87MG xenograft, untreated (left) or lambda phosphatase-treated (right), using Phospho-Akt (Ser473) (D9E) XP ® Rabbit mAb.

The serine/threonine protein kinase B (PKB, also known as Akt) constitutes an important node in diverse signaling cascades downstream of growth factor receptor tyrosine kinases. Akt plays an essential role in cell survival, growth, migration, proliferation, polarity, and metabolism (lipid.

• Confocal immunofluorescent analysis of C2C12 cells, LY294002-treated (left) or insulin-treated (right), using Phospho-Akt (Ser473) (D9E) XP ® Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor ® 555 phalloidin #8953 (red). Blue pseudocolor = DRAQ5 ®#4084 (fluorescent DNA dye). • Flow cytometric analysis of Jurkat cells, untreated (green) or treated with LY294002 #9901, wortmannin #9951 and U0126 #9903 (blue), using Phospho-Akt (Ser473) (D9E) XP ® Rabbit mAb compared to a nonspecific negative control antibody (red).

Western Blotting Protocol For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween ® 20 at 4°C with gentle shaking, overnight. NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution. Solutions and Reagents From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: Western Blotting Application Solutions Kit NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

• 20X Phosphate Buffered Saline (PBS): () To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH 2O, mix. • 10X Tris Buffered Saline (TBS): () To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH 2O, mix. • 1X SDS Sample Buffer: Blue Loading Pack () or Red Loading Pack () Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH 2O. • 10X Tris-Glycine SDS Running Buffer: () To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH 2O, mix. • 10X Tris-Glycine Transfer Buffer: () To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH 2O, mix. • 10X Tris Buffered Saline with Tween ® 20 (TBST): () To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH 2O, mix.

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