Anti AKT, phospho Ser473  

Immunofluorescence Microscopy of Mouse Anti-AKTpS473 antibody using STED nanoscopy to evaluate AKT activation and migration. Tissue: A431 cells. Antigen retrieval: Panel A: serum starved, unstimulated cells. Panel B: serum starved, EGF stimulated for 15 mins. A massive increase in AKT-pS473 activation, as measured by intensity signal, peaked at 15 minutes and was associated with depolymerized tubulin. Staining: Panel A shows STED data (AKT-pS473, red channel) collected simultaneously with confocal signal (a-tubulin, green channel). Upon stimulation of cells with EGF, a rapid activation of AKT is observed (Panel B) along with a coincident change in the tubulin organization (yellow signal), as well as an extensive cell shape-change (cell membrane folding) and accumulation of AKTpS473 at the cell periphery.

Immunofluorescence confocal microscopy of Mouse Anti-AKT pS473 antibody. Tissue: EGF treated A431 cells. Fixation: 0.5% PFA. Antigen retrieval: EGF 15 min. Primary antibody: AKT pS473 antibody at 10 μg/mL for 1 h at RT. Secondary antibody: DyLight 488^TM Goat anti-Rabbit IgG, MAb anti-AKT pS473, atto-647N anti-Mouse IgG (Active Motif). at 1:10,000 for 45 min at RT. Localization: AKT pS473 is nuclear and occasionally cytoplasmic. Staining: AKT pS473 as red signal with tubulin (cyan).

Immunohistochemistry of Mouse anti-AKT pS473 antibody. Tissue: human prostate tissue. Fixation: formalin fixed paraffin embedded. Antigen retrieval: not required. Primary antibody: AKT pS473 antibody at 20 μg/mL for 1 h at RT. Secondary antibody: Dako's Techmate streptavidin-biotin reagents at 1:10,000 for 45 min at RT. Localization: AKT pS473 is nuclear and occasionally cytoplasmic. Staining: AKT pS473 as precipitated red signal with hematoxylin purple nuclear counterstain.

High resolution STED immunofluorescence nanoscopy of Mouse anti-AKT pS473 antibody. Tissue: A431 cells. The merge images (A) and at high magnification (B) show phosphorylated AKT colocalized with the distal microtubules. Fixation: 4% paraformaldehyde for 5 min and after washes blocked with 10% NGS/0.2% Triton X-100 for 30 min. Antigen retrieval: serum deprivation for 12 h. Primary antibody: AKT pS473 antibody at 10 μg/mL and α-tubulin (cyan) (p/n 600-401-880) at 1.4 μg/mL for 1 h at RT. Secondary antibody: Atto 647N anti-Mouse IgG (ATTO TEC GmbH), and DyLight^TM488 anti-Rabbit IgG (p/n 611-141-122) were used at 1.0 μg/mL for 1h at RT for indirect detection. Localization: AKT pS473 is in the cytoplasm and also organized at the periphery of the cell. Staining: AKT pS473 as red signal with bis-benzimide (blue) nuclear counterstain.

Western Blot of Mouse anti-AKT antibody. Lane 1: unstimulated NIH/3T3 cell lysates (p/n W10-000-358). Lane 2: PDGF stimulated NIH/3T3 cell lysates (p/n W10-001-377). Load: 10 μg per lane. Primary antibody: AKT antibody at 1:400 for overnight at 4°C. Secondary antibody: HRP conjugated Gt-a-Mouse IgG (p/n 610-103-121) was used at a 1:40,000 dilution for 1 h at 4° C with FemtoMax^TM enhanced chemiluminescent reagent (p/n FEMTOMAX-100). Block: 5% BLOTTO (p/n B501-0500 in TBS for 2h at RT. Observed size: ~56 kDa for AKT. Other band(s): none.

Western Blot of Mouse Anti-Akt pS473 antibody. Lane 1: unstimulated NIH/3T3 lysates (p/n W10-000-358) contain inactive unphosphorylated Akt1, green band. Lane 2: PDGF stimulated NIH/3T3 lysate (p/n W10-001-377) contains both inactive (green band) and activated phosphorylated Akt1 (red band). Load: 10 μg per lane. Primary antibody: rabbit anti-Akt (pan) (p/n 100-401-401) and mouse anti-Akt pS473 (p/n 200-301-B19) specific antibodies at 1:400 for overnight at 4°C. Secondary antibody: anti-rabbit IgG DyLight^TM 549 (green) and anti-mouse IgG DyLight^TM 649 conjugated (red) secondary antibodies at 1:10,000 for 45 min at RT. Block: 5% BLOTTO overnight at 4°C.

Western Blot of Mouse Anti-AKTpS473 antibody. Lane 1: A431 cell lysate (p/n W09-000-361). Lane 2: A431 cells stimulated for 15 min with EGF (p/n W09-000-362). Load: 35 μg per lane. Primary antibody: AKTpS473 antibody at 1:400 for overnight at 4°C. Secondary antibody: DyLight^TM649 Conjugated Anti-AKT pS473 Monoclonal Antibody (p/n 200-343-268) at 1:10,000 for 45 min at RT. Block: Blocking Buffer for Fluorescent Western Blotting (p/n MB-070) overnight at 4°C. Predicted/Observed size: 56kDa. Other band(s): none.

A forward flow component is required for flow-induced Akt-1 activation. A: Pulsatile (Puls) and oscillatory (Osc) flow were applied at similar frequency and amplitude of flow (10 dyn cm−2,？T = 1 s). B: Akt-1 (S473) and Src (Y416) phosphorylation levels respectively in non-adapted HUVEC monolayers after 5 min of pulsatile or oscillatory flow or in sham-mounted slides (Sh). Ratio pAkt-1/Akt-1: Puls: 1.82 ±0.25, Osc: 0.15 ±0.01,？P= 0.032. Representative blots with duplicates are shown above. Fig 4. PMID: 23913776

Cell morphology and spatiolocalization of phosphorylated Akt-1 in the mouse aorta.？En face？immunostaining of pAkt-1S473？(green) and PECAM-1 (red) in the brachiocephalic area (A), the renal artery bifurcation (B), the distal part of the descending aorta (C), and the adjacent intercostals artery (D). Scale bar is 20？μm. E: Sites along the aorta where samples were derived. F: Bar graph illustrating the fraction of EC expressing perinuclear pAkt-1S473？staining in regards to cell diameter from areas shown in a, b, and c. Fig 1. PMID: 23913776