Flow cytometry analysis of c-RAF [pS621] was done on A549 cells treated with EGF (200ng/ml, 10 minutes). Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with c-RAF [pS621] Rabbit Polyclonal Antibody (44504G, red histogram) at a dilution of 1:20 or a matched isotype control (pink histogram) in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.
|Tested species reactivity||Human|
|Published species reactivity||Rat, Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||The antiserum was produced against a chemically synthesized phosphopeptide derived from a region of human c-Raf that contains serine 621. The sequence is conserved in mouse and rat.|
|Purification||Antigen affinity chromatography|
|Storage buffer||Dulbecco's PBS, pH 7.3, with 50% glycerol, 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1:20|
|Western Blot (WB)||Assay Dependent|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
Raf-1 is a MAP kinase kinase kinase (MAP3K) which functions downstream of the Ras family of membrane associated GTPases to which it binds directly. Once activated Raf-1 can phosphorylate to activate the dual specificity protein kinases MEK1 and MEK2 which in turn phosphorylate to activate the serine/threonine specific protein kinases ERK1 and ERK2. Activated ERKs are pleiotropic effectors of cell physiology and play an important role in the control of gene expression involved in the cell division cycle, apoptosis, cell differentiation and cell migration.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
6-Formylindolo(3,2-b)Carbazole (FICZ) Modulates the Signalsome Responsible for RA-Induced Differentiation of HL-60 Myeloblastic Leukemia Cells.
44-504G was used in western blot to study the molecular signaling responsible for retinoic acid-induced differentiation.
|Bunaciu RP,Jensen HA,MacDonald RJ,LaTocha DH,Varner JD,Yen A||PloS one (10:null)||2015|
Heterogeneous effects of calorie restriction on in vivo glucose uptake and insulin signaling of individual rat skeletal muscles.
44-504G was used in western blot to study the effects of calorie restriction on glucose uptake and insulin signaling in muscle in vivo.
|Sharma N,Sequea DA,Castorena CM,Arias EB,Qi NR,Cartee GD||PloS one (8:null)||2013|
Preventing the calorie restriction-induced increase in insulin-stimulated Akt2 phosphorylation eliminates calorie restriction's effect on glucose uptake in skeletal muscle.
44-504G was used in western blot to examine the effect of Akt2 phosphorylation on on glucose uptake in skeletal muscle
|Sharma N,Arias EB,Sequea DA,Cartee GD||Biochimica et biophysica acta (1822:1735)||2012|
|Rat||Not Cited||Mechanisms for increased insulin-stimulated Akt phosphorylation and glucose uptake in fast- and slow-twitch skeletal muscles of calorie-restricted rats.||Sharma N,Arias EB,Bhat AD,Sequea DA,Ho S,Croff KK,Sajan MP,Farese RV,Cartee GD||American journal of physiology. Endocrinology and metabolism (300:E966)||2011|
|Rat||Not Cited||Nitric oxide increases p21(Waf1/Cip1) expression by a cGMP-dependent pathway that includes activation of extracellular signal-regulated kinase and p70(S6k).||Gu M,Lynch J,Brecher P||The Journal of biological chemistry (275:11389)||2000|
||Mechanisms for increased insulin-stimulated Akt phosphorylation and glucose uptake in fast- and slow-twitch skeletal muscles of calorie-restricted rats.||Sharma N,Arias EB,Bhat AD,Sequea DA,Ho S,Croff KK,Sajan MP,Farese RV,Cartee GD||American journal of physiology. Endocrinology and metabolism (300:E966)||2011|