Quantitative Protein Analysis in Brain Slices

Brain slices B- E were collected and flash frozen. Brain tissue was then homogenized and protein expression was measured as described previously [9 (link)]. Non-specific binding was blocked by incubating the membranes in 5% milk in TBST for 60 minutes prior to overnight incubation with primary antibody against VEGF-B, (1:1000, Abcam, Cambridge, MA), rabbit polyclonal VEGF receptor 1 (VEGFR1) antibody (ab2350; abcam,1: 100, Cambridge, MA), anti-phospho-Flt (Tyr 1213) (07–758; Millipore; 1;750, Billerica, MA), VEGF-A and phosphor-VEGFR2 (1:200, Millipore, Billerica, MA). Beta-actin (Sigma-Aldrich, St. Louis, MO) was used as an endogenous loading control. Protein levels were analyzed densitometrically, using Image J software and were normalized to loading controls.

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Ishrat T., Soliman S., Eldahshan W., Pillai B., Ergul A, & Fagan S.C. (2018). Silencing VEGF-B diminishes the neuroprotective effect of candesartan treatment after experimental focal cerebral ischemia. Neurochemical research, 43(10), 1869-1878.

Publication 2018

VEGF-B ab2350 VEGF-A phosphor-VEGFR2 Beta-actin

Antibody Beta actin Brain Frozen Membranes Milk Phosphor Protein Rabbit Tissue Vegf Vegf b Vegf receptor 1 Vegfr2

Corresponding Organization : University of Tennessee Health Science Center

Other organizations : Charlie Norwood VA Medical Center, University of Georgia, Augusta University

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Variable analysis

independent variables

Brain slices B-E

dependent variables

Protein expression
VEGF-B protein levels
VEGFR1 protein levels
Phospho-Flt (Tyr 1213) protein levels
VEGF-A protein levels
Phospho-VEGFR2 protein levels

control variables

Beta-actin (used as endogenous loading control)

controls

Non-specific binding was blocked by incubating the membranes in 5% milk in TBST for 60 minutes prior to overnight incubation with primary antibody.

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