Western analysis was performed as described previously (20 (link)). Cell lysates were prepared using complete lysis buffer (EMD Millipore, San Diego, CA) with protease and phosphatase inhibitor cocktails (Roche Diagnostics, Indianapolis, IN). Protein quantification was performed using DC protein assay from Bio-Rad (Hercules, and CA). Western blot analysis was performed as described previously (21 (link),22 (link)). Antibodies used include stromelysin1 (cat. No. 14351-S) dilution 1:1,000 in milk, vascular endothelial cadherin (VE-cadherins; cat No. 2158) dilution 1:1,000 in BSA, P-P38 MAPK (cat No. 9112-S) dilution 1:1,000 in BSA, T-P38MAPK (cat No. 9212-S) dilution 1:1,000 in BSA, P-SRC Tyr-416 (cat No. 6943-S) dilution 1:1,000 in BSA and T-SRC (cat No. 2109-S) dilution 1:1,000 in BSA all from Cell Signaling Technology (Danvers, MA). β-actin (dilution in milk, primary antibodies 1:10,000 and secondary antibodies 1:20,000) from Sigma (St. Louis, MO) and Claudin-5 antibodies (cat No. ab15106) 1:1,000 and secondary antibodies 1:5,000 dilution in milk from Abcam (Cambridge, MA). Band densitometry was done using NIH Image J software.
Protocol detail
Western Blotting Protein Analysis
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complete lysis buffer
protease and phosphatase inhibitor cocktails
DC protein assay
P-P38 MAPK
T-P38MAPK
P-SRC Tyr-416
T-SRC
β-actin
Claudin-5 antibodies
Actin
Antibodies
Assay
Buffer
Cell
Claudin 5
Densitometry
Diagnostics
Dilution
Milk
P38mapk
Phosphatase
Protease
Protein
Rad protein
Ve cadherins
Western blot analysis
Corresponding Organization :
Other organizations : University of Georgia, Charlie Norwood VA Medical Center
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Variable analysis
independent variables
- Western analysis protocol used previously (20)
- Cell lysis buffer and inhibitor cocktails used
- Antibodies used for Western blot analysis
- Protein expression levels of stromelysin1, VE-cadherin, P-P38 MAPK, T-P38MAPK, P-SRC Tyr-416, T-SRC, Claudin-5, and β-actin
- Protein quantification method (DC protein assay from Bio-Rad)
- Band densitometry using NIH Image J software
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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