Evaluation of common housekeeping proteins under ischemic conditions and/or rt-PA treatment in bEnd.3 cells

Thrombolysis with recombinant tissue plasminogen activator (rt-PA) is the only pharmacological approved treatment for ischemic stroke, despite its associated increasing risk of hemorrhagic transformation. Since many of rt-PA effects in blood-brain barrier (BBB) are not well characterized, the study of protein changes in BBB cells after rt-PA administration may help to understand its adverse effects. Our aim was to analyze protein levels of four commonly used housekeeping proteins: β-Actin, α-Tubulin, GAPDH and HPRT in bEnd.3 endothelial cell line subjected to oxygen and glucose deprivation (OGD) conditions and rt-PA treatment to determine their reliability as Western blot loading controls. bEnd.3 monolayers were subjected to 2.5 h of OGD and reperfusion with/without 20 μg/ml of rt-PA. At 3, 6, 24 and 72 h post-OGD, protein levels were analyzed by Western blot using Stain-Free technology. OGD significantly decreased β-Actin, α-Tubulin, GAPDH and HPRT protein levels at 3, 6, 24 and 72 h post-OGD without significant rt-PA treatment effects except for the GAPDH levels increase in control condition at 3 h post-OGD. The present study clearly demonstrated that β-Actin, α-Tubulin, GAPDH and HPRT proteins are not suitable as loading controls for Western Blot analysis in bEnd.3 cells after OGD.We reported altered levels of β-Actin, α-Tubulin, GAPDH and HPRT housekeeping proteins in bEnd.3 endothelial cell line after an ischemic insult. Therefore, we demonstrated that these proteins are not suitable as loading controls for Western Blot analysis in our experimental conditions and we recommended the use of Stain-Free gels as an alternative to traditional housekeeping proteins normalization.