Small portions of the snap frozen resected brain tissue from EPI and NON-EPI regions were homogenized in radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, USA) with protease inhibitor (Sigma-Aldrich, USA). The tissue suspension was centrifuged at 14000G (Avanti-J25I, Beckman Coulter, USA), thereafter supernatant was collected and concentration of protein was measured by Bradford Method.
P-gp/MDR1 was separated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and later transferred to PVDF (polyvinylidene fluoride) membranes (EMD Millipore Corp., Billerica, MA, USA) in semi-dry transfer (trans-Biot™ SD, Bio-Rad, USA). All other protein targets were separated by 10% SDS-PAGE. In brief, the membranes were probed overnight with primary antibody (see target proteins listed in Supplemental Table 1a). After incubation with primary antibody the membranes were probed with appropriate secondary antibody (Supplemental Table 1b) as previously described [14 (link)]. For the target proteins either the PVDF membranes were incubated in stripping buffer in 50 °C for 30 minutes followed by blocking of the membranes or a fresh gel was simultaneously repeated with the samples. In each case, the protein expression was normalized by β-actin (as loading control) and quantified by ImageJ software.