T40044

Total proteins were extracted from the right cerebral cortex in the ischemic penumbra and quantitated using a BCA kit (Beyotime, Shanghai, China). Equal amounts of protein were separated with 8–12% SDS-PAGE gels and electrically transferred to PVDF membranes (Millipore, Burlington, MA, USA). The membranes were incubated with primary antibodies against Bcl-2 (1:1000, T40056, Abmart, Shanghai, China), Beclin1 (1:2000, ab207612, Abcam, Cambridge, UK), Bax (1:10,000, 50599-2-Ig, Proteintech, Wuhan, China), caspase3 (1:1000, T40044, Abmart, Shanghai, China), LC3B (1:2000, T40044, Abcam, Cambridge, UK), ZO-1(1:2000, 21773-1-AP, Proteintech, Wuhan, China), Occludin (1:10,000, 66378-1-Ig, Proteintech, Wuhan, China), β-actin (1:100,000, AC026, Abclonal, Wuhan, China), and GAPDH (1:10,000, 60004-1-Ig, Proteintech, Wuhan, China) at 4 °C overnight. They were then incubated in Peroxidase-Conjugated Goat Anti-Rabbit IgG(H+L) or Goat Anti-Mouse IgG(H+L) (Yeasen, Shanghai, China) diluted at 1:10,000 for 1 h at room temperature. After chemiluminescence detection had been performed using ECL reagents (Yeasen, Shanghai, China) and a Fusion Edge Multi-function Imaging System (Vilber, Collégien, France), the relative optical densities of the protein bands were analyzed using ImageJ software.

Lenvatinib (HY-10981), gefitinib (HY-50895), and copanlisib (HY-15346A) were purchased from MedChemExpress (Shanghai, China), and elacridar (S7772) was purchased from Selleck Chemicals (Shanghai, China). Stock solutions of 20 mM Lenvatinib, 100 mM elacridar, and 20 mM gefitinib were dissolved in 100% dimethyl sulfoxide (DMSO), and the stock solution of 10 mM copanlisib was dissolved in Milli-Q water. Antibodies against total epidermal growth factor receptor (EGFR; A11577, ABclonal), phospho-EGFR (AP0820, ABclonal), total PI3K (ab32089, Abcam), phospho-PI3K (4228, CST), total AKT (9272, CST), phospho-AKT (4060, CST), total MEK1/2 (A4868, ABclonal), phospho-MEK1/2 (AP0209, ABclonal), total ERK1/2 (4695, CST), phospho-ERK1/2 (4376, CST), caspase-3 (T40051, Abmart), Bcl-2-associated X (Bax; T40044, Abmart), multidrug resistance protein 1 (MDR1; 13978, CST), breast cancer resistance protein (BCRP; 130244, Abcam), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 5174, CST) were used. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse antibodies were purchased from Beyotime Biotechnology (Shanghai, China). Alexa Fluor-conjugated goat anti-rabbit (647 nm) and goat anti-mouse (488 nm) antibodies were purchased from Invitrogen (Shanghai, China).