Ab189139

The proteins were extracted from glioma tissues or cell lines utilizing lysis buffer of protease inhibitor PMSF. The BCA Kit was applied to analyze the protein concentration. Then proteins (20 μg) were separated on SDS-PAGE gels and blotted on PVDF membrane. After blocking in 5% skim milk, the primary antibody was added for incubation. After cleaning the membrane, secondary antibody was incubated before exposure. Then the membrane incubated with primary antibodies targeting PLK1 (ab189139; Abcam, Cambridge, MA, USA) or GAPDH (1:1000; Beyotime, Nantong, China) overnight at 4 °C. Next, the membranes were washed using TBS for three times and incubated with an HRP-linked goat antirabbit secondary antibody (ab205718; Abcam) at room temperature for 2 h. The ECL Western Blotting Substrate Kit (ab65623; Abcam) was used to detect protein signals.

Total protein was extracted with the RIPA buffer (Beyotime, USA). The protein samples of mitochondria were extracted with the commercial kits (Invitrogen, USA). BCA method was used for determining the concentration of protein. After that, these proteins were separated with 10% SDS-PAGE gel (Beyotime, China) and transferred to PVDF membranes (Millipore, USA). Then, proteins were blocked with the BSA (Beyotime, China) and incubated with the primary antibodies at room temperature for 2 hours followed by an incubation with the secondary antibodies for 2 hours at room temperature. The primary antibodies used in this research were PLK1 (Abcam, ab189139), Bax (Abcam, ab32503), Bcl-2 (Abcam, ab32124), cleaved caspase3 (Abcam, ab32042), LC3 I (Abcam, ab63817), LC3 II (Abcam, ab51520), Beclin1 (Abcam, ab207612), p-AMPK (Abcam, ab133448), AMPK (Abcam, ab32047), FUNDC1 (Abcam, ab224722) and β-actin (Abcam, ab8226). The primary antibodies were diluted with the BSA with the ratio (1:1000). Finally, the Pierce Western Blotting Substrate (Thermo Fisher Scientific, USA) was used for the detection of the immunoreactive signals. The quantification of bands was performed with the ImageJ (National Institutes of Health, USA) [28 (link)].

At 48 h post transfection, cells were collected and lysed in 200 μL of lysis buffer. By 12% sulfate–polyacrylamide gel electrophoresis to separate the total cell lysates and blotted with rabbit anti-human PLK1 (ab189139, Abcam, Cambridge, UK) and anti-β-actin (20536-1-AP, Proteintech, Los Angeles, CA, USA). After incubation with DyLight fluorescent dye-conjugated secondary antibodies (A23720, Abbkine, Wuhan, China), images were obtained using the Odyssey CLx Imaging System (LI-COR Biosciences, Lincoln, NE, USA).