Protein extraction and western blotting

Stable U87 cells transfected by pCL19A and pCDNA3.1( +) vectors were lysed using Ripa buffer (BioBasic, Canada) on ice for 30 min according to the manufacturer’s protocol. The lysates were centrifuged at 13,000 rpm for 30 min, and the supernatant was collected and stored at -80 °C. The protein concentration was measured by the Bradford assay. To prepare the Bradford reagent, coomassie blue G-250 (Merck, Germany) was completely dissolved in methanol (Merck, Germany) and 85% phosphoric acid (H3PO4) (Merck, Germany). Next, the acid solution mixture was slowly added to H2O, and the solution was filtered with Whatman paper No1 (Merck, Germany) [45]. To evaluate the concentration of standard protein (BSA) and protein samples, the Bradford reagent was added to each sample, and absorbance was measured at 630 nm using a spectrophotometer. Total protein was subjected to SDS-PAGE using a 10% polyacrylamide gel and transferred to a PVDF membrane (Thermo Fisher Scientific, USA). Next, membrane blocking was performed at 4 °C for 2 h using 5% skim milk (Sigma, USA) diluted in PBST (Bio basic, Canada). Subsequently, the membrane was incubated with the primary antibodies to GFP (Santa Cruz, USA, sc-9996, 1:300), MMP2 (Santa Cruz, USA, sc-10736, 1:300), RECK (Santa Cruz, USA, sc-373929, 1:300), BAX (Santa Cruz, USA, sc-7480, 1:300), BCL2 (Santa Cruz, USA, sc-492, 1:300), IκB-α (Santa Cruz, USA, sc-1643, 1:300), TIMP3 (Santa Cruz, USA, sc-373839, 1:300), PI3K (Santa Cruz, USA, sc-67306, 1:300), and β-Actin (Santa Cruz, USA, sc-47778, 1:300) overnight at 4 °C. The anti-mouse IgG-Horse Radish Peroxidase (HRP) (Santa Cruz, USA, sc-516102, 1:1000) or anti-rabbit IgG-HRP (Santa Cruz, USA, sc-2357, 1:1000) secondary antibodies were diluted according to the manufacturer’s instructions and was incubated with the membrane for 1 h after three times washes with PBST. Signals were detected with an ECL detection reagent (Thermo Fisher Scientific, USA). β-Actin protein was used for normalization.