Western blotting imaging system

Human bladder cancer cell lines T24 and 5637 were provided by the cell bank of the Chinese Academy of Sciences in Shanghai. The present study used metformin (Shanghai Beyotime Biotechnology Co., Ltd., Shanghai, China), Roswell Park Memorial Institute (RPMI)-1640 medium (Cellmax Company, Groningen, Netherlands), fetal bovine serum (FBS) (Sigma, St. Louis, MO, USA), phosphate-buffered saline (PBS) (Beijing Solarbio Company, Beijing, China), Cell counting Kit-8 (CCK-8) (Shanghai Beyotime Biotechnology Co., Ltd., Shanghai, China), and an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Assay Kit (BD Biosciences, San Jose, CA, USA). Antibodies recognizing PI3K/Akt/mTOR signaling pathway associated proteins (phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), phosphorylated (p)-PI3K, protein kinase B (Akt), p-Akt, mechanistic target of rapamycin (mTOR), p-mTOR), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and Apoptosis-related protein (cleaved-caspase 3 and cleaved-poly(ADP-ribose) polymerase (PARP)) were all purchased from Affinity Biosciences (Cincinnati, OH, USA). We also used a microplate analyzer (Thermo Scientific, Waltham, MA, USA), a flow cytometer (Bio-Rad, Hercules, CA, USA), and a western blotting imaging system (Bio-Rad).

After the behavior tests, the contralateral hippocampal tissues from the six remaining mice in every group were collected for western blotting. Briefly, the tissues were placed in RIPA lysis buffer containing protease inhibitors and homogenized. After centrifugation, the supernatant was collected. The BSA assay was used to quantitate protein concentration in the supernatant. Protein samples were denatured at 95°C for 10 min. Then, 20 μg were used for SDS-PAGE and transferred onto a nitrocellulose (NC) membrane. The membrane was incubated with blocking solution at room temperature for one hour. Then, the membrane was incubated with primary antibodies at 4°C overnight: GAD76 (1:500, Proteintech, IL, USA), GA1B (1:1000, Abcam, MA, USA), and N-methyl-D-aspartate receptor 2B (NR2B) (1:500, Proteintech, IL, USA). The membrane was then washed and then incubated with horseradish peroxidase-(HRP-)-conjugated secondary antibodies at room temperature for 1 hour. An enhanced chemiluminescence (ECL) reagent kit was used for detection of western blotting results. A western blotting imaging system (Bio-Rad, Hercules, CA) was used to measure the fluorescence intensity of protein bands, and ImageJ was used for quantitation.

Seven days after osteoinduction, Western blotting was conducted as described in our previous study,²⁸ using primary antibodies against DMP 1 (ab103203, Abcam), DSPP (sc‐73632, Santa Cruz Biotechnology, Dallas, TX), BSP (ab52128, Abcam), OSX (ab94744, Abcam), and VEGF (sc‐57496, Santa Cruz Biotechnology), with GAPDH as an internal control. Membranes were visualized using a chemiluminescent reagent (Sigma‐Aldrich) under a Western blotting imaging system (Bio‐Rad, Hercules, CA), and protein expression was quantified using ImageJ software.