Serpinb2

Proteins from cells lysed in RIPA buffer were separated using SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked using 5% skim milk in Tris-buffered saline containing Tween-20, incubated overnight at 4°C with primary antibodies directed against SerpinB2 (Abcam, Cambridge, UK), SerpinE1 (Abcam), uPA (Abcam), c-Jun (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or phospho-c-Jun (Santa Cruz Biotechnology), and β-actin (Sigma-Aldrich, St. Louis, MA, USA), and then incubated with HRP-conjugated secondary antibodies (Santa Cruz Biotechnology). Blotted membranes were visualized using enhanced chemiluminescence reagents (GE Healthcare, Piscataway, NJ, USA) and protein expression was depicted as intensity of each protein relative to that of β-actin using ImageJ software.

hBMSCs were purchased from Cyagen Biosciences (Guangzhou, China). hBMSC growth medium were purchased from Cyagen Biosciences. Cells cultured in a atmosphere of 5% CO₂ at 37 °C. Osteogenic induction medium was prepared according to previous methods [49 (link)].
SERPINB2 recombinant protein was purchased from Amyjet Scientific (Abnova, wuhan, china). Antibodies used for western blotting including GAPDH (1:1000, Beyotime, shanghai, china), RUNX2 (1:1000, Beyotime), COL1A1 (1:1000, Beyotime), SERPINB2 (1:1000, Abcam), SP7/Osterix (1:1000, Abcam, shanghai, china), non-phosphorylated (active) β-catenin (1:1000; Cell Signaling Technology), or total β-catenin (1: 1000; Beyotime), phosphorylated and total ERK (1:1000; Cell Signaling Technology), phosphorylated and total AKT (1:1000; Cell Signaling Technology). Second antibodies Alexa Fluor 555 was purchased from Beyotime. Lipo6000 Transfection Reagents were purchased from Beyotime (Shanghai, china).
The siRNAs were purchased from Genepharma (Hangzhou, china).
hSERPINB2 sense: GCAAAGAAUCAAGUUGCAATT
hSERPINB2 antisense: UUGCAACUUGAUUCUUUGCTT
hNC sense: UUCUCCGAACGUGUCACGUTT
hNC antisense: TTAAGAGGCUUGCACAGUGCA
mSERPINB2 sense: GGAGAGAAGUCUGCAAGAUTT
mSERPINB2 antisense: AUCUUGCAGACUUCUCUCCTT
mNC sense: GGCUCUGTTCUUGUCACGUAA
mNC antisense: UUTTGAGGCAAGCACAGUGCA