Chemcam imager

Cell lysates of hOBs were prepared by using RIPA buffer, and micro Lowry assay was used to quantify total protein of hOBs. Cell lysates were diluted to obtain a protein content of 1 ng/ μL in ddH₂O. 20 ng protein was transferred to a nitrocellulose membrane via a Dot blotter (Carl Roth, Karlsruhe, Germany). Ponceau staining was performed to confirm the transfer of proteins. Membranes were washed with Tris-buffered saline with Tween 20 (TBS-T) to remove ponceau and then blocked with 5 % BSA in TBS-T for 1 h at ambient temperature. Membranes were incubated with primary antibodies (see detailed information in Table 2(Tab. 2)) overnight at + 4 °C, and GAPDH was used for the housekeeper. The next day membranes were incubated with the secondary antibodies for 2 h at ambient temperature after washing with TBS-T. ECL substrate solution was used for chemiluminescent signal development of membranes. Signals of proteins were detected by a ChemCam imager (INTAS, Göttingen, Germany), and the intensity of signals was quantified by using the ImageJ software (Weng et al., 2020[52 ]).

Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 5% fat-free dried skimmed milk in TBS-T (10 mM Tris HCl pH 7.6, 150 mM NaCl, 1% Tween 20) and probed with the indicated primary antibodies in 5% BSA in TBS-T at 4°C overnight. After washing, membranes were incubated with secondary peroxidase-conjugated antibodies (1:2,500) diluted in 5% fat-free dried skimmed milk. The Immobilon™ Western Reagents (Millipore Corporation, Billerica, MA, United States) and the ChemCam Imager (INTAS Science Imaging Instruments GmbH, Göttingen, Germany) were used for signal detection.