Chemiluminescence reagent

Cells were lysed by RIPA buffer supplemented with protease inhibitor. The proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were incubated with primary antibodies, then with horseradish peroxidase (HRP)-conjugated secondary antibodies, and visualized by chemiluminescence reagent (Proteintech, Wuhan, Shanghai). The primary antibodies used included antibodies produced by Abcam (Cambridge, MA, USA): anti-osterix, anti-ALP, and anti-osteopontin (OPN); antibodies by Cell Signaling Technology (Danvers, MA, USA): anti-PPARγ, anti-C/EBPα, anti-phospho-src homology 2 domain-containing transforming protein C1 (SHC1) (Tyr317), anti-ERK1/2, and anti-phospho-ERK1/2 (Thr202/Tyr204); antibody by ABclonal (Wuhan, China): anti-nuclear factor of activated T cells 1 (NFATC1); antibody by Beyotime (Shanghai, China): anti-cathepsin K (CTSK); antibodies by Proteintech (Wuhan, China): anti-OSMR, anti-fatty acid-binding protein 4 (FABP4), anti-microtubule-associated protein 1 light chain 3 (LC3), anti-autophagy-related gene 5 (ATG5), anti-autophagy-related gene 7 (ATG7), anti-Beclin 1, anti-P62, anti-SHC1, and anti-β-actin. The expression levels of the target genes were analyzed by dividing the grayscale of the target bands with that of β-actin.

Western blotting was conducted as described before 28 (link). In brief, RIPA buffer (APPLYGEN, China) containing 1% protease/phosphatase inhibitor was used for protein extraction. Then 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and polyvinylidene difluoride (PVDF) membranes were used to separate and transfer proteins. Then the membranes were subjected to primary antibodies incubation after blocking with 5% milk. Primary antibodies against ZFP91 were purchased from Abcam and β-actin antibodies were purchased from Proteintech. Followed by an hour incubation with secondary antibodies, the bands were observed by the chemiluminescence reagent (Proteintech).

The tissues and cells were subjected to protein isolation after lysis in RIPA buffer (Applygen Tech Inc., China) containing 1% protease/phosphatase inhibitor. Then, a 10% ExpressCast PAGE Gel Preparation Kit (New Cell and Molecular Biotech, China) was used to separate the proteins. The polyvinylidene difluoride membranes were used for proteins transfer, and the membranes were then blocked with 5% milk. Subsequently, antibodies against LRP8 (NB100-2216, Novus Biologicals, USA), GAPDH (60,004-1-Ig; Proteintech, USA), β-catenin (8480s, Cell Signaling Technology, USA), c-Myc (5605s, Cell Signaling Technology), cyclin D1 (2978s, Cell Signaling Technology), E-cadherin (ab1416; Abcam), N-cadherin (ab98952, Abcam), and vimentin (5741s, Cell Signaling Technology) were used to incubate the membranes overnight. The bands were observed and analyzed using the chemiluminescence reagent (Proteintech) after 1 h incubation with the corresponding secondary antibodies (Proteintech).