G box chemi xx9

For western blotting, a portion of frozen liver was dissected and ground in liquid nitrogen. The proteins were extracted with RIPA lysis buffer (#P0013B, Beyotime, China) supplemented with 1× Protease Inhibitor Cocktail (#P8849, Sigma, USA). Protein concentrations were determined by Pierce™ BCA Protein Assay Kit (#23225, Thermo, USA). Equal amounts of proteins from different mice liver samples were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were then blocked with 5% skim milk in TBST for 1 h and incubated with primary antibodies overnight at 4 °C. The antibodies included anti-alpha smooth muscle actin (α-SMA) [1:1000; #19245s, Cell Signaling Technology (CST), USA], anti-phospho-Akt (Thr308) (1:1000; #13038S, CST, USA), anti-pan-Akt (1:1000; #4691T, CST, USA) and anti-GAPDH (1:50,000; #60004-1-Ig, Proteintech, China), and corresponding secondary antibody was incubated with membrane for 1 h after washed thrice with TBST. The protein bands were then washed thrice with TBST and visualized with Clarity Western ECL Substrate (#170-5061, Bio-Rad, USA) by using G:BOX Chemi XX9 (Syngene, UK) and semi-quantitative analysis was performed by ImageJ (National Institutes of Health, USA).

TMSCs were washed twice with PBS and lysed in RIPA lysis buffer supplemented with cocktail of protease/phosphatase inhibitors (Thermo Fisher Scientific). Supernatants were collected after centrifugation at 12,000 rpm for 10 min. Humerus was frozen rapidly using liquid nitrogen immediately after dissection. Frozen humerus was homogenized using a mortar and pestle on dry ice. Whole protein of bone was extracted in ice by RIPA lysis buffer supplemented with cocktail of protease/phosphatase inhibitors for 20 min. Supernatants were collected after centrifugation at 12,000 rpm for 10 min and protein concentration was measured using BCA protein assay kit (Pierce). Primary antibodies used were anti-Wnt16 (Thermo Fisher Scientific), anti-RUNX2, anti-osteocalcin, anti-CathepsinK (Santa Cruz Biotechnology Inc., Dallas, TX, USA), and anti-beta actin (Sigma-Aldrich) antibodies. Secondary antibodies used were HRP conjugated antibodies. Protein bands were visualized with ECL™ Prime Western Blotting Detection Reagent (Amersham) in GBOX chemi XX9 (Syngene).

HeLa 229 cells (ATCC, CCL-2.1, USA) were transfected with either pcDNA3 or pcDNA3/FlaB3 using the Lipofectamine^TM 2000 Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Transfected HeLa cells were harvested and detected using immunoblotting analysis with antibodies against FlaB3. The primary anti-FlaB3 antibodies were provided by the Institute of Pathogen Biology at the University of South China. The secondary antibody was horseradish peroxidase-conjugated secondary goat anti-rabbit IgG (GeneTex, San Antonio, TX, USA). Finally, the ECL Prime Western Blotting Detection Reagent (Thermo Fisher Scientific, Fremont, CA, USA) was used as recommended to visualize the protein bands with a G:BOX Chemi XX9 (Syngene, Cambridge, UK) digital imager.

Western blotting was performed on frozen tissue samples preserved at − 80 °C. We used RIPA lysis buffer, proteinase inhibitors and phosphatase inhibitors as the protein extraction reagents. A bicinchoninic acid (BCA) protein analysis kit was employed to examine the protein concentration of each sample. The proteins of the samples were loaded onto sodium dodecyl sulfate (SDS) polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes after electrophoresis. In general, the membranes were subsequently blocked by using 5% skimmed milk and incubated for 1 h at room temperature under agitation. For phosphorylated protein detection, we used 5% bovine serum albumin (BSA) for blocking. Then, the membranes were incubated overnight at 4 °C with the following primary antibodies: pan-AKT (CST), p-AKT^Ser473 (CST), m-TOR (CST), p-mTOR^Ser2448 (Abcam), S6 (CST), p-S6^Ser240/244 (CST), 4E-BP1 (CST) and p-4E-BP1^Thr37/46 (CST) at a dilution of 1:1000. β-Actin was used as an internal reference. After that, the membrane was incubated with HRP-linked secondary antibodies at room temperature for 1 h. Finally, the membrane was exposed to film using GBox-Chemi XX9 (Syngene) after being incubated with enhanced chemiluminescence substrate. We used GeneTools software to evaluate the grayscale intensity and perform quantitative analysis of the extracted protein.

SDS PAGE on Novex™ 4–12% Tris-Glycine Mini Gels (Invitrogen) and Western blotting was performed, as previously described [35 (link),37 (link)]. Briefly, following electro transfer, the membranes were incubated with 1 μg/mL anti-DPE or a mixture of anti-CDAG/anti-DLS, each at 1 μg/mL (see Supplementary Materials Figure S3 for location of the epitopes), and developed using the Pierce ECLPlus Kit (Thermo Fisher Scientific, Waltham, MA, USA). Reprobes with anti-PRG4 (MAb 9G3, 0.05 µg/mL; Millipore Sigma, Burlington, MA, USA) [37 (link),38 (link)] were performed by washing membranes three times for ten minutes each with 8 mL of TBS containing 0.1% Tween 20 (Bio-Rad, Hercules, CA, USA). The membranes were incubated with and were developed using the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Waltham, MA, USA). To confirm equivalent loading amounts, western membranes from media samples and cell extracts were reprobed with anti-HC1 (1 μg/mL) [30 ] or anti-β-actin (1:5000 MAbAC-15; Novus Biologicals, Littleton, CO, USA). Imaging was performed with GBox Chemi-XX9 (Syngene, Frederick, MD, USA).

A total of 10 μg protein per sample were separated by SDS-PAGE (12% w/v) followed by transfer to activated PVDF membranes. Membranes were blocked in 5% (w/v) skimmed-milk dissolved in TBST (20 mM Tris-HCl, pH 7.6, 17 mM NaCl, 0.1% (v/v) Tween-20) and incubated with polyclonal primary antibodies (1:1000 dilution) for 12 h at 4 °C. For detection, goat anti-rabbit IgG conjugated to horseradish peroxidase (1:10,000 dilution, BioRad, 1706515) and Clarity Western ECL Substrate (BioRad, 1705061) were used according to the manufacturers’ instructions. Syngene G:BOX Chemi XX9 was used for chemiluminescent detection.