Irdye infrared dye

Whole cell extracts (WCE) were prepared by freezing the cell pellet overnight at −80°C. The pellet was then resuspended in 3 volumes of WCE buffer (20 mM HEPES, 0.42 M NaCl, 0.2 M EDTA, and 25% glycerol; pH 7.4) plus protease inhibitor cocktail and incubated on ice for ten min followed by 100,000 ×g centrifugation at 4°C. Protein samples were resolved by SDS polyacrylamide gel electrophoresis and electrophoretically transferred to Immobilon-FL membranes. Membranes were blocked at room temperature for 1 hour in TBS [TBS; 10 mM Tris-HCl (pH 7.4) and 150 mM NaCl] containing 3% BSA. Subsequently, the membrane was incubated overnight at 4°C with antibodies to PPARγ (Santa Cruz, 7273), C/EBPα (Santa Cruz, 365318), or heat-shock protein 90 (HSP90) (Santa Cruz, 13119) (Santa Cruz Biotechnology, Dallas, Texas). After three washes in TBST (TBS plus 0.1% Tween 20), the membrane was incubated with an infrared anti-rabbit (IRDye 800, green) or anti-mouse (IRDye 680, red) secondary antibody labeled with IRDye infrared dye (LI-COR Biosciences) for 2 hours at 4°C. Immunoreactivity was visualized and quantified by infrared scanning in the Odyssey system (LI-COR Biosciences).

Transient transfections were performed using GeneFect (Alkali Scientific Inc., Pompano Beach, FL, USA) in the HEK293 cells for 48 h. Cells were harvested via centrifugation and resuspended in 200 μM HEPES. The His-Tagged proteins were extracted using the HisLink™ Protein Purification Resin (Promega, Madison, WI, USA). Proteins were subsequently dialyzed overnight to remove excess elution compounds. The proteins were quantified using the BCA Protein Assay Kit (Thermofisher Scientific, Waltham, MA, USA). To confirm that the correct protein was purified, protein extracts were resolved by SDS polyacrylamide gel electrophoresis and electrophoretically transferred to Immobilon-FL membranes. Membranes were blocked at room temperature for 1 h in Odyssey Blocking buffer (LI-COR Biosciences, Lincoln, NE, USA). Subsequently, the membrane was incubated overnight at 4 °C with PPARα (sc-1982), PPARγ (sc-7273), or His-Probe Antibody (sc-8036). After three washes in TBST (TBS plus 0.1% Tween 20), the membrane was incubated with an infrared anti-goat (IRDye 800, green) or anti-mouse (IRDye 680, red) secondary antibody labeled with IRDye infrared dye (LI-COR Biosciences) (1:15,000 dilution in TBS) for 2 h at 4 °C. Immunoreactivity was visualized and quantified by infrared scanning in the Odyssey system (LI-COR Biosciences, Lincoln, NE, USA).

Western blots derived from whole cell lysates or immunoprecipitated proteins were visualized after incubation with primary antibodies (listed in Supplementary Table S2) using IRDye infrared dye (Li-Cor Biosciences) conjugated secondary antibodies and the signal was collected with a Li-Cor Odyssey infrared imager.
Co-immunoprecipitation was performed as previously described (21 (link)), and anti-FOXK2 antibody (Bethyl Laboratories A301–729A) was used for immunoprecipitation. Whole cell lysates were from H1-FOXK2-DHFR ESCs cultured either in the presence of TMP (10 μM) in the media to maintain protein stability or in the absence of TMP to trigger FOXK2 degradation.

OA-MSCs and OACs were collected and treated with 1 × radioimmuno-precipitation lysis buffer (Thermo Fisher Scientific) supplemented with Halt protease inhibitor (Cat#78430, Thermo Fisher Scientific). Cell lysis was then sonicated for 15 s, heated to 95°C for 5 min, and stored at −20°C until use for the sodium dodecyl sulfatepoly acrylamide gel electrophoresis; 20-μg protein samples were loaded and transferred to nitrocellulose membrane probed with anti-SHH antibody (Cat#2207, at 1:1,000 dilution, Cell Signaling Technology, Danvers, MA, United States), anti-SMO antibody (Cat#ab235183, at 1:500 dilution, Cambridge, MA, United States), anti-RUNX2 antibody (Cat# ab23981, at 1:5,000 dilution, Abcam), anti-SOX9 (Cat# ab185230, at 1:2,000 dilution, Abcam), and anti-MMP13 antibody (Cat#39012, at 1:3,000 dilution, Abcam) and normalized with β-Actin (Cat#ab108349, at 1:1,000 dilution, Abcam, United States). After the primary antibody incubation, the membranes were labeled with secondary anti-rabbit IgG antibody (at a dilution of 1:15,000, IRDye infrared dye, LI-COR Biosciences, Lincoln, NE, United States) and were then imaged using the Licor Odyssey CLx scanner (LI-COR Biosciences). Quantification of Western blot data by evaluating the gray values of protein expression level was performed using software in the Odyssey Infrared Imaging system.

Western blot was performed using a quantitative Western-Blot System (LI-COR Bioscience, Lincoln, NE, USA) following the manufacturer’s instructions. Cells were lysed in RIPA buffer (Sigma-Millipore) for 30 min on ice. Samples containing identical amounts of protein (25–40 µg) were resolved by NOVEX 4–12% Tris-glycine gradient gel (Thermo Fisher Scientific), transferred to Amersham Protran nitrocellulose membrane (GE HealthCare), and blocked in Li-COR blocking buffer. Membranes were probed with antibodies listed in Supplementary Table 6. Secondary antibodies were labeled with IRDye infrared dyes (LI-COR Biosciences) and protein levels were quantified using the Odyssey Infrared Imager (LI-COR Biosciences). Densitometry analysis was performed using the Image Studio™ acquisition software from LI-COR imaging systems. Protein expression was normalized to the loading control (i.e., Actin).

Proteins were extracted with RIPA buffer (RIPA buffer, 1 mM DTT, 1M NaF, 100 mM sodium orthovanadate and protease and phosphatase inhibitors). After SDS PAGE separation, and transfer onto a PVDF membrane by iBlot dry blotting system (Invitrogen), membranes were incubated overnight at 4°C with primary antibodies: HER2 (GTX50425; Genetex), βIII-tubulin (clone TUJ1), βII-tubulin (clone 7B9) α-tubulin (T6199), β-tubulin (T4026) and β-actin (A5441) from Sigma-Aldrich, and 1h at room temperature with secondary antibodies (IRDye Infrared Dyes from LI-COR Biosciences). Membranes were scanned using Odyssey infrared imaging system (LI-COR Biosciences) and densitometric quantification was performed with Odyssey software. Quantification of Western Blot was performed using Image J software (Rasband, W.S., National. Institutes of Health). Expression levels of proteins were normalized against β-actin.