Protease phosphatase inhibitor cocktail

Total lung proteins were prepared from lung homogenates in RIPA buffer and protease/phosphatase inhibitor cocktail (Solarbio Co., Ltd.) and the protein concentration was determined by BCA method. Protein (30 µg/lane) was separated via SDS-PAGE on 10% gels and transferred to PVDF membranes. PVDF membranes were incubated with 5% non-fat milk at 25°C for 1 h with gentle agitation for blocking. Then, the membranes were incubated overnight with the aforementioned antibodies (1:1,000) at 4°C. Following incubation with secondary antibodies (1:3,000) at 25°C for 1 h, the blots were visualized using enhanced chemiluminescence reagent (cat. no. PE0010; Solarbio Co., Ltd.). ImageJ version 2.1.4.7 (National Institutes of Health) was used for densitometric quantification of expression.

Cells were lysed in radioimmunoprecipitation assay buffer containing a protease/phosphatase inhibitor cocktail (Beijing Solarbio Science & Technology Co., Ltd.). The protein concentration estimated using the BCA method, and total proteins (30 µg) were separated in 10% sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (MilliporeSigma). The membranes were then blocked with 5% skim milk for 2 h at room temperature and probed with primary antibodies over-night at 4°C and horseradish-peroxidase-labeled secondary antibodies for 2 h at room temperature. Following three washes the membranes using Tris-buffered saline with Tween-20 (TBST) buffer, the signal was detected using an enhanced chemiluminescence detection system (ECL; Biosharp) and analyzed using the FluorChem™ E system (FluorChem F, ProteinSimple). The band intensities were quantified using ImageJ software (version 1.52a; National Institutes of Health). The primary and secondary antibodies used in the present study are listed in Table SIII.

Cells were lysed using radioimmunoprecipitation assay (Solarbio) containing protease phosphatase inhibitor cocktail (Solarbio) for protein extraction. Protein lysates (20 μg) were separated by SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes for antibody incubation. After blocked with Tris-buffered saline, 0.1% Tween 20 (TBST) containing 5% skim milk powder, the membrane was probed overnight with primary antibodies at 4°C and washed with TBST. After that, the membranes were incubated with secondary antibodies at room temperature for 1 hour. Afterward, the protein bands were visualized using the ECL kit (Monad) and recorded with the Alpha-FluorChemQ imaging system.
The following primary antibodies were used: RUNX2 (sc-390351, Santa Cruz Biotechnology), OSX (ab209484, Abcam), CREB (381013, ZenBio), p-CREB (380697, ZenBio), JUNB (sc-8051, Santa Cruz Biotechnology), PKA (251816, ZenBio), CALCRL (bs-1860R, Bioss), RAMP1 (R25544, ZenBio), SHH (BF0146, Affinity), and glyceraldehyde-3-phosphate dehydrogenase (BS65483M, BIOGOT; 380626, ZenBio). The following secondary antibodies were used: goat anti-mouse immunoglobulin G (IgG) H&L [horseradish peroxidase (HRP)] (511103, ZenBio) and goat anti-rabbit IgG H&L (HRP) (511203, ZenBio).