Horseradish peroxidase conjugated secondary antibodies bs 0293m

Cell proteins lysates from MC3T3-E1 cells were partitioned by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (EMD Millipore, Billerica, MA, USA). Blotting was carried out with spectific antibodies [anti-B-cell lymphoma-2 (Bcl-2) associated X protein (Bax), 1:5,000 dilution, cat. no. ab32503; Abcam; anti-Bcl-2, 1:1,000 dilution, cat. no. ab692; Abcam; anti-OPN, 1:1,000 dilution, cat. no. ab8448; Abcam; anti-OCN, 1:1,000 dilution, cat. no. ab13418; Abcam; anti-Osterix, 1:1,000 dilution, cat. no. ab94744; Abcam; anti-BMP2, 1:500 dilution, cat. no. ab14933; Abcam; anti-Smad1, 1:1,000 dilution, cat. no. ab33902; Abcam; anti-Smad5, 1:1,000 dilution, cat. no. ab194661; Abcam; anti-Smad8, 1:5,000 dilution, cat. no. ab13723; Abcam; anti-RunX2, 1:500 dilution, cat. no. ab23981; Abcam; anti-β-actin, 1:1,000 dilution, cat. no. ab8227; Abcam]. After that, horseradish peroxidase-conjugated secondary antibodies (bs-0293M; Bioss, Beijing, China) were added and maintained at room temperature for 1 h. The results were assessed by enhanced chemiluminescent reagents (EMD Millipore) using an ECL system (Amersham Pharmacia, Piscataway, NJ, USA).

Proteins from 16HBE cells were obtained and bicinchoninic acid assay was performed to detect the protein concentration. After that, equal quantity of proteins (50 µg) were solubilized in 5× sodium dodecyl sulfate (SDS)-sample buffer and separated on the SDS polyacrylamide gels (Thermo Fisher Scientific, Inc.). Separated proteins were then transferred onto a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). After blocking, the membranes were incubated with anti-IDO (dilution, 1:500; ab55305); anti-p-Raf-1 (dilution, 1:500; ab208449); anti-Raf-1 (dilution, 1:1,000; ab50858); anti-p-Mek1/2 (dilution, 1:1,000; ab194754); anti-Mek1/2 (dilution, 1:1,000; ab215263); anti-p-Erk1/2 (dilution, 1:1,000; ab201015); anti-Erk1/2 (dilution, 1:1,000; ab17942); anti-GAPDH (dilution, 1:1,000, ab8245; all from Abcam) antibodies overnight at 4°C. After that, horseradish peroxidase-conjugated secondary antibodies (bs-0293M; BIOSS, Beijing, China) were added and incubated at room temperature for 1 h. The results of all the assessments were evaluated by enhanced chemiluminescent reagents (EMD Millipore).