Ab45381

MG-63 and Saos-2 cells infected with LV-shCNN3 or LV-NC were seeded (4 × 10⁵ cells/well) in 6-well plates. After being cultured for 48 h, cells were harvested, and the total protein content was isolated with RIPA buffer. After protein quantification, 30 μg of total protein was used for western blotting, which was conducted according to the methods described previously [33 (link)]. The primary antibodies used in this study were purchased from Abcam (Cambridge, MA, USA) and their details are as follows: anti-CNN3 (1:2000, ab151427), anti-MMP9 (1:1500, ab76003), anti-VEGF (1:5000, ab52917), anti-vimentin (1:1000, ab92547), anti-E-cadherin (1:1000, ab231303), p-p38 (1:1000, ab45381), p38 (1:1000, ab32142), ERK1/2 (1:1000, ab115799), p-ERK1/2 (1:800, ab214362), and anti-GAPDH (1:5000, ab181602).

Cells were lysed by NP-40 with protease inhibitors and phosphatase inhibitors; the proteins were separated via SDS-PAGE electrophoresis, transferred onto nitrocellulose membranes, and then incubated with primary and secondary antibodies, respectively. Primary antibodies against JNK1/2/3 (YT2441) (1 : 1000) and phospho-JNK1/2/3 (phospho Thr183/Y185) (1 : 1000) were obtained from ImmunoWay Biotechnology (Newark, DE, USA). Primary antibodies against ERK (ab196883, 1 : 1000), p-ERK (ab50011, 1 : 5000), p38 (ab27986, 1 : 1000), and p-p38 (ab45381, 1 : 5000) were purchased from Abcam (Abcam, Cambridge, MA, United States). The JNK inhibitor SP600125 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The immunoreactive bands were detected with an enhanced chemiluminescence detection system from Pierce (Thermo Fisher Scientific Inc., Rockford, IL, USA). Protein band was analyzed with Quantity One software (Bio-Rad Laboratories, Life Science Research, CA, USA). Protein levels were normalized to that of the internal control β-actin purchased from Abcam (Cambrige, MA, USA) (ab8226, 1 : 1000).