Ab19348

An Abcam type II collagen protein staining kit (America) and an Abcam N-cadherin (NCAD) protein staining kit (America) were employed to evaluate type II collagen and NCAD protein expressed in chondrocytes. Firstly, chondrocytes were cultured with the ionic extracts of BGC and Cu-BGC for 3 days, and then fixed with 2.5% gluteraldehde (Sinopharm Group Co., Ltd). And then the chondrocytes were incubated for 1 h by using 1% bovine testicular hyaluronidase. Afterwards, the primary antibody (COL II, Abcam: ab3092; NCAD, Abcam: ab19348, respectively) was used to incubate the chondrocytes for 12 h at 4°C, and second antibody (COL II: Abcam: ab150105; NCAD, Abcam: ab150107, respectively) was applied to incubate the cells at 37°C for 1 h. Finally, the cell cytoskeleton was stained by a fluorescein isothiocyanatephalloidin (FITC, Sigma-Aldrich, America) solution, and the nuclei were stained by a 4', 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, America) solution. Fluorescent photographs were captured by a confocal laser scanning microscopy (CLSM, Leica TCS SP8). The contents of proteins were analyzed using Image pro-plus 6.0 (Media Cybernetics, US) software.

Immunohistochemical staining was utilized to measure the protein expression levels of E-cadherin and N-cadherin in the paraffin sections of mice lung metastases, and the detailed primary antibody information is as follows: anti-E-cadherin (1:500; Abcam, ab40772) and anti-N-cadherin (1:1000; Abcam, ab19348).

Western blot analysis was performed as earlier described [17 (link)]. Briefly, cell lysates were prepared and proteins were separated by 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto polyvinylidene fluoride (PVDF) filters. The probing antibodies used were as against the following antigens: E-cadherin (ab1416, mouse monoclonal antibody, 1:1000, Abcam, Cambridge, UK), N-cadherin (ab19348, mouse monoclonal antibody, 1:1000, Abcam), Vimentin.
SNAIL (ab53519, goat polyclonal antibody, 1:1000, Abcam), SLUG (sc166476, mouse monoclonal antibody, 1:1000, Santa Cruz), Twist.
OPN (ab8448, rabbit polyclonal antibody, 1:1000, Abcam), RUNX2 (ab115899, mouse monoclonal antibody, 1:4000, Abcam), SRSF1 (32–4500, mouse monoclonal antibody, 1:250, Thermo Fisher Scientific, Waltham, MA), HDAC1 (CY5154, rabbit monoclonal antibody, 1:1000, Abways, Shanghai, China), HDAC2 (CY5062, rabbit monoclonal antibody, 1:1000, Abways), HDAC3 (CY5595, rabbit monoclonal antibody, 1:1000, Abways), DDDK (M185-3L, mouse monoclonal antibody, 1:1000, MBL, Beijing, China) and GAPDH (TA-08, mouse monoclonal antibody, 1:3000, ZSGB-BIO, Beijing, China).

Immunohistochemistry is used to detect the protein expression of ZNF582, TJP2 and ERK2 in 60 pairs of ccRCC and adjacent normal renal (AN) tissues and the protein expression of E-cadherin and N-cadherin in the lung metastases of mice kidney tumors. The details of these antibodies are as follows: anti-ZNF582 (1:300, ab254814, Abcam), anti-TJP2 (1:200, 18900-1-AP, Proteintech), anti-ERK2 (1:100, ab32081, Abcam), anti-E-cadherin (1:500, ab40772, Abcam) and anti-N-cadherin (1:1000, ab19348, Abcam).