Anti c kit antibody

Crude cell extracts were prepared by lysing cells using RIPA lysis buffer (Pierce) supplemented with phosphatase and protease inhibitor cocktails (Roche). The extracted proteins (20–50 μg) were resolved with 10% SDS polyacrylamide gels and transferred onto a nitrocellulose membrane (Millipore) according to standard protocols. Polyclonal anti-CXCR4 antibody (1:1000, Abcam), anti-c-kit antibody (1:500, Abcam), anti-CXCR4-phospho-serine 339 antibody (1:500, Abcam), anti-ERK1/2 antibody (1:1000, Cell Signalling Technology), anti-phospho-ERk1/2 antibody (1:1000, Cell Signalling Technology), anti-p38 antibody (1:1000, Cell Signalling Technology), anti-phospho-p38 antibody (1:1000, Cell Signalling Technology), anti-GRK6 antibody (1:1000, Santa Cruz), and anti-GRK2 antibody (1:1000, Santa Cruz) were used overnight at 4 °C. This was followed by incubation with a horseradish peroxidase-conjugated secondary antibody (1:10,000; Pierce). Peroxide activity was detected using the enhanced chemiluminescence Supersignal West Dura system (Pierce). As a loading control, mouse anti-beta-actin antibody was used at a concentration of 1:1,000.

The MCF-10A, SKBR-3, MCF-7, and MDA-MB-231 cell lines were purchased from ATCC (Manassas, VA, USA). The medium were obtained from HyClone supplemented with 10% FBS (Gibco, USA). ASCs were prepared as our previous description [3 (link)]. Briefly, the pelleted cells being washed twice using PBS were labeled with rabbit polyclonal anti-c-kit antibody (Abcam, USA) and goat anti-rabbit IgG magnetic beads (Miltenyi Biotech Inc., USA). Anti-c-kit-labeled cells were further purified by fluorescence-activated cell sorting. The cells were incubated in 37°C humidifed incubator containing 5% CO₂, and the medium was replaced for 2-3 days.
The scrambled nontarget control shRNA- or hTERT-expressing lentiviruses and the lentivirus particles for CBP short-hairpin RNA (shRNA) were purchased from GenePharma (Shanghai, China). The breast cancer cell lines were coinfected with CBP shRNA and the hTERT-expressing lentivirus to rescue hTERT expression.
Selumetinib and SP600125 (Selleckchem, USA) were diluted using DMSO to the final concentrations of 1 μM and 10 μM, respectively. SCF (R&D, USA) was used at a final concentration of 100 ng/ml.

Mouse colon tissues were cut and fixed in 4% paraformaldehyde (PFA) for 24 h. Then fixed tissues were embedded in paraffin, sectioned, and stained using hematoxylin and Eosin Staining Kit (Beyotime Biotechnology). Immunohistochemistry (IHC) was performed as we described previously [35 (link)]. Briefly, tissue sections were incubated by appropriate primary antibodies overnight at 4 °C, followed by incubation with GTVisionTM III Detection System/Mo & Rb/including DAB (gene tech, Shanghai). The cell nucleus was stained with hematoxylin (Sigma-Aldrich). The stained sections were scanned with Leica versa 8. Anti-IL1β antibody was purchased from Cell Signaling Technology. Anti-c-Kit antibody was obtained from Abcam. Anti-TNFα antibody were purchased from Santa Cruz biotechnology. Anti-IL6 antibody were purchased from Servicebio.