Informed consent was obtained from the patients before surgery. The malignant skin tumor tissues, including 8 MM, 8 SCC, and 8 BCC, were obtained from patients who were treated with excisional surgery. All tumor tissues were examined using both conventional histopathological confirmation and immunohistochemical studies to confirm

the diagnosis. Clinical and histopathological data are shown in Table 1. A portion of the specimens were frozen in liquid nitrogen immediately after resection and https://www.selleckchem.com/products/Everolimus(RAD001).html stored at -80°C degrees for subsequent western blot analysis. The human malignant melanoma cell line G361, obtained from the American Type Culture Collection (CRL 1424; Rockville, MD, 7-Cl-O-Nec1 ic50 USA), served as a positive control for c-Src and c-Yes expression. Table 1 Clinicopathological features of 24 malignant skin tumors Case No. Sex/Age Site Tumor type 1 M-1 F/53 Foot MM(ALM) 2 M-2 F/51 Lower back MM(NM) 3 M-3 M/70 Foot MM(NM) 4 M-4 M/66 Foot

MM(NM) 5 M-5 M/54 Thigh MM(ALM) 6 M-6 M/65 Thumb MM(NM) 7 M-7 M/58 Foot MM(ALM) 8 M-8 M/63 Foot MM(SSM) 9 S-1 F/86 Temple SCC 10 S-2 F/76 Cheek SCC 11 S-3 M/51 Buttock SCC 12 S-4 F/86 Face SCC 13 S-5 F/87 Cheek SCC 14 S-6 F/74 Scalp SCC 15 S-7 F/82 Temple SCC 16 S-8 F/77 Cheek SCC 17 B-1 F/67 Cheek BCC 18 B-2 M/75 Nose BCC 19 B-3 M/52 Nose BCC 20 B-4 M/64 Nose BCC 21 B-5 F/68 Nose BCC 22 B-6 F/71 Lower lid BCC 23 B-7 F/65 Nose BCC 24 B-8 M/56 Cheek BCC Abbreviations: MM, malignant melanoma; ALM, acral lentiginous melanoma; NM, nodular melanoma; SSM, superficial spreading melanoma; SCC, squamous cell carcinoma; BCC, basal cell carcinoma. Levels of invasion of MM (M-1~M-7) were Clark’s Level IV, M-8 was Level I. Western blot analysis Tissue samples Unoprostone were homogenized in WCE buffer [25 mM HEPES (pH 7.7), 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM ethylenediamine tetraacetic acid (EDTA), 0.1% Triton X-100, 0.5 mM dithiothreitol

(DTT), 20 mM-glycerolphosphate, 0.1 mM Na3VO4, 2 g per mL leupeptin, 2 g per mL aprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF), and a protease inhibitor cocktail tablet (Boehringer Mannheim)]. The tissue suspension was rotated at 4°C for 10 minutes. Supernatants were collected and then kept at -70°C and used for western blotting. Proteins from the tissue were separated by SDS-PAGE using NuPAGE 4-12% bis-Tris gels (Invitrogen, NP0335Box) and then transferred to Immobilon-P membranes. The membrane was blocked using 5% BSA in TBS-T (20 mM Tris, pH 7.6, 130 mM NaCl, and 0.1% Tween 20) solution. 6 MM, 6 SCC, 6 BCC and 6 normal skin tissues were then reacted with the primary antibody, Src (36D10) rabbit mAb (Cell Signaling technology®, #2109) and Yes antibody (Cell Signaling technology®, #2734) diluted to 1:1,000 concentration, at 4°C for 16 hours.