Kindlin 2

Surgically removed colonic cancer tissues and adjacent normal tissues were collected from three patients at Peking University Third Hospital (Beijing, China) and used for Western blot analysis. The experiments were approved by the Ethics Committee of Peking University Third Hospital.
Immunohistochemical staining for specific protein expression was performed on mouse tissue sections. In brief, sections (4 mm thick) were deparaffinized with xylene, followed by rehydration in ethanol. Hydrogen peroxide (3%) was used to eliminate endogenous peroxidase. Sections were incubated overnight at 4°C with primary antibodies against Kindlin-2 (Sigma-Aldrich), 1:200; Smurf1 (Abcam), 1:100; and Smad1 (Bioss), 1:100. After extensive washing in PBS buffer, sections were incubated for 30 min with secondary antibodies (Dako). Immunostaining was examined with a BX51 microscope (Olympus), and images were photographed with a 40× objective.

Immunohistochemistry was performed to examine subcellular localizations and expression patterns of Kindlin proteins in osteosarcoma and noncancerous bone tissues. All fresh, resected tissue samples were fixed in 10% formalin, embedded in paraffin and cut into 4 μm-thick sections, which were subsequently dewaxed in xylene and rehydrated with graded alcohol. Following rinsing with distilled water, the antigen was incubated in ethylenediaminetetraacetic acid buffer at 100°C for 20 min and then cooled to room temperature. The slides were then incubated at 4°C overnight with the primary antibodies: Kindlin-1 (rabbit polyclonal antibody, Millipore; 1:100 dilution), Kindlin-2 (rabbit polyclonal antibody, Millipore; 1:150 dilution) and Kindlin-3 (rabbit polyclonal antibody, Abcam; 1:100 dilution). After washing with phosphate-buffered solution (PBS) three times, the biotin-labeled secondary antibody was added and the sections were incubated at room temperature for 10 min. After washing with PBS three times, 3,3′-diaminobenzidine chromogenic reagents were added. Then, sections were counterstained with hematoxylin, dehydrated and mounted. The negative controls were processed in a similar manner with PBS instead of the primary antibodies.

Total proteins of osteosarcoma and adjacent noncancerous tissues were extracted using radioimmunoprecipitation assay buffer containing phenylmethanesulfonyl fluoride and were quantified by a BCA kit (Thermo, Waltham, MA, USA). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Then, the membranes were blocked with 5% nonfat milk (Bio-Rad) in Tris-buffered saline containing Tween 20 (TBST) solution, overnight at 4°C. After that, the following primary antibodies were used: Kindlin-1 (rabbit polyclonal antibody; Millipore, Billerica, CA, USA; 1:100 dilution), Kindlin-2 (rabbit polyclonal antibody, Millipore; 1:150 dilution), Kindlin-3 (rabbit polyclonal antibody; Abcam, MA, USA; 1:100 dilution) and GAPDH (rabbit polyclonal antibody, Abcam; 1:150 dilution). Secondary antibodies were incubated after washing with TBST at room temperature for 1 h (horse radish peroxidase labeled goat anti-mouse IgG, 1:1000; Sangon Biotech, Shanghai, China). The membranes were detected by ECL system (Pierce, Rockford, IL, USA), and gray assay was performed by ImageQuant 5.2 software. GAPDH protein levels were used as a control to verify equal protein loading.