Ab220962

The proteins were extracted by using Radio-Immunoprecipitation Assay (RIPA) (Beyotime Institute of Biotechnology, CHN) lysis buffer adding with PMSF (KeyGEN, CHN) and protease inhibitor cocktail. Forty micrograms of protein lysates were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel (KeyGEN, CHN) and transferred to a polyvinylidene fluoride (PVDF) membrane (KeyGEN, CHN). After blocking with skimmed milk for 1 hour, the membrane was incubated with the specific primary antibodies overnight. The membranes were then washed and incubated with secondary antibodies (ZSGB-BIO, CHN) at room temperature for 2 hours. Antibodies against Wnt1 (1500, ab85060), cyclin D1 (1100, ab16663), SPINDOC (1:50, ab220962) were purchased from Abcam (UK). β-catenin (1:500, 610153), GAPDH (1:5000, 60004-1-Ig) were purchased from BD sciences (USA). AXIN2 (1:50, K006954P) and SPIN1 (1:50, K006346P) were purchased from Solarbio (CHN).

Fresh nude mouse tumor specimens were converted into wax blocks by routine fixation, and standardized operation was carried out according to the kit instructions (Beijing Dingguo Changsheng Biotechnology, CHN). The primary antibody was added and incubated at 4°C overnight. The specific information of primary antibodies used is as follows: Wnt1, 1:500, ab85060, Abcam, (UK); cyclin D1, 1:100, ab16663, Abcam, (UK); SPINDOC, 1:50, ab220962, Abcam, (UK); β-catenin,1:500, 610153, BD sciences, (USA); AXIN2, 1:50, K006954P, Solarbio, (CHN); SPIN1, 1:50, K006346P, Solarbio, (CHN). The blocks were washed with PBS 3 times, 2min each, and biotin-labeled sheep anti-rabbit IgG solution (1:100, Z021006Y, ZSGB-BIO, CHN), was added followed by incubation at room temperature for 30min. Thereafter, DAB chromogenic solution was added after PBS washing, and the blocks were placed under the microscope for observation. The reaction time was controlled, and the blocks were then washed with the distilled water to stop the reaction. Hematoxylin (Beijing Dingguo Changsheng Biotechnology, CHN) was added for redyeing, followed by washing, routine dehydration, spin drying, and observation of the results under the microscope after sealing with the neutral adhesive.