β catenin sc 7963

BMDM lysates were run in 12% SDS-polyacrylamide gels under denaturing conditions and blots were stained with anti-pY-STAT3 (9131;Cell Signaling Technology), anti-total-STAT3 (8768; Cell Signaling Technology), β-catenin (sc-7963; Santa Cruz), and anti-GAPDH antibodies (G8795; Sigma-Aldrich). Immunoreactive bands were visualized using the Odyssey imaging system (LI-COR Biosciences) by using horseradish peroxidase-conjugated secondary antibodies (ab205718; Abcam and 626520; Thermo Fisher Scientific) and chemiluminescence.

HeLa cells in 10 cm dishes were grown to 80% confluence, and transfected with 5 µg of Myc-KCTD1. After 30 h, the cells were harvested and lysed as previously described [36] (link). The whole cell extracts were immunoprecipitated using rabbit polyclonal antibodies against Myc-tag (C3956) (Sigma, St. Louis, MO, USA) or β-catenin (sc-7199) (Santa Cruz Biotech, CA, USA) and protein A/G plus agarose (sc-2003) (Santa Cruz Biotech), the immunoprecipitates were separated by 10% SDS-polyacrylamide gels and detected with mouse monoclonal antibodies against β-catenin (sc-7963) or Myc-tag (sc-40) (Santa Cruz Biotech). Rabbit preimmune IgG (sc-66931) (Santa Cruz Biotech) was served as negative control.

Primary VSMCs were extracted from rat aorta. Cells were maintained with complete Dulbecco's modified Eagle's medium (DMEM), containing 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml). Culture conditions were set at 37ºC and 5% CO₂. Primary antibodies for TGF-β1 (sc-130348), COX-2 (sc-376861), c-Myc (sc-40), p-GSK3β (sc-373800), Runx2 (sc-390715), GAPDH (sc-47724), and β-catenin (sc-7963) were ordered from Santa Cruz Biotechnology. Primary antibodies for GSK3β (22104-1-AP), α-SMA (55135-1-AP), and sclerostin (21933-1-AP) were ordered from Proteintech (Wuhan, China). Primary antibodies for p-Smad2/3 (8828s) and p-CREB (ab32096) were ordered from CST and Abcam, respectively. NS-398 (Cat. no. N194-25MG) was ordered from Sigma-Aldrich. Meloxicam (S67664) was ordered from Yuanye Biotech (Shanghai, China).

Serial four micron sections of xenograft tumors have been immunostained for Ki67 (MoAb, Dako, Denmark), VEGF-A165 (A-20, Santa Cruz biotechnology, U.S.), Acetyl Histone H4 (Upstate, NY), α-SMA (Dako, Denmark), CD31 (ab124432, Abcam), β-catenin (sc-7963, Santa Cruz Biotechnology, Inc.), and HIF-1α (MA1-16504, Affinity Bioreagents) following the streptavidin-biotin method, as previously described (29 (link)). Slides were independently examined by three pathologists. All cases were digitally scanned by iScan (BioImagene, Now Roche-Ventana) with Scanning Resolution 0.46 μm/pixel at 20× or 40×. Tissue staining was semi-quantitatively graded for intensity as negative/weak: 0, moderate: 1 and strong: >2. The cell positivity was also scored as <10% (0), from 10 to 25% (1 ) from 26 to more than 50% (2 (link)). The final score was calculated by adding both partial scores. For the evaluation of results, the Chi square test was performed. The density of vessels, immunostained by CD31 and α-SMA, was determined by calculating the tube number per 40× field of view. Five areas were chosen and an arithmetic mean was obtained. The microvessel density quantification was performed manually, by three independent experts in the field. The image processing was performed with Lucia G System and the statistical analysis was performed with SPSS Software ver. 20.

Whole-cell lysate extracts were prepared with RIPA Buffer (Sigma Aldrich) supplemented with protease inhibitor and phosphatase inhibitor cocktail (Thermo Fisher Scientific). The protein content was determined by Bradford analysis (Bio-Rad). Equal amounts of proteins (30–50 μg) were separated on 4–15% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad) and transferred onto nitrocellulose membrane (Bio-Rad). After blocking in TBS-T (0.05% Tween 20) containing 5% nonfat dry milk for 1 h at room temperature, the membrane was incubated overnight at 4 °C with primary antibodies against Nanog (PA1-097, Thermo Fisher Scientific), Oct4 (60093, STEMCELL Technologies), β-catenin (sc-7963, Santacruz), phospho-Akt Ser473 (9271, Cell Signalling), phospho-Akt Thr308 (4056, Cell Signalling), Akt1 (2967, Cell Signalling), mTOR (2983, Cell Signalling), phospho-mTOR Ser2448 (5536, Cell Signalling), S6K1 (2708, Cell Signalling), phospho-S6K1 Thr389 (9206, Cell Signalling), and Actin (sc-1616, Santa Cruz).
The membrane was then washed three times with TBS-T, incubated with a 1:10,000 dilution of horseradish peroxidase-linked secondary antibodies (Jackson ImmunoResearch), and the immunoreactive proteins were visualized with the enhanced chemiluminescence detection system (Bio-Rad).

Aliquots of 30 µg protein extracts were subjected to SDS-PAGE electrophoresis. Proteins on the gels were blotted onto a nitrocellulose membrane. Proteins of interest on the blots were, respectively, probed by GSK3β (#9315, Cell signaling, Danvers, MA, USA), Dkk1 (sc-25516, Santa Cruz Biotechnology, Dallas, MA, USA), GAPDH (GTX100118, GeneTex, Irvine, CA, USA), phospho-Smad3 (p-Smad3; ab51451, abcam, Cambridge, UK), Smad 3 (ab40854, abcam, Cambridge, UK), phospho-β-catenin (p-β-catenin ; sc-16743-R, Santa Cruz Biotechnology, Dallas, MA, USA), and β-catenin (sc-7963, Santa Cruz Biotechnology, Dallas, MA, USA). After washing, the blots were further incubated with secondary antibodies horseradish peroxidase-coupled anti-mouse immunoglobulin-G antibodies. The proteins designated were visualized by an enhanced chemiluminescence detection (GE Healthcare Biosciences AB, Little Chalfont, UK). Intensities of protein bands were quantified by a gel densitometry system. Six animals were used for detection.