Anti β catenin 51067 2 ap

Immunoprecipitation and Western blotting were performed as described previously.^{35 (link)} GAPDH protein levels were used as loading controls. We used the following primary antibodies: anti-Non-phospho (Active) β-catenin (Ser33/37/Thr41) (8814, Cell Signaling Technology), anti-phospho-β-catenin (Ser33/37/Thr41) (9561, Cell Signaling Technology), anti-β-catenin (51067-2-AP, Proteintech), anti-β-TrCP (4394, Cell Signaling Technology), anti-FAM83A (A15201, ABclonal), anti-c-myc (10828-1-AP, Proteintech), anti-CyclinD1 (60186-1-Ig, Proteintech), anti-AXIN2 (20540-1-AP, Proteintech), anti-mouse HA (M180-3, EMD Millipore), anti-Rabbit HA (51064-2-AP, Proteintech), anti-mouse DYKDDDDK (M185, MBL), anti-Rabbit DYKDDDDK (80010-1-RR, Proteintech), anti-GAPDH (60004-1-Ig, Proteintech), anti-GFP (598, EMD Millipore), anti-phosphotyrosine (P4110, Sigma), anti-phosphoserine (05-1000x, EMD Millipore), anti-phospho-threonine (9386 S, Cell Signaling Technology), anti-GSK3β (22104-1-AP, Proteintech), anti-AXIN1 (16541-1-AP, Proteintech), anti-TCF4 (22337-1-AP, Proteintech), anti-BLK (10510-1-AP, Proteintech), anti-HDAC1 (10197-1-AP, Proteintech), anti-HDAC2 (12922-2-AP, Proteintech), anti- Acetyl-Histone H3 (8173, Cell Signaling Technology).

Protein extracts were run on 10–15% SDS–polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes. After blocking with 5% skim milk, the membranes were probed with primary antibodies, afterwards incubated with horseradish peroxidase-conjugated secondary antibodies. Finally, the bands were visualized using a chemiluminescence reagent (Proteintech, Wuhan, China). The primary antibodies included antibodies from Cell Signaling Technology (Danvers, MA, USA): anti-Runx2 (#12,556), anti-phospho-LRP6 (Ser1490) (#2568), anti-non-phospho-β-catenin (#8814), anti-C/EBPα (#8178), and anti-PPARγ (#2443); antibodies from Abcam (Cambridge, MA, USA): anti-NDRG1 (ab124689), anti-osterix (ab94744), anti-LRP6 (ab134146), anti-transcription factor 7 like 2 (TCF7L2) (ab76151), and anti-alkaline phosphatase (ALP) (ab108337); antibodies from Proteintech (Wuhan, China): anti-fatty acid binding protein 4 (FABP4) (12802-1-AP), anti-osteopontin (25715-1-AP), anti-β-catenin (51067-2-AP), and anti-β-actin (66009-1-Ig).

The total protein of HaCaT cells was extracted with a Total Protein Extraction Kit (Millipore, Burlington, MA, USA). The protein concentration was determined using a bicinchoninic acid (BCA) method with a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Protein samples were separated on a 10% SDS-PAGE and then transferred to a PVDF membrane. After blocking with 5% (w/v) skimmed milk at room temperature for 1 h, protein samples were incubated with the corresponding primary antibody at 4°C overnight. The following antibodies were used: anti-β-catenin (51067-2-AP; Proteintech, Wuhan, China), anti-c-Myc (10828-1-AP, Proteintech), and anti-VEGF (66828-1-Ig, Proteintech). Then, protein samples were incubated with the secondary antibody. Band signals were visualized by the enhanced chemiluminescence (ECL, Bio-RAD, Hercules, CA, USA) method according to the manufacturer's instructions. Relative protein expression was normalized to actin.

Immunohistochemical staining was conducted as previously described [24 (link)]. The antibodies used for immunochemistry staining were as follows: anti-SETD1A (A300-289A; Bethyl Lab, Montgomery, TX, USA), anti-EZH2 (#5246; Cell Signaling Technology), anti-β-catenin (51067–2-AP; Proteintech), anti-Ki67 (27309–1-AP; Proteintech) and normal rabbit IgG (#2729; Cell Signaling Technology). Both the staining intensity and proportion of positive cells were taken into consideration: 0–25%, 26–50%, 51–75%, and 76–100% SETD1A positive cells were scored as 1, 2, 3, and 4, respectively; non-significant staining, light staining, moderate staining and strong staining were scored as 1, 2, 3, and 4, respectively. Then the two scores were multiplied to acquire a final score ranged from 1 to 16.

Cellular or tissue protein was extracted with RIPA lysis buffer (Servicebio, Wuhan, China), and western blot was performed as previously described [27 (link)]. Antibodies use for western blot: anti-PABPN1 (66807-1-Ig, Proteintech, USA), anti-GAPDH (60004-1-Ig, Proteintech, USA), anti-ATP6AP2 (10926-1-AP, Proteintech, USA), anti-TMEM97 (26444-1-AP, Proteintech, USA), anti-ZNF777 (NBP1-03344, Novus Biologicals, USA), anti-CDCA2 (17701-1-AP, Proteintech, USA), anti-CCND3 (26755-1-AP, Proteintech, USA), anti-AACS (ab237802, Abcam, UK), anti-β-catenin (51067-2-AP, Proteintech, USA), anti-Histone H3 (17168-1-AP, Proteintech, USA), and anti-α-Tubulin (66031-1-Ig, Proteintech, USA).