Anti β trcp

Genipin was purchased from Calbiochem (San Diego, CA, USA). GANT61 was purchased from Selleckchem (Houston, TX, USA). Protein G PLUS-Agarose and anti-BAX, anti-BCL2, anti-MCL-1, anti-UB, anti-BCL-Xl, anti-GLI3, anti-PCAF, anti-SHH, anti-PTCH, and anti-p53 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-XIAP, anti-NOXA, anti-PUMA, anti-BIM, anti-SURVIVIN, anti-BID, anti-cleaved PARP, anti-CASP3, anti-CASP9, anti-β-TrCP, and anti-GLI1 antibodies were purchased from Cell Signaling (Beverly, MA, USA). The anti-actin antibody was obtained from Sigma (St. Louis, MO, USA), the anti-SMO antibody was obtained from Abcam (Cambridge, UK), and the anti-ITCH antibody was purchased from BD science (San Jose, CA, USA). The secondary antibodies, anti-mouse IgG HRP and anti-rabbit IgG HRP, were obtained from Cell Signaling.

Cells were harvested and lysed. The lysate was centrifuged, then incubated with anti-β-TrCP antibody (#4394, Cell signaling Technology) overnight at 4°C. For each IP sample, 500 µl of the lysate was incubated overnight at 4°C with 1 µg of the indicated antibody and 10 µl of Protein A/G Plus-Agarose (sc-2003, Santa Cruz). After incubated with Protein A/G Plus-Agarose for 1h on a rocking platform, the complex was washed three times with cold IP buffer and 1 X SDS loading buffer was added. Finally the whole-cell lysates and immunoprecipitated proteins were subjected to Western blotting analysis. Western blotting assay was performed as previously described. Primary antibodies were used at dilutions of 1:1000 for anti-CD147 (ab108308, Abcam), anti-Nrf2 (ab180845, Abcam), anti-NQO-1 (ab28947, Abcam), anti-HO-1 (ab13248, Abcam), anti-GCLC-1 (ab190685, Abcam), anti-β-TrCP (#4394, Cell signaling Technology), anti-Phospho-GSK-3-beta (Ser9) (#9322, Cell Signaling Technology), anti-GSK-3-beta (#12456, Cell Signaling Technology), anti-Phospho-Akt (Ser473) (#4051, Cell Signaling Technology), anti-Akt (#9272, Cell Signaling Technology), and anti-β-actin (#A5441, Sigma) antibody, anti-Lamin B(#13435, Cell Signaling Technology), respectively.

MG132-treated transfected cells were collected and treated overnight with Protein-A/G. MagBeads (Yeasen) and anti-ELAVL1 (Cell Signaling Technology) or anti-β-TrCP (Cell Signaling Technology) antibodies at 4 °C. The immune complexes were centrifuged and washed, and the proteins were assessed by western blotting with anti-ELAVL1 or anti-βTrCP antibodies [24 (link)].

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).

Antibodies purchased from Cell Signaling Technologies: Anti-pS33/37 β-catenin #2009S (1:1000); Anti-pT41/S45 β-catenin #9565S (1:1000); Anti-β-catenin (C-ter) #9587S (1:2000); Anti-FLAG #2368 (1:2500); Anti-β-TrCP #4394 (1:1000). Anti-Actin was from Sigma, Catalog #A5441 (1:20,000), and Anti-pS33 β-catenin from GeneTex, Catalog #GTX50255 (1:1000)