Research Paper Volume 12, Issue 12 pp 11812—11834

Figure 9. FAM83H and SCRIB are involved in the stabilization of β-catenin from proteasomal ubiquitin degradation. (A) Western blot for β-catenin and active β-catenin after overexpression of FAM83H and/or knock-down of SCRIB. (B) The protein lysate obtained from MKN-45 cells after immunoprecipitation with FAM83, SCRIB, or β-catenin. Thereafter, the immunoprecipitated protein was immunoblotted for FAM83H, SCRIB, and β-catenin. (C) Western blot for FAM83H, SCRIB, β-catenin, actin, and lamin B1 with the cytoplasmic membrane, cytoplasm, and nuclear protein lysates fractionated according to subcellular localization after inducing knock-down or overexpression of FAM83H or SCRIB in MKN-45 cells. (D) The MKN-45 cells were transfected with empty vector, shRNA for FAM83H, or hSCRIB CRISPR/Cas9 KO plasmid and treated with 30 μM cycloheximide or 30 μM MG132 for 0.5 to 4.0 hours. Thereafter, the total protein was immunoblotted for β-catenin and actin. (E) The MKN-45 cells were transfected with empty vector, shRNA for FAM83H, or hSCRIB CRISPR/Cas9 KO plasmid, and were treated with 30 μM MG132 for two hours. The protein lysate was immunoprecipitated with anti-β-catenin antibodies and immunoblotted with anti-ubiquitin antibodies. The immunoblot was performed on total protein lysate. (F) The MKN-45 cells were transfected with empty vectors, shRNA for FAM83H, or an overexpression vector for FAM83H. The protein lysate was immunoprecipitated with anti-β-catenin antibodies and immunoblotted with anti-β-TrCP, anti-USP47, or anti-GAPDH antibodies. The immunoblots were performed on total protein lysate.