Fig. 6: PSMD2 interacts with ubiquitinated β-catenin. | Cell Death & Disease

Fig. 6: PSMD2 interacts with ubiquitinated β-catenin.

From: RACK1 facilitates breast cancer progression by competitively inhibiting the binding of β-catenin to PSMD2 and enhancing the stability of β-catenin

Fig. 6

A Diagram of full-length HA-tagged β-catenin and its two mutants, S33A/S37A/T41A/S45A (4A) β-catenin and N-terminal truncated (ΔN) β-catenin. B Co-IP analysis of the interaction pattern between PSMD2 and the indicated β-catenin mutants in HEK-293T cells. C PSMD2 interacts with ubiquitinated β-catenin. HEK-293T cells were co-transfected with PSMD2-Flag, UB-myc and β-catenin-HA or its mutants and then treated with MG132, followed by immunoprecipitation of cell lysates with anti-HA antibodies and detection with anti-HA, -myc and -Flag antibodies. D Diagram of full-length myc-tagged β-catenin and S33E/S37E/T41E/S45E (4E) β-catenin. E Co-IP analysis of the interaction pattern between PSMD2 and β-catenin-WT-myc or β-catenin-4E-myc in HEK-293T cells. F PSMD2 interacts with ubiquitinated β-catenin. HEK-293T cells were co-transfected with PSMD2-Flag, HA-UB and β-catenin-WT-myc or β-catenin-4E-myc and then treated with MG132, followed by immunoprecipitation of cell lysates with anti-myc antibodies and detection with anti-HA, -myc and -Flag antibodies. G, H Knockdown of RACK1 increased the interaction between PSMD2 and β-catenin in MDA-231 cells. Cell lysates from control or RACK1-silenced cells were immunoprecipitated with ant-PSMD2 (G) or anti-β-catenin (H) antibodies and analyzed by Western blotting with anti-PSMD2, RACK1, or β-catenin antibodies. I, J Restoration of RACK1 expression in RACK1-silenced cells attenuated the interaction between PSMD2 and β-catenin. Cell lysates from RACK1-silenced or RACK1-rescued cells were immunoprecipitated with ant-PSMD2 (I) or anti-β-catenin (J) antibodies and analyzed by Western blotting with anti-PSMD2, RACK1, or β-catenin antibodies in MDA-231 cells. The protein levels of the IP products were quantified by grayscale analysis and the results are shown below the bands (E-J). All data are expressed as the mean ± SD; *p < 0.05 and ns p > 0.05 versus control, N = 3. K Knockdown of RACK1 increased the binding ability of PSMD2 to ubiquitinated β-catenin in breast cancer cells. Control and RACK1-silenced cells were transfected with UB-HA and then treated with MG132. Afterwards, cell lysates were immunoprecipitated with anti-β-catenin antibodies and analyzed by Western blotting using anti-PSMD2, RACK1, HA or β-catenin antibodies.

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