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. 2021 Jan 1;11(7):3150–3166. doi: 10.7150/thno.52848

Figure 6.

Figure 6

PHB2 interacts with RACK1 and regulates the stability of RACK1 in A549 cells. (A) Flowchart to identify proteins interacting with PHB2. Whole A549 cell lysates were prepared for immunoprecipitation using an anti-PHB2 antibody or a control IgG antibody, and the immunocomplexes were analyzed using LC-MS/MS. A total of 117 potential interacting proteins with high fidelity were identified. RACK1 was identified as a potential PHB2 target protein. (B) Endogenous Co-IP assays between PHB2 and RACK1 in A549 cells. (C) Representative immunofluorescence images of PHB2 and RACK1 colocalization in A549 cells. (D)-(E) Representative immunoblots for RACK1 and β-actin and their densitometric quantification in A549 cells with stable PHB2 knockdown. (F) mRNA level of RACK1 in A549 cells with stable PHB2 knockdown. (G)-(I) Representative immunoblots for RACK1, PHB2, and β-actin and their densitometric quantification in A549 cells transfected for 48 h with a specific siRNA targeting RACK1. Western blot confirming RACK1 knockdown efficiency at the protein level. (J) mRNA level of PHB2 in A549 cells with RACK1 inhibition. (K) Representative immunoblots for RACK1, PHB2, and β-actin in A549 cells with stable PHB2 overexpression treated with CHX (a protein synthesis inhibitor, 10 µg/mL) at the indicated time points. (L) Evaluation of the half-life of RACK1. (M)-(N) Representative immunoblots for RACK1, PHB2, and β-actin and their densitometric quantification in A549 cells with stable PHB2 knockdown treated with MG132 (a proteasome inhibitor, 10 μΜ) for 8 h before collection. (O) Immunoprecipitation with RACK1 antibody and western blotting with anti-HA antibody to detect ubiquitinated RACK1 in whole-cell lysates of A549 cells with or without PHB2 overexpression transfected with HA-Ub and then treated with MG132 (10 μΜ) for 8 h before collection. These tests were repeated three times independently. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. Abbreviations: CHX, cycloheximide; Co-IP, coimmunoprecipitation; DAPI, 4',6-diamidino-2-phenylindole; LC-MS/MS, liquid chromatography and tandem mass spectrometry; siRNA, small interfering RNA.