ha phlpp1 plasmid  (Addgene inc)

Journal: Cell death & disease

Article Title: FKBP51 plays an essential role in Akt ubiquitination that requires Hsp90 and PHLPP.

doi: 10.1038/s41419-023-05629-y

Figure Lengend Snippet: Fig. 4 PHLPP improves K63-ubiquitination of Akt. a IB assay of A375 cells transfected with siTRAF6 RNA and NS RNA as negative control. Cells were collected after 24 h from transfection. IB shows that silencing of TRAF6 decreased pAkt levels. b IB assay of A375 cells transfected with siPHLPP RNA and NS RNA as negative control. Cells were collected after 24 and 48 h from transfection. IB shows that silencing of PHLPP decreased TRAF6 levels. c IB analysis of A375 cells transfected with HA-Akt, HA-K63-Ub, HA-PHLPP and siPHLPP RNAs and immunoprecipitated with Akt. IgG served as control for non-specific binding. IB showed that PHLPP increased Akt K63-Ub binding, whereas silencing of the phosphatases decreased it. IB of whole lysates is also shown. d IP assay of A375 cells transfected with Flag-FKBP51, HA-PHLPP, HA-K63-Ub and siPHLPP RNAs. Cells were immunoprecipitated with a Flag antibody and IgG served as control for non-specific binding. Immunoprecipitated protein was then assayed by IB with anti-K63-Ub antibody. PHLPP increased K63-Ub binding to FKBP51. IB of whole lysates is also shown. e IP assay of A375 melanoma cells treated with 0, 0.5 and 1 µM of the HSP90 inhibitor 17-AAG for 16 h. Endogenous FKBP51 was immunoprecipitated with anti-FKBP51 antibody, while IgG served as control for a not-specific binding. IB showed that inhibition of HSP90 did not affect the binding of FKBP51 to PHLPP. Immunoblot of whole lysates is also shown. f IP assay of A375 melanoma cells transfected with Flag-FKBP51, FKBP51-mutPPIase or Flag-FKBP51-mutTPR. Flag-FKBP51 was immunoprecipitated with anti-Flag antibody, while IgG served as control for non-specific binding. Immunoprecipitated proteins were then assayed by IB, and anti-PHLPP antibody revealed that mutated TPR did not affect the binding of FKBP51 to PHLPP. IB of whole lysates is also shown.

Article Snippet: PcDNA3 HA-tagged TRAF6 was a gift of Prof. Shao-Cong Sun (MD Anderson Cancer Center, Houston, TX, USA), while PcDNA3 HA-tagged PHLPP1 full length was purchased from Addgene (#37100) [37].

Techniques: Ubiquitin Proteomics, Transfection, Negative Control, Immunoprecipitation, Control, Binding Assay, Inhibition, Western Blot

Journal: Cell death & disease

Article Title: FKBP51 plays an essential role in Akt ubiquitination that requires Hsp90 and PHLPP.

doi: 10.1038/s41419-023-05629-y

Figure Lengend Snippet: Fig. 5 PHLPP sustains the oncogenic function of FKBP51. a IP assay of A375 cells transfected with EV, Flag-FKBP51, Flag-FKBP51 + HA-PHLPP. Cells were immunoprecipitated with a Flag antibody and compared to EV as control for non-specific binding. Immunoprecipitated protein was then assayed by IB with the indicated antibodies to validate selected FKBP51 interactors identified by proteomic analysis. PHLPP increased binding of such interactors to FKBP51. IB of whole lysates is also shown. b FKBP51-protein interaction network, as deriving from STRING analysis of components identified by proteomic analysis of IB samples from A375 cells in basal and PHLPP-over-representing conditions, respectively (Supplementary Information, Table S1). FKBP51 is highlighted in red; direct/indirect interactors identified in basal, PHLPP-over- representing or both conditions are reported in blue, yellow and green, respectively. c Functional annotation of the global network based on Gene Ontology-Biological Process terms. d Functional annotation of the global network based on Gene Ontology-Molecular function terms.

Article Snippet: PcDNA3 HA-tagged TRAF6 was a gift of Prof. Shao-Cong Sun (MD Anderson Cancer Center, Houston, TX, USA), while PcDNA3 HA-tagged PHLPP1 full length was purchased from Addgene (#37100) [37].

Techniques: Transfection, Immunoprecipitation, Control, Binding Assay, Functional Assay

Journal: Cell death & disease

Article Title: FKBP51 plays an essential role in Akt ubiquitination that requires Hsp90 and PHLPP.

doi: 10.1038/s41419-023-05629-y

Figure Lengend Snippet: Fig. 6 Proposed mechanism for the interaction of FKBP51 with Akt and PHLPP in melanoma cells. Left, FKBP51 binds to PHLPP thus stabilizing TRAF6 and allowing the formation of a K63 polyubiquitin chain and the full activation of Akt. Right, PHLPP is not kept into the complex by FKBP51s, which hampers Akt ubiquitination and phosphorylation. The same occurs when PHLPP is subtracted from the FKBP51 complex.

Article Snippet: PcDNA3 HA-tagged TRAF6 was a gift of Prof. Shao-Cong Sun (MD Anderson Cancer Center, Houston, TX, USA), while PcDNA3 HA-tagged PHLPP1 full length was purchased from Addgene (#37100) [37].

Techniques: Activation Assay, Ubiquitin Proteomics, Phospho-proteomics