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. Author manuscript; available in PMC: 2017 Feb 18.
Published in final edited form as: Mol Cell. 2016 Feb 11;61(4):625–639. doi: 10.1016/j.molcel.2016.01.013

Figure 2. PRAS40 binds to iβ precursors during their folding and suppresses de novo assembly of the 20S core.

Figure 2

(A) Endogenous PRAS40 binds to β5i precursor. An untagged form of β5i was expressed in HEK293T cells, and its binding to PRAS40 was analyzed by co-IP. GAPDH IP was analyzed as control.

(B) β5i-flag was expressed with HA-PRAS40 in HEK293T cells.

(C) PRAS40 binds to β5i and β6 precursors during their folding. For coexpression (co), myc-tagged β subunit or raptor was coexpressed with HA-PRAS40 in HEK293T cells. Otherwise, myc-tagged construct was expressed in HEK293T cells then combined with cell lysate containing HA-PRAS40.

(D) PRAS40 can interact with β5i precursor without other components of mTORC1. For coexpression (co), β5i-flag was expressed with myc-PRAS40 in E. coli. Otherwise, flag-tagged β subunits were expressed in E. coli then combined with E. coli extracts containing myc-PRAS40.

(E) PRAS40 suppresses de novo biogenesis of proteasomes. β5i-flag was expressed with or without HA-PRAS40 in HEK293T cells. Proteasomal components associated with β5i-flag were analyzed by WB.

(F) PRAS40 enhances binding of β5i to β1i. Proteins were transiently expressed in HEK293T cells.

See also Figure S2.