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. 2013 Dec 13;23(9):2428–2439. doi: 10.1093/hmg/ddt638

Figure 3.

Figure 3.

TMEM127 potentiates Rab5 inhibition of mTOR signaling. (A) Lysates from 293T cells expressing the Flag-Rab5 WT, Q79L or S34N and HA-TMEM127 or an HA-EV constructs were immunoprecipitated (IP) with Flag antibody and immunoblotted with TMEM127 antibody. Input lysates are shown after probing with Flag (for the Rab5 constructs), TMEM127 or β-actin as a loading control. (B) Lysates from 293T cells expressing the indicated Flag-Rab5 constructs or empty vector were immunoprecipitated (IP) with an endogenous TMEM127 polyclonal antibody and probed with Flag antibody and β-actin. (C) 293T cells were transfected with GFP-TMEM127, Flag-Rab5Q79L, both constructs or their respective empty vectors. Whole cell lysates were harvested at 48 h, run on western blots and probed with antibodies for both phosphorylated (P) and total (T) mTOR targets, S6K, S6 or 4EBP1, and for GFP, Flag antibody and β-actin (asterisk indicates non-specific band). (D) 293T cells were transfected with empty GFP-vector (EV), GFP-TMEM127WT (WT) or each of the four RCC TMEM127 mutations, along with Rab5Q79L, and harvested after 48 h. Blots were probed with antibodies targeting phosphorylated (P) and total (T) S6K, S6, Flag, TMEM127 and β-actin. (E) Relative levels of P/T S6 from lysates shown in (d) were quantified by Image J and normalized to cells expressing empty vector (−) .