Skip to main content
. 2012 Sep 1;125(17):4090–4102. doi: 10.1242/jcs.105171

Fig. 2.

Fig. 2.

APPL1 enhances activity of the NF-κB pathway and acts synergistically with TRAF2. (A) APPL1 knockdown inhibits the NF-κB pathway. HEK293T cells were cotransfected with reporter vectors and APPL1 siRNA #1 and #2 or non-targeting siRNA (φ). The luciferase reporter activity was measured as described in the Materials and Methods. Knockdown of the APPL1 level was confirmed by immunoblotting (lower panel) with GAPDH as a loading control. (B) Overexpression of APPL1 or its PTB domain overcomes the inhibitory effect of its depletion on the NF-κB reporter. HEK293T cells were cotransfected with reporter vectors together with APPL1 siRNA #1 and #2 or non-targeting siRNA (φ) alone or in combination with vectors encoding the HA-tagged PTB of human APPL1 (PTB/APPL1) or full-length mouse HA–APPL1 (mAPPL1), as indicated (all values are in µg of DNA). The luciferase assay was performed as in A (upper panel). The levels of PTB/APPL1, mAPPL1, TRAF2 and GAPDH were analyzed by western blotting (bottom panel). mAPPL1 and PTB/APPL1 were detected with anti-APPL1 and anti-HA antibodies, respectively. (C) Dose–response effect of APPL1 overexpression on the NF-κB reporter activation. HEK293T cells were cotransfected with reporter vectors as well as 0.5 or 1 µg of APPL1 expression vector. APPL1 and GAPDH (loading control) levels were analyzed by western blotting (bottom panel). (D) APPL1-induced increase in cell viability is dependent on NF-κB. HEK293T cells were transfected with APPL1 expression vector and treated with different concentrations of BAY 11-7082 or DMSO, as a control, for 48 h. Cell viability was measured as described in the Materials and Methods, and expressed as OD450. (E) APPL1 and TRAF2 act synergistically to induce the NF-κB reporter. HEK293T cells were cotransfected with reporter vectors, together with the different combinations of APPL1 and TRAF2 expression vectors, as indicated (all values are in µg of DNA). (F) APPL1 acts upstream of IKK2. HEK293T cells were cotransfected with reporter vectors and different combinations of APPL1 and IKK2-K44M expression vectors, as indicated (all values are in µg of DNA).