Fig. 2
From: A designer polyQ fusion protein modulates NF-κB signaling by sequestering P65/RelA into aggregates
The polyQ-LDEL fusion sequesters endogenous P65 into aggregates. (A) S/P fractionation for characterizing the distribution of endogenous P65 in supernatant and pellet affected by the polyQ-LDEL fusion. HEK 293T cells were transfected with FLAG-tagged Atx793Q-N172 or Atx793Q-N172-LDEL, followed by culturing for 48 h. The P65 levels in the supernatant and pellet fractions were then detected and analyzed. Sup., supernatant; Pel., pellet. Vec., vector; Atx793Q-N172, the N-terminal 172-residue fragment of Atx793Q; LDEL, Atx793Q-N172-LDEL, a polyQ fusion combined Atx793Q-N172 with LDEL. Data are shown as Mean ± SD (n = 3). *, p < 0.05; **, p < 0.01; N.S., no significance. (B) Immunofluorescence imaging for characterizing the co-localization of endogenous P65 with the inclusions formed by the polyQ-LDEL fusion. HEK 293T cells were transfected with FLAG-tagged Atx793Q-N172 or Atx793Q-N172-LDEL, and after cultured for 48 h, the cells were fixed and immunostained with anti-FLAG (green) and anti-P65 (red) antibodies. Nuclei were stained with Hoechst (blue). Scale bar = 10 μm. Bottom: co-localization analysis of the fluorescence signals for the distance represented by white lines.
