Fig. 3.

TBK1 mediates SFA-induced p62 phosphorylation and aggregation into ubiquitinated protein inclusions. (A,B) HepG2 cells were treated with BSA (Con), PA (500 μM), BSA + BX795 (20 μM), PA + BX795, BSA + Amlexanox (Amx, 100 μM), or PA + Amx for 9 h. (A) The cells were lysed, fractionated into 1% Triton X-100-soluble and insoluble fractions, and then subjected to the immunoblotting (left panels) and immunoblot quantification (right panels). (B) Cells with indicated treatments were subjected to immunostaining (upper panel). Boxed areas in fluorescence images are magnified in rightmost panels. Amount of aggregated proteins was quantified (lower panel). Scale bars, 5 μm. (C) HepG2 cells were transduced with shRNA lentiviruses targeting luciferase (sh-Con) or TBK1 (sh-TBK1 #1 and #2) and incubated for 48 h. Then the cells were treated with BSA or PA (500 μM) for 9 h, followed by subcellular fractionation, immunoblotting (upper panel) and immunoblot quantification (lower panels). TBK1 was analyzed from soluble fractions while p62, ubiquitin and β-actin were analyzed from insoluble fractions. All data are shown as mean ± s.e.m. **P < 0.01, ***P < 0.001 (Student’s t-test). Arrowheads indicate the exact or nearest position of the protein molecular weight markers (kD).