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. 2014 Apr 8;124(5):1914–1927. doi: 10.1172/JCI72327

Figure 4. TP53INP2 promotes degradation of ubiquitinated proteins in skeletal muscle.

Figure 4

(A) p62 and NBR1 protein levels in total homogenates of gastrocnemius muscles from WT and SKM-Tg mice. Representative images are shown. (B) Western blot analysis of ubiquitinated protein content in total homogenates of gastrocnemius muscles from WT and SKM-Tg mice with or without chloroquine treatment. Representative images are shown. (C) Western blot analysis of p62, NBR1, TP53INP2, and ubiquitinated protein (FK2) content in protein extracts from day 5 C2C12 myotubes overexpressing TP53INP2 or LacZ (control). Cells were treated with bafilomycin (Baf.) as indicated. Thin black lines indicate that lanes were run on the same gel but were noncontiguous. Representative images are shown. (D) Immunofluorescence against p62 and LC3 in control (LacZ) or TP53INP2-overexpressing C2C12 day 5 myotubes. Cells were treated with bafilomycin at 200 nM during 3 hours as indicated. p62 is stained in red and LC3 is stained in green. Scale bar: 20 μm. (E) Immunofluorescence against ubiquitin (FK2) and TP53INP2 in C2C12 day 5 myotubes. Cells were treated with puromycin at 50 μg/ml during 4 hours as indicated. Ub (FK2) is stained in red and TP53INP2 is stained in green. Scale bar: 20 μm. (F) HEK293T cells were transfected with plasmids coding for GFP-Ub and/or mouse TP53INP2 or mouse TP53INP2 3KR mutant. Pull-down was performed using GFP-Trap beads and inputs and pellets were probed in Western blot assays with anti-GFP and anti-TP53INP2 antibodies.