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. 2020 Aug 27;219(9):e201908078. doi: 10.1083/jcb.201908078

Figure 6.

Figure 6.

ALAL-1 interactions regulate USP4 localization and deubiquitinase activity. (A) Co-immunoprecipitation of USP4 and SART3 in HCC95 transfected with HA-SART3 and siRNAs targeting ALAL-1. (B and C) Ubiquitination of TAK1 and PRP3 in cells depleted or not of ALAL-1 by RNAi evaluated by immunoprecipitation and Western blot. (D) Co-immuno–FISH of endogenous SART3 and ALAL-1 with and without TNF treatment. Graphs show SART3, ALAL-1, and colocalization signal normalized to DAPI. Graphs of median (± SD) for three independent experiments are shown. Significance was determined by two-tailed unpaired t test and represented as *, P ≤ 0.05. (E) SART3 localization evaluated by Western blot of cell fractions (Nucleus ([Nuc]/Cytoplasm [Cyt]). (F) Co-immuno–FISH of endogenous USP4 and ALAL-1 with and without TNF treatment. Graphs show USP4, ALAL-1, and colocalization signal normalized to DAPI. Graphs of median (± SD) for three independent experiments are shown. Significance was determined by two-tailed unpaired t test and represented as *, P ≤ 0.05; ***, P ≤ 0.001. (G) USP4 localization evaluated by Western blot of cell fractions (Nucleus/Cytoplasm). ns, not significant; siALAL-1, ALAL-1 siRNA; siCtrl, control siRNA; IP, immunoprecipitation; Nuc, nucleus; Cyt, cytoplasm.