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. 2020 May 16;34:101571. doi: 10.1016/j.redox.2020.101571

Fig. 5.

Fig. 5

Ibuprofen activates both proteasomal and lysosomal degradation of EFHD2. A) EFHD2 mRNA levels of H1299 cells with or without 600 μM ibuprofen treatment for indicated time were determined by qPCR. GAPDH, 18S rRNA, and β-actin mRNA individually served as internal control of gene expression, respectively. B) After 10 mg/mL cycloheximide (CHX) reaction for 1 h, H1299 and F4 cells were treated with or without 600 μM ibuprofen for indicated time. EFHD2 expression in ibuprofen-treated and the control cells was determined by Western blot assay. C) After reaction with 1 nM MG132 and/or 5 nM Baf-A1 for 0.5 h, H1299 cells were treated with 600 μM ibuprofen for indicated time. EFHD2 expression was determined by Western blot assay. The relative signal intensities were analyzed by ImageJ software. D) H1299 cells were treated with 600 μM ibuprofen for indicated time, and then autophagy-related proteins were examined by Western blot assay. E) Both H1299 and F4 cells were treated with 600 μM ibuprofen for 2 h, and then confocal images of antibody to LC3B were analyzed. Scale bar, 10 μm. F) After 600 μM ibuprofen treatment for indicated time, EFHD2 proteins were immunoprecipitated from 1 mg total protein of H1299 cells with EFHD2-specific antibody. The immunoprecipitated EFHD2 and ubiquitin (Ub)-conjugated proteins were examined by Western blot assay. β-actin, loading control. G) After 600 μM ibuprofen treatment for indicated time, proteasome activities of H1299 cells were determined by the Proteasome Activity Fluorometric Assay Kit (BioVision).