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Fig. 5 | Epigenetics & Chromatin

Fig. 5

From: DNA methylation modifier LSH inhibits p53 ubiquitination and transactivates p53 to promote lipid metabolism

Fig. 5

LSH is a novel PKM2-interacting protein. a A549 cells were treated with 100 ng/ml EGF or untreated for 6 h, stained with anti-LSH and PKM2 antibodies and subsequently visualized by confocal microscopy. The subcellular localizations of LSH and PKM2 were detected by immunochemistry using the indicated antibodies (scale bars, 10 μm). Representative images from three independent experiments are presented. b Analysis of the mean fluorescence intensity of PKM2 in the cytosol and nucleus. Mean values from 12 independent cells from three preparations were determined by the ImageJ. The relative fluorescence intensity in the nucleus is expressed as percentage of MFI [N/(N + C)]. Statistical significance was evaluated using the paired Student t test. *P < 0.05; **P < 0.01. c, d Endogenous PKM2 or LSH was immunoprecipitated from A549 cell lysates and separated by 10% SDS-PAGE followed by Western blotting with anti-PKM2 and LSH antibodies. A549 cells were treated with 100 ng/ml EGF or untreated for 6 h. Representative images from three independent experiments are presented. e FLAG-LSH or control plasmid was stably transfected into HK1 cells. Total protein extracts were incubated with FLAG magnetic beads and subsequently separated by SDS-PAGE followed by Western blotting with an anti-FLAG, PKM2, or β-actin antibody. Representative images from three independent experiments are presented. f, g) Determination of the minimal PKM2–LSH interaction region. Co-IP assays were performed with an anti-His antibody in HEK293T cells transfected with His-LSH plus one of a series of N-terminal or C-terminal FLAG-PKM2 mutants. Schematic of PKM2 and the four FLAG PKM2 constructs used for the mapping. Representative images from three independent experiments are presented. h, j Determination of the minimal PKM2–LSH interaction region. Co-IP assays were performed with an anti-GST antibody in HEK293T cells transfected with GST-PKM2 plus one of a series of N-terminal or C-terminal FLAG-LSH mutants. Schematic of LSH and the three FLAG LSH constructs used for the mapping. Representative images from three independent experiments are presented

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