Fig. 3

CCHE1 interacted with LDHA and regulated LDHA activity. A partial list of proteins identified by mass spectrometry that specially bound CCHE1. The gene name, molecular weight, number of unique peptides, and log₂ fold change were shown. B RIP assay was performed using anti-LDHA antibody and significantly enriched CCHE1 abundance was found, demonstrating the specific binding CCHE1 with LDHA in melanoma cells. C, D His-tagged recombinant LDHA (C) or LDHA in whole cell lysates (D) was pull-down by biotin-labeled CCHE1 but not the antisense CCHE1. E, F LDHA activity was determined in melanoma cells with CCHE1 overexpression (E) or knock-down (F) using the lactate dehydrogenase activity assay kit. Overexpressed CCHE1 significantly increased the LDHA activity (E), while CCHE1 depletion reduced the activity of LDHA (F). The signals were normalized to cell numbers. AS, antisense; S: sense. ***p < 0.001 vs. control