Fig 1: ACLY associates with F-actin. (A) Fluorescence micrographs (epifluorescence microscopy) of cells immunostained for ACLY showing nucleocytosolic and peripheral labeling of ACLY. (B) Fluorescence micrographs (epifluorescence microscopy) of ACLY WT and KO T24 cells immunostained for ACLY showing antibody specificity. (C) Fluorescence micrographs (confocal microscopy) of cells immunostained for ACLY showing colocalization between ACLY and F-actin (phalloidin). (D) Immunoblot analysis of ACLY in detergent-soluble (S) and -insoluble (I) fractions in detergent-extracted cells. a-tubulin was used to assess fraction purity and ß-actin was used as a loading control. (E) Fluorescence micrographs (confocal microscopy) of ACLY immunostaining and colabeled with phalloidin in detergent-extracted T24 cells treated with Lat A (1 h). (F) Immunoblots of biotinylated phalloidin F-actin pulldown in HeLa-KRAS(G12V) and T24. ARP3 and ACTN1 (known actin-binding proteins) and a-tubulin and proliferating cell nuclear antigen (PCNA) (nonactin-binding proteins) were used as positive and negative controls, respectively. (G) Immunoblot analysis of supernatant and pellet fractions of actin cosedimentation assay using purified F-actin incubated with either recombinant ACLY tetramer, recombinant alpha-actinin (ACTN) as a positive control, or purified ALB (albumin) as a negative control. Reactions without F-actin serve as background sedimentation controls. (Scale bar for all images, 10 µm.) All images and immunoblots are representative.