Journal: Nature Communications
Article Title: The UFL1-AKT positive feedback loop promotes breast cancer progression by enhancing lipid synthesis
doi: 10.1038/s41467-026-68492-3
Figure Lengend Snippet: A IB analysis of AKT1 phosphorylation at S473 (p-AKT1 (S473)) and T308 (p-AKT1 (T308)) in MDA-MB-231 and MDA-MB-468 cells expressing shSC or shUFL1. B Phosphorylation of AKT1-WT and AKT1-3K/R was determined by co-IP and IB assays in MDA-MB-231 and MDA-MB-468 cells. C Immunofluorescence (IF) colocalization analysis of AKT1 and the endoplasmic reticulum (ER) marker Calreticulin in MDA-MB-231 cells expressing shSC or shUFL1. Scale bars, 20 μm. D Protein levels of total and phosphorylated AKT1 in the cytosol or ER of MDA-MB-231 and MDA-MB-468 cells treated with or without 100 nM insulin for 30 min before harvest, as determined by cell fractionation and IB analysis. E The ER fraction was isolated from breast cancer cells treated with or without 100 nM insulin for 30 min and subjected to co-IP assays using PIP3-coated beads, followed by IB analysis. F The interaction between the pleckstrin homology (PH) domain and kinase domain (KD) of AKT1 was detected by co-IP and IB analysis in MDA-MB-231 and MDA-MB-468 cells. G The binding of AKT1 to its upstream regulators PDK1 and Rictor was investigated by co-IP and IB analysis in MDA-MB-231 and MDA-MB-468 cells. H A long exposure (l.e.) was used to capture UFMylated AKT1 bands in shSC and shUFSP2 MDA-MB-231 and MDA-MB-468 cells. s.e., short exposure. I UFMylation of AKT1-WT or T308A/S473A mutant AKT1 (AKT1-DM) was detected by IP and IB analysis in MDA-MB-231 and MDA-MB-468 cells. J The association of UFSP2 with AKT1-WT or T308A/S473A mutant AKT1 (AKT1-DM) was detected by co-IP and IB analysis in MDA-MB-231 and MDA-MB-468 cells cultured in medium in the presence or absence of 100 nM insulin for 30 min. K IB analysis of the impact of UFL1 ablation on proteins involved in lipid de novo synthesis in MDA-MB-231 and MDA-MB-468 cells. Asterisks (*) indicated mature SREBP1 (mSREBP1). All experiments were repeated three times independently with similar results.
Article Snippet: For immunoprecipitation assays, cell lysates were incubated with the indicated antibodies and protein A/G magnetic beads (Bio-Rad) or PIP3 coated magnetic beads (Echelon Biosciences, P-B345A) at 4 °C overnight.
Techniques: Phospho-proteomics, Expressing, Co-Immunoprecipitation Assay, Immunofluorescence, Marker, Cell Fractionation, Isolation, Binding Assay, Mutagenesis, Cell Culture
