Fig 1: Interactions of 14-3-3 (YWHA) protein with PLIN1 and specific role of YWHAB in Fsk-stimulated lipolysis. A) Western blot probed with antibody recognizing YWHAB/Z/G/E of proteins biotinylated by PLIN1-APEX2 enriched by neutravidin beads compared with whole-cell lysate. B) Immunofluorescence analysis of primary adipocytes using antibody recognizing YWHAB/Z/G/E (green), DAPI (blue), and LipidTOX (red). C) RT-PCR of RNA obtained 48 h after transfection of adipocytes with two siRNAs targeting YWHAB, YWHAE, YWHAZ, and YWHAG, as indicated in the y-axis. D) RT-PCR of RNA obtained 48 h after transfection of adipocytes transfected with two siRNAs targeting YWHAB, YWHAE, YWHAZ, or YWHAG, as indicated in the legend, probed for the transcript indicated in the x-axis. E) RT-PCR analysis of RNA obtained 48 h after transfection of adipocytes with siRNAs targeting YWHAB, YWHAE, YWHAZ, and YWHAG, probed for expression of PLIN1 and ADIPOQ. For C, D, and E, values are expressed as the fold relative to siRNA control values. Bars represent means and SD of n = 3 biological replicates. Statistical differences were assessed using two-way ANOVA, and exact P-values are shown. F) Glycerol accumulation over 6 h from cells treated with vehicle or Fsk, 48 h after transfection with siRNAs targeting YWHAB, YWHAE, YWHAZ, and YWHAG. Bars represent the mean and SD of n = 2 independent experiments, each conducted with 3-fold technical replication. Statistical differences were assessed using two-way ANOVA, and exact P-values are shown.