Fig. 5. Localization of SPRING to the Golgi is required for regulation of SREBP.
a Schematic depiction of SPRING’s predicted domains and post-translational modifications (www.uniprot.org). b HeLa cells were transfected with a SPRING-GFP expression construct. Subsequently, cells were counterstained against the Golgi-resident protein GM130. DAPI was used to stain the nuclei; scale bar, 10 μm. c HEK293T cells were transfected with expression constructs encoding SPRING WT or a N67Q mutant. Total cell lysates were immunoblotted as indicated (N = 3). d HEK293T cells were co-transfected with expression constructs encoding LDLR-HA and SPRING-V5. Cellular membranes were isolated and subjected to tryptic digestion in the presence or absence of the permeabilizing detergent Triton-X-100. An equal amount of protein was immunoblotted as indicated. e Immunofluorescence staining of Hap1 cells transfected with expression constructs encoding SPRING-mCherry or SPRING-mCherry-KDEL and nuclei were counterstained with DAPI; scale bar, 25 μm. f Hap1-WT and Hap-SPRINGKO cells were transfected as indicated and total cell lysates immunoblotted as shown (N = 2). b, d, e Representative images of three independent experiments are shown.