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. 2014 Aug 1;289(38):26021–26037. doi: 10.1074/jbc.M114.561134

FIGURE 4.

FIGURE 4.

Nrbf2 forms cytoplasmic puncta that are positive for isolation membrane markers in RPE-1 cells, particularly under starvation conditions. Subcellular fractionation revealed that both stably expressed Nrbf2-EGFP (a) and endogenous Nrbf2 (b) were primarily localized in the cytosol in HEK293 cells under nutrient-rich conditions. A1, cytosolic fraction as marked by SOD1; A2, membrane fraction as marked by EGFR; A3, soluble nuclear fraction as marked by SP1; A4, chromatin-bound nuclear fraction as marked by histone H3; A5, cytoskeletal fraction; A6, pellet fraction. c, ectopically expressed Nrbf2-EGFP formed cytoplasmic punctate structures under both nutrient-rich and Hanks' buffer starvation conditions. The numbers of cells used for quantification were 18 (for EGFP, nutrient-rich), 24 (for EGFP, Hanks' buffer starvation), 22 (for Nrbf2-EGFP, nutrient-rich), and 22 (for Nrbf2-EGFP, Hanks' buffer starvation). d, ectopically expressed mCherry-Nrbf2 and EGFP-Atg5 colocalized on punctate structures under Hanks' buffer starvation conditions. The numbers of cells used for quantification were 14 (for mCherry) and 12 (for mCherry-Nrbf2). e, ectopically expressed Nrbf2-EGFP and myc-tagged Ulk1 WT colocalized on punctate structures under Hanks' buffer starvation conditions. The numbers of cells used for quantification were nine (for EGFP) and four (for Nrbf2-EGFP). f, ectopically expressed mCherry-Nrbf2 and endogenous FIP200 colocalized on punctate structures under Hanks' buffer starvation conditions. The numbers of cells used for quantification were three (for mCherry) and eight (for mCherry-Nrbf2). g, ectopically expressed Nrbf2-EGFP and myc-tagged Ulk1 K46I mutant colocalized on punctate structures under Hanks' buffer starvation conditions. The numbers of cells used for quantification were six (for EGFP) and nine (for Nrbf2-EGFP). d–g, note that mCherry or EGFP was primarily diffused, whereas mCherry-Nrbf2 or Nrbf2-EGFP formed puncta under Hanks' buffer starvation conditions. h, ectopically expressed Nrbf2-EGFP formed cytoplasmic puncta in both FIP200+/+ and FIP200−/− MEFs under Hanks' buffer starvation conditions. The numbers of cells used for quantification were six (for EGFP, FIP200+/+), 11 (for Nrbf2-EGFP, FIP200+/+), eight (for EGFP, FIP200−/−), and ten (for Nrbf2-EGFP, FIP200−/−). NS, not significant. Scale bars = 10 μm (c–f) and 20 μm (g and h).