Journal: The Journal of Cell Biology

Article Title: TIP47 functions in the biogenesis of lipid droplets

doi: 10.1083/jcb.200812042

Figure Lengend Snippet: Endogenous TIP47 and Rab9 do not colocalize. HeLa cells were fixed and stained for the indicated proteins. GFP-Rab7 and GFP-Lamp1 were transiently transfected 48 h prior to fixation. Colocalization is indicated in yellow. The inset displays a cell with small LDs at steady state. Bar, 15 µm.

Article Snippet: Anti-ADRP was obtained from Progen, anti-Lamp1 and anti-Na + /K + -ATPase from the Hybridoma Bank, antitransferrin receptor from Invitrogen, antibodies for EEA1, α-catenin, α-adaptin and GM130 from Transduction, anti-Rab9 from Enzo Biochem, Inc. or Thermo Fisher Scientific, anti-TGN46 from AbD Serotec, anti-GAPDH from Millipore, anti-VPS26 and anti-VPS35 from M. Seaman (Cambridge Institute for Medical Research, Cambridge, England, UK), monoclonal anti-His antibody from QIAGEN, and monoclonal anti-HA from Babco.

Techniques: Staining, Transfection

Journal: The Journal of Cell Biology

Article Title: TIP47 functions in the biogenesis of lipid droplets

doi: 10.1083/jcb.200812042

Figure Lengend Snippet: CFP-Rab9 does not colocalize with YFP-TIP47. (A–H) HeLa cells were transiently transfected with YFP-TIP47 (A and E, green), CFP-Rab9 (B and F, green), CFP-Rab9, and YFP-TIP47 (C and G; green, CFP; red, YFP) or with CFP-ADRP and YFP-TIP47 (D and H; green, CFP; red, YFP). Cells depicted in the bottom panels were fixed after oleic acid (OA) feeding. A neutral lipid stain is shown in red (E and F) or blue (G and H). Brightness and contrast of TIP47 staining in A and E were enhanced to visualize the otherwise faint cytoplasmic staining. Colocalization is indicated in yellow. Bar, 15 µm.

Article Snippet: Anti-ADRP was obtained from Progen, anti-Lamp1 and anti-Na + /K + -ATPase from the Hybridoma Bank, antitransferrin receptor from Invitrogen, antibodies for EEA1, α-catenin, α-adaptin and GM130 from Transduction, anti-Rab9 from Enzo Biochem, Inc. or Thermo Fisher Scientific, anti-TGN46 from AbD Serotec, anti-GAPDH from Millipore, anti-VPS26 and anti-VPS35 from M. Seaman (Cambridge Institute for Medical Research, Cambridge, England, UK), monoclonal anti-His antibody from QIAGEN, and monoclonal anti-HA from Babco.

Techniques: Transfection, Staining

Journal: The Journal of Cell Biology

Article Title: TIP47 functions in the biogenesis of lipid droplets

doi: 10.1083/jcb.200812042

Figure Lengend Snippet: TIP47 does not bind to Rab9 in vitro. (A) Pull-down assay is shown. His 6 -GST-TIP47 was loaded on beads, and Rab9 either complexed to GDP (lanes a), GTP (lanes b), or GppNHp (lanes c) was added in equimolar amounts. After 2 h, the flow through and the wash fractions were collected before the beads were eluted. All fractions were analyzed by SDS-PAGE. The first two lanes show free His 6 -GST-TIP47 and Rab9 for size comparison. M, marker. (B) Analytical gel filtration of an equimolar mixture of TIP47 and Rab9 complexed to GppNHp that had been incubated for 2 h. (C) Western blot detection of the indicated proteins in cytosol (C) and membranes (M) from control (HeLa), TIP47 KD, and Rab9 KD cells. (D) Detection of the indicated proteins in immunoprecipitates of a nonspecific serum or a TIP47-specific rabbit serum. An aliquot (1%) of a PNS served as a control for the protein detection. Values on blots are shown in kilodaltons.

Article Snippet: Anti-ADRP was obtained from Progen, anti-Lamp1 and anti-Na + /K + -ATPase from the Hybridoma Bank, antitransferrin receptor from Invitrogen, antibodies for EEA1, α-catenin, α-adaptin and GM130 from Transduction, anti-Rab9 from Enzo Biochem, Inc. or Thermo Fisher Scientific, anti-TGN46 from AbD Serotec, anti-GAPDH from Millipore, anti-VPS26 and anti-VPS35 from M. Seaman (Cambridge Institute for Medical Research, Cambridge, England, UK), monoclonal anti-His antibody from QIAGEN, and monoclonal anti-HA from Babco.

Techniques: In Vitro, Pull Down Assay, SDS Page, Marker, Filtration, Incubation, Western Blot