Lipotransin Molecular Cell Li-Jyun Syu, Alan R Saltiel  Molecular Cell  Volume 4, Issue 1, Pages 109-115 (July 1999) DOI: 10.1016/S1097-2765(00)80192-6

Figure 1 Expression of the Lipotransin and HSL Genes (A) A human multiple-tissue Northern blot, containing approximately 2 μg of poly(A+) RNA in each lane, was hybridized at 65°C for overnight using a [α-32P]dCTP-labeled probe of lipotransin (LPT) spanning from 74 to 697 in the cDNA sequence. The same blot was stripped and reprobed with [α-32P]dCTP-labeled HSL (mouse) spanning from 900 to 1848 in the coding sequence, and with [α-32P]dCTP labeled β-actin (human) as control. The positions of RNA size markers in kilobases are shown on the left. (B) HEK293T cells were transiently transfected (TransF) with an expression vector containing amino-terminal Flag-tagged lipotransin (L) or the vector (V) alone. Triton X-100 soluble lysates (T) and Triton-insoluble pellets (TIP) prepared from transfected cells were then subjected to SDS-PAGE and immunoblotted with anti-lipotransin (LPT) antibodies. The SDS-soluble lysates (SDS) prepared from 3T3-L1 cells were also analyzed. (C) Triton X-100 soluble lysates from HEK293T cells described in (B) were immunoprecipitated with either anti-Flag antibodies or anti-lipotransin (LPT) crude serum. The immunoprecipitates were eluted without any reducing agent and followed by separation and immunoblotting with anti-Flag antibodies. Whole-cell lysates (WCL) from both transfected cells were analyzed. Molecular mass markers (kilodaltons) are on the left. Molecular Cell 1999 4, 109-115DOI: (10.1016/S1097-2765(00)80192-6)

Figure 2 Temporal and Spatial Expression of Lipotransin in 3T3-L1 Adipocytes (A) Triton X-100-solubilized lysates were prepared from 3T3-L1 adipocytes (samples in the left column). Samples in the right column were prepared from SDS-solubilized total protein lysates. The blot was probed with anti-HSL or anti-lipotransin (LPT) antibodies. Differentiation of preadipocytes into adipocytes was initiated on day 0 (PreA, 2 days postconfluence) and completed on day 4 (A). Adipocytes were maintained in FBS-containing medium after completion of differentiation. (B) Fully differentiated 3T3-L1 adipocytes (day 8 in 10% FBS) were fractionated into N/M/ER (nuclei, mitochondria, and endoplasmic reticulum), CYTO (cytolic), PM (plasma membrane), HDM (high-density microsomes), LDM (low-density microsomes), FAT (washed fat), and FATP (195K × g pellets from fat) fractions as described in Experimental Procedures. Pellets were resuspended with a 23-gauge needle in homogenization buffer. Proteins in fat were extracted as described in Experimental Procedures. Equal cell equivalents were subjected to SDS-PAGE, transferred to nitrocellulose, and probed with anti-HSL or anti-lipotransin antibodies. Immunoreactive proteins were detected by ECL. (C) HEK293T cells were transfected (TransF) with vector alone (V) or Flag-lipotransin together with HA-HSL (L/H). Cells were lysed in HNTG buffer with the addition of either 1 mM EDTA or 5 mM MgATP. The lysates were immunoprecipitated with anti-Flag. Following SDS-PAGE, the immunoprecipitates were immunoblotted with anti-HA or Flag antibodies. Whole-cell lysates (WCL) are also shown. (D) HA-HSL was immunoprecipitated from transfected HEK293T cells, which were serum starved overnight. The recovered HA-HSL beads were either subjected to in vitro phosphorylation with PKA or left untreated. Pulldowns were performed with 3T3-L1 lysates prepared from adipocytes at day 3 in insulin medium, serum-deprived for 2 hr, and lysed in HNTG buffer with the addition of 1 mM EDTA/5 mM ATP, 1 mM EDTA/5 mM AMP-PNP, 10 mM Mg2+/5 mM ATP, or 10 mM Mg2+/5 mM AMP-PNP. After 30 min, beads were collected in each pulldown and washed. The bound proteins were boiled in Laemmli sample buffer, separated on SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-lipotransin (LPT). The same immunoblot was stripped and reprobed with anti-HSL antibodies. The 32P-HSL band reflects the amount of phosphorylated HSL in each pulldown. Beads alone without lysates are also shown. Molecular Cell 1999 4, 109-115DOI: (10.1016/S1097-2765(00)80192-6)

Figure 3 The Association of Lipotransin with HSL in Intact 3T3-L1 Adipocytes Requires Treatment with Insulin (A) Fully differentiated 3T3-L1 adipocytes were serum and phosphate deprived for 3 hr before labeling with [32P]orthophosphate for 1 hr. Following a 15 min treatment with 100 nM insulin (Ins) or 10 μg/ml of forskolin (Forsk), cells were lysed in HNTG buffer and immunoprecipitated with anti-lipotransin (LPT) antiserum. As a control, mixed lysates were also immunoprecipitated with preimmune (Pre) serum. Following SDS-PAGE, the immunoprecipitates were immunoblotted with anti-HSL antibodies. (B) Fully differentiated 3T3-L1 adipocytes were serum starved for 2 hr. Following a 20 min treatment with either 50 nM isoproterenol (IPT), 100 nM insulin (Ins), or both, cells were lysed in HNTG buffer plus 5 mM EDTA and 5 mM ATP, and immunoprecipitated with anti-HSL antiserum. Following SDS-PAGE, the immunoprecipitates were immunoblotted with anti-lipotransin (LPT) antibodies. (C) HA-HSL beads were prepared as described above. Pulldowns were performed in 3T3-L1 lysates, which were prepared from adipocytes at day 10 in 10% FBS medium, serum deprived for 2 hr before insulin stimulation for 15 min, and lysed in HNTG buffer with the addition of 5 mM EDTA and 5 mM ATP. Lysates were incubated with the fusion proteins for 30 min, and the beads were collected and washed. The bound proteins were eluted and separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-lipotransin (LPT). The same immunoblot was stripped and reprobed with anti-HSL antibodies to show the relative amount of HA-HSL in each pulldown. Beads alone without lysates are also shown. Molecular Cell 1999 4, 109-115DOI: (10.1016/S1097-2765(00)80192-6)

Figure 4 Translocation of HSL and Lipotransin (A) Fully differentiated 3T3-L1 cells (day 14 in 10% FBS) were serum starved overnight before 15 min stimulation with either 100 nM insulin (Ins), 50 nM isoproterenol (IPT), or both. Cells were homogenized and fractionated as described in Experimental Procedures. Equal cell equivalents were subjected to 10% SDS-PAGE, transferred to nitrocellulose, and probed with anti-HSL antibodies. The same immunoblot was stripped and reprobed with anti-lipotransin (LPT) antibodies. Protein staining using Ponceau S (Sigma) is also shown to demonstrate the equal loading of proteins. (B) Fully differentiated 3T3-L1 adipocytes (day 11 in 10% FBS) were serum starved overnight before 30 min stimulation with either 100 nM insulin (Ins), or 10 μM isoproterenol (IPT) and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX). Cells were homogenized as described in Experimental Procedures, except for the addition of either 5 μM ATP or 5 mM ATPγS in the homogenization buffer. Fractionation was performed with 195 K × g centrifugation for 75 min. Equal cell equivalents were subjected to 4%–15% gradient SDS-PAGE (Bio-Rad), transferred to nitrocellulose, and probed with anti-HSL antibodies. Only data from cytosolic fractions are shown. Protein staining using Ponceau S is also shown to demonstrate the equal loading of proteins. Molecular Cell 1999 4, 109-115DOI: (10.1016/S1097-2765(00)80192-6)