1. Volume 11, Issue 1, Pages 25-33 (January 2003)
Accelerated Degradation of HMG CoA Reductase Mediated by Binding of Insig-1 to Its Sterol-Sensing Domain 
Navdar Sever, Tong Yang, Michael S Brown, Joseph L Goldstein, Russell A DeBose-Boyd 
Molecular Cell 
Volume 11, Issue 1, Pages (January 2003)
DOI: /S (02)

2. Figure 1
Insig-1 Confers Sterol-Mediated Degradation upon HMG CoA Reductase in Transfected CHO Cells
CHO-K1 cells were set up for experiments on day 0 at 5 × 105 cells per 60 mm dish in medium A supplemented with 5% FCS. On day 1, cells were transfected in 2 ml of medium A containing 5% lipoprotein-deficient serum and 2 μg pCMV-HMG-Red-T7 in the absence (lanes 1 and 2) or presence (lanes 3–12) of 10 ng pCMV-Insig-1-Myc. The total DNA in each lane was adjusted to 3 μg/dish by addition of pcDNA3 mock vector. Three hours after transfection, cells received a direct addition of 2 ml of medium A containing (final concentration) 5% lipoprotein-deficient serum, 50 μM sodium compactin, 50 μM sodium mevalonate, and sterols (1 μg/ml 25-hydroxycholesterol and 10 μg/ml cholesterol) plus 10 mM sodium mevalonate as indicated. After 16 hr, each dish received 4 μl of dimethyl sulfoxide containing the indicated final concentration of MG-132 (lanes 1–10) or 65 μM ALLN (lanes 11 and 12). After incubation for 5 hr, cells were harvested, membrane fractions were prepared, and aliquots (33 μg) were subjected to SDS-PAGE and immunoblot analysis with 1 μg/ml monoclonal anti-T7 IgG (against HMG CoA reductase) and 5 μg/ml monoclonal anti-Myc IgG (against insig-1). Filters were exposed to film for 2 s.
Molecular Cell  , 25-33DOI: ( /S (02) )

3. Figure 2
Transfected Insig-1 Enhances Turnover of 35S-Labeled HMG CoA Reductase in CHO Cells
(A and B) CHO-K1 cells were set up for experiments on day 0 at 5 × 105 cells per 60 mm dish in medium A supplemented with 5% FCS. On day 1, cells were transfected in 3 ml of medium A containing 5% FCS, 2 μg pCMV-HMG-Red-T7, and the indicated amount of pCMV-Insig-1-Myc (A) or 10 ng pCMV-Insig-1-Myc (B). Twenty hours after transfection, cells were preincubated for 1 hr in methionine/cysteine-free medium C supplemented with 1% hydroxypropyl β-cyclodextrin, after which they were pulse labeled for 1 hr with 250 μCi/ml 35S-methionine in medium C. Cells were then chased in medium C supplemented with 0.5 mM unlabeled methionine and 1 mM unlabeled cysteine, in the absence (−) or presence (+) of sterols (1 μg/ml 25-hydroxycholesterol and 10 μg/ml cholesterol) plus 10 mM sodium mevalonate as indicated. Following the indicated chase period, cells were harvested, lysed, and subjected to immunoprecipitation with monoclonal anti-T7 IgG-coupled agarose beads as described in Experimental Procedures. Aliquots of immunoprecipitates (normalized for equal 35S radioactivity incorporated into total cell protein) were subjected to SDS-PAGE and transferred to nylon membranes. Filters were exposed for 48 hr to an imaging plate at room temperature and scanned in a Fuji X Bas 1000 Phosphorimager, and the image was photographed.
(C) 35S radioactivity in scanned gels corresponding to HMG CoA reductase was quantified by densitometry. The intensity of HMG CoA reductase during the pulse (lane a of both gels in [B]) was arbitrarily set at 100%.
Molecular Cell  , 25-33DOI: ( /S (02) )

4. Figure 3
Overexpression of Wild-Type, but Not Mutant, SCAP(TM1-6) Prevents Insig-1-Mediated Degradation of Transfected HMG CoA Reductase in CHO Cells
CHO-K1 cells were set up for experiments on day 0 at 5 × 105 cells per 60 mm dish in medium A supplemented with 5% FCS. On day 1, cells were transfected in 2 ml of medium A containing 5% lipoprotein-deficient serum with 2 μg pCMV-HMG-Red-T7, 10 ng pCMV-Insig-1-Myc (lanes 7–12), and 10 ng of either wild-type (lanes 3, 4, 9, and 10) or mutant Y298C (lanes 5, 6, 11, and 12) versions of pCMV-CBP-Flag-SCAP(TM1-6)-Myc. The total DNA in each lane was adjusted to 3 μg/dish by addition of pcDNA3 mock vector. Six hours after transfection, cells received a direct addition of 2 ml of medium A containing (final concentration) 5% lipoprotein-deficient serum, 50 μM sodium compactin, 50 μM sodium mevalonate in the absence (−) or presence (+) of sterols (1 μg/ml 25-hydroxycholesterol and 10 μg/ml cholesterol) plus 10 mM sodium mevalonate. After 16 hr, cells were harvested, membrane fractions were prepared, and aliquots (20 μg) were subjected to SDS-PAGE and immunoblot analysis with 1 μg/ml monoclonal anti-T7 IgG (against HMG CoA reductase) and 5 μg/ml monoclonal anti-myc IgG (against SCAP(TM1-6) and insig-1). Filters were exposed to film for 10 s (top blot) and 1 s (middle and bottom blots).
Molecular Cell  , 25-33DOI: ( /S (02) )

5. Figure 4
Overexpression of Wild-Type, but Not Mutant, SCAP(TM1-6) Prevents Sterol-Mediated Inhibition of SREBP Processing and Degradation of Endogenous HMG CoA Reductase in HEK-293S Cells
HEK-293S cells, stably transfected with pcDNA3 empty vector (“mock”) or with either wild-type or mutant Y298C versions of pCMV-CBP-Flag-SCAP(TM1-6 or TM1-5)-Myc as indicated, were set up on day 0 at 6 × 105 cells per 100 mm dish in medium B supplemented with 10% FCS. On day 2, cells were switched to medium B containing 10% lipoprotein-deficient serum, 50 μM sodium compactin, and 50 μM sodium mevalonate. After 16 hr, the cells were re-fed with identical medium containing 10 mM sodium mevalonate and the indicated concentration of 25-hydroxycholesterol (25-HC). After incubation for 5 hr, cells were harvested, and membrane and nuclear fractions were prepared as described in Experimental Procedures. Aliquots of nuclear fractions (120 μg) and membranes (25 μg) were subjected to SDS-PAGE. Immunoblot analysis was then carried out with one of the following antibodies: 5 μg/ml of monoclonal IgG-1D2 (against SREBP-2), 5 μg/ml of polyclonal IgG-R139 (against endogenous SCAP), 5 μg/ml of monoclonal IgG-A9 (against endogenous HMG CoA reductase), and 5 μg/ml of monoclonal anti-Myc IgG (against transfected SCAP). Filters were exposed to film for 2.5 min (top two blots) and 1 s (bottom three blots). “N” and “P” denote the cleaved nuclear and precursor forms of SREBP-2, respectively.
Molecular Cell  , 25-33DOI: ( /S (02) )

6. Figure 5
Sterol-Dependent Binding of HMG CoA Reductase to Insig-1 Mediated by the Membrane Domain of Reductase
(A) CHO-K1 cells were set up for experiments on day 0 at 5 × 105 cells per 60 mm dish in medium A supplemented with 5% FCS. (A) On day 1, cells were transfected in 2 ml of medium A containing 5% lipoprotein-deficient serum with 5 or 10 ng pCMV-Insig-1-Myc (lanes 1, 2, and 4–7) and 2 μg pCMV-HMG-Red-T7 (lanes 3–7). The total DNA in each lane was adjusted to 3 μg/dish by the addition of pcDNA3 mock vector. Three hours after transfection, cells received a direct addition of 2 ml of medium A adjusted to produce final concentrations of 5% lipoprotein-deficient serum, 50 μM sodium compactin, 50 μM sodium mevalonate, and sterols (1 μg/ml 25-hydroxycholesterol and 10 μg/ml cholesterol) plus 10 mM sodium mevalonate as indicated. After 16 hr, each dish received a direct addition of 4 μl of dimethyl sulfoxide to give a final concentration of 65 μM ALLN. After incubation for 5 hr, cells were harvested, lysed, and subjected to immunoprecipitation with monoclonal anti-T7 IgG-coupled agarose as described in Experimental Procedures. Equal fractions of resuspended pellet and supernatant (“Sup.”) solutions were subjected to SDS-PAGE and immunoblotting with 5 μg/ml monoclonal anti-Myc IgG (against insig-1) and 1 μg/ml monoclonal anti-T7 IgG (against HMG CoA reductase). Filters were exposed to film for 45 s (top panel) or 10 s (bottom three panels).
(B) On day 1, cells were transfected in 3 ml of medium A containing 5% FCS, 1 μg pCMV-HMG-Red-T7(TM1-8), and 10 ng pCMV-Insig-1-Myc. Six hours after transfection, cells were incubated for 16 hr in 3 ml of medium A containing 5% lipoprotein-deficient serum, 50 μM sodium compactin, 50 μM sodium mevalonate, and sterols (1 μg/ml 25-hydroxycholesterol and 10 μg/ml cholesterol) plus 10 mM sodium mevalonate as indicated. Four hours prior to harvest, each dish received a direct addition of 3 μl of ALLN to give a final concentration of 65 μM. Cells were harvested, lysed, and subjected to immunoprecipitation with polyclonal anti-Myc IgG as described in Experimental Procedures. Equal fractions of resuspended pellet (lanes 1–6) and supernatant (lanes 7–12) solutions were subjected to SDS-PAGE and immunoblotting with 1 μg/ml monoclonal anti-T7 IgG (against HMG CoA reductase) and 1 μg/ml monoclonal anti-Myc IgG (against insig-1). Filters were exposed to film for 10 s (top panel) or 1 s (bottom panel).
Molecular Cell  , 25-33DOI: ( /S (02) )