SUPPLEMENTAL MATERIALS AND METHODS. Puromycin-synchronized metabolic labelling - Transfected HepG2 cells were depleted of

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1 SUPPLEMENTAL MATERIALS AND METHODS Puromycin-synchronized metabolic labelling - Transfected HepG2 cells were depleted of cysteine and methionine and then treated with 10 μm puromycin in depletion medium for 10 minutes at 37ºC. Cells were washed in depletion medium three times on ice (30 minutes total) to completely remove the puromycin and then incubated in [ 35 S]cysteine/methionine containing media (with or without 25 μm MG132) for 5 minutes at 37ºC. Following the pulse, the medium was removed and chase medium (also with or without MG132) was added and cells were harvested at 5 minute intervals up to 20 minutes. Cell lysis and analysis of apob-100 was performed as described in Materials and Methods. Immunoblot Analysis - Cells were treated for 16 h with U0126 or transfected with NT or sigp78 and harvested by lysis in 1% SDS 72 hours post-transfection. Total cell lysates were resolved by SDS-PAGE in 10% (w/v) polyacrylamide gels and the indicated proteins were visualized by immunoblotting. Primary antibodies to diacylglycerol acyltransferase 1 (DGAT1) (H-255, Santa Cruz Biotechnology, Santa Cruz, CA), microsomal triglyceride transfer protein (MTP)(Novus Biologicals, Littleton, CO), hydroxymethylglutaryl coenzyme A reductase (HMGCoAR)(Upstate Cell Signalling Solutions, Lake Placid, NY), sterol regulatory element binding protein 1a (SREBP1a)(Chemicon, Temecula, CA), peroxisome proliferator activated receptor (PPAR (Chemicon), peroxisome proliferator activated receptor (PPAR (Santa Cruz Biotechnology), ERK1/2 (#9102; Cell Signaling Technology, Danvers, MA) and Phospho- ERK1/2 (#9101; Cell Signaling Technology, Danvers, MA) were incubated overnight and secondary antibodies and chemiluminescence were performed as described in Materials and Methods. 1

2 Trypsin digestion in radiolabelled cells Transfected HepG2 cells were depleted of cysteine and methionine and then incubated in [ 35 S]cysteine/methionine containing media for one hour. The cells were then permeabilized with digitonin and trypsin digestion was performed as described in Materials and Methods. ApoB and apoai were then collected by immunoprecipitation, visualized by SDS-PAGE with autoradiography and quantified by liquid scintillation counting. SUPPLEMENTAL FIGURE LEGENDS Supplemental Figure 1. Gp78 knockdown reduces the cellular accumulation of nascent apob-100 in puromycin-synchronized HepG2 cells. Seventy-two hours following transfection with either NT or gp78-targeting sirna, HepG2 cells were treated with 10 μm puromycin for 5 minutes and then washed for 3 x 10 minutes on ice. Cells were immediately pulse-labeled for 5 minutes at 37ºC in medium containing [ 35 S]cysteine/methionine and then chased for up to 20 minutes. Where indicated, 25 μm MG132 was included in the pulse and chase medium. ApoB was recovered from the cells by immunoprecipitation and visualized by autoradiography. Supplemental Figure 2. Gp78 sirna does not alter levels of proteins involved in lipid metabolism. Cells were transfected with NT or and harvested by lysis 72 hours posttransfection. Total cell lysates were resolved by SDS-PAGE and the indicated proteins were visualized by immunoblotting. 2

3 Supplemental Figure 3. Reduced gp78 expression protects radiolabelled apob-100 from trypsin digestion in digitonin-permeabilized HepG2 cells. A. Cells were transfected with either non-targeting (NT) or gp78-targeting sirna. Seventy-two hours post-transfection the cells were labelled with [ 35 S]cysteine/methionine for 1 hour. Cells were then permeabilized with digitonin and the supernatant was discarded. The remaining monolayers were then treated with trypsin at the indicated concentration for 30 minutes on ice. Soybean trypsin inhibitor was then added and the monolayers were collected. ApoB-100 and apoai were recovered from cells by immunoprecipitation and visualized by autoradiography. B. ApoB-100 and apoai bands were excised and quantified by liquid scintillation counting. The ratio of apob-100 to apoai is expressed as percent of 0 µg/ml trypsin control (NT and ) to demonstrate the relative level of digestion. Supplemental Figure 4. U0126 does not affect global ubiquitination during MG132 treatment and does not affect phospho-erk. A. Cells were pre-treated for 16 hours with either DMSO or 10 μm U0126. Cells were incubated for one hour with 360 μm oleic acid prior to the 25 μm MG132 treatment. From total cell lysates, 6% input was loaded per lane for the total ubiquitin blot (upper left panel) and 2% input was loaded for the actin blot (lower left panel). ApoB immunoprecipitates (right panel) were performed on total cell lysates with a polyclonal anti-apob and the elutions analyzed by Western blot with anti-ubiquitin antibody. B. Cells were harvested 72 hours post-transfection or after 16 h incubation with 10 μm U0126. Total cell lysates were resolved by SDS-PAGE and total ERK and phospho-erk were visualized by immunoblot analysis. 3

4 NT - MG132 + MG132 - MG132 + MG Chase (min) apob100 incomplete apob polypeptides Fisher et al, Supplemental Figure 1

5 NT DGAT1 MTP HMGCoAR SREBP1a PPARα PPARγ actin Fisher et al, Supplemental Figure 2

6 A NT apob-100 apoai Trypsin (µg/ml) B apob-100/apoai (% of control) NT Trypsin ( g/ml) Fisher et al, Supplemental Figure 3

7 A Total cell lysate anti-apob IP MG132 (min) U Total ubiquitin Ub-apoB actin B DMSO U0126 NT - phospho-erk1/2 - total ERK1/2 Fisher et al, Supplemental Figure 4