Journal: eLife

Article Title: Schnyder corneal dystrophy-associated UBIAD1 inhibits ER-associated degradation of HMG CoA reductase in mice

doi: 10.7554/eLife.44396

Figure Lengend Snippet: MEFs from WT and Ubiad1 Ki/Ki mice were set up for experiments on day 0 at 2 × 10 5 cells per 10 cm dish in MEF medium supplemented with 10% fetal calf serum (FCS). ( A ) On day 3, cells were harvested for subcellular fractionation. Aliquots of resulting membrane and nuclear extract fractions (35–50 µg total protein/lane) were subjected to SDS-PAGE, followed by immunoblot analysis using antibodies against the indicated proteins. ( B ) On day 3, cells were harvested for measurement of Hmgcr mRNA levels by quantitative RT-PCR and total cholesterol levels using a colorimetric assay as described in ‘Materials and methods.’ ( C and D ) On day 2, cells were depleted of isoprenoids through incubation for 16 hr at 37°C in MEF medium containing 10% lipoprotein-deficient serum, 10 µM sodium compactin, and 50 µM sodium mevalonate. The cells were subsequently treated with 1 µg/ml 25-HC as indicated; in ( D ), the cells also received 10 µM MG-132. ( C ) After 4 hr at 37°C, cells were harvested for preparation of membrane and nuclear extract fractions (35–50 µg total protein/lane) that were analyzed by immunoblot with antibodies against the indicated protein. ( D ) Following incubation for 1 hr at 37°C, cells were harvested, lysed in detergent-containing buffer, and immunoprecipitated with 30 µg polyclonal anti-HMGCR antibodies. Immunoprecipitated material was subjected to SDS-PAGE and immunoblot analysis with IgG-A9 (against HMGCR) and IgG-P4D1 (against ubiquitin).

Article Snippet: Antibody , Rabbit polyclonal anti-Calnexin , Novus Biologicals , Cat#NB100-1965; RRID: AB_10002123 , used at 1–5 μg/ml for immunoblots.

Techniques: Fractionation, Membrane, SDS Page, Western Blot, Quantitative RT-PCR, Colorimetric Assay, Incubation, Immunoprecipitation, Ubiquitin Proteomics

Journal: eLife

Article Title: Schnyder corneal dystrophy-associated UBIAD1 inhibits ER-associated degradation of HMG CoA reductase in mice

doi: 10.7554/eLife.44396

Figure Lengend Snippet: ( A ) Male and female mice (15 WT, 24 Ubiad1 Ki/Ki , 50 weeks of age) consuming an ad libitum chow diet were analyzed by stereomicroscopic examination. Corneal opacification is indicated by white arrows. ( B–E ) Mice analyzed in ( A ) were sacrificed, corneas were then harvested and analyzed by immunohistochemical staining with anti-HMGCR polyclonal antibodies ( B ), quantitative RT-PCR ( C ), and LC-MS/MS ( D and E ) as described in the legend to and ‘Materials and methods.’ Error bars , S.E. The p value was calculated using Student’s t test: *, p < 0.05; **, p < 0.01; ***, p 0.005. Dhcr7 , 7-dehydrocholesterol reductase; Dhcr24 , 24-dehydrocholesterol reductase; 7-DehyDes., 7-dehydrodesmosterol; 8-Dehydrochol., 8-dehydrocholesterol; 7-Dehydrochol., 7-dehydrocholesterol.

Article Snippet: Antibody , Rabbit polyclonal anti-Calnexin , Novus Biologicals , Cat#NB100-1965; RRID: AB_10002123 , used at 1–5 μg/ml for immunoblots.

Techniques: Immunohistochemical staining, Staining, Quantitative RT-PCR, Liquid Chromatography with Mass Spectroscopy

Journal: eLife

Article Title: Schnyder corneal dystrophy-associated UBIAD1 inhibits ER-associated degradation of HMG CoA reductase in mice

doi: 10.7554/eLife.44396

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit polyclonal anti-Calnexin , Novus Biologicals , Cat#NB100-1965; RRID: AB_10002123 , used at 1–5 μg/ml for immunoblots.

Techniques: Knock-In, Residue, Western Blot, Recombinant, Sequencing, Reverse Transcription, SYBR Green Assay, Quantitation Assay, Software