Additionally, the effects of androgens, known to play an important role in the development of MGD in patients, on PPAR signaling are also needed. Finally, Raman spectral analysis of CN-G2 lipid detected an elevated vibrational peak around 2940 cm?1 in addition to the two main peaks at 2846 cm?1 and 2886 cm?1 associated with wax and cholesterol esters. up-regulated of PPAR, ADP and ADFP mRNA. Conclusions This study confirms the loss of cytoplasmic/vesicular PPAR localization in older, atrophic mouse meibomian glands. Furthermore, PPAR stimulates lipid synthesis in mouse meibocytes, associated with PPAR sumoylation and translocation to the cytoplasm. Taken together these data suggest that lipid synthesis in older mice is down regulated by a PPAR mediated pathway. subroutine in Metamorph and the area recorded. For each image the number of cells was also determined by counting the nuclei using the Manual Count subroutine. To calculate the lipid content per cell in each image, the thresholded lipid pixel area was then divided by Butein the total number of cells in each image. E. Protein Isolation and Western Blotting For mouse meibomian glands, 5 young (2-month-old) and 5 old (2-year-old) mice were sacrificed and the meibomian glands isolated from the upper and lower eyelids and pooled together to obtain enough protein for multiple western blots. Tissues were then homogenized using a Polytron (PT-1200, Kinematic, Bohemia, NY). For extraction of protein from tissue cultured cells, 100 mm dishes were first rinsed 3 times in Dulbecco’s phosphate buffered saline, and then cells were scraped from the dish using a polyethylene Cell Lifter (Corning Inc., Corning, NY). Proteins from tissues and cells were fractionated using the NE-PER Nuclear & Cytoplasmic Extraction Reagent kit from Thermo Scientific (Rockford, IL). All extraction buffers were supplemented with the Calbiochem Protease Inhibitor Cocktail Set III (EMD Chemical Inc, Gibbstown, NJ) and Phosphatase Inhibitor Cocktail 2 (Sigma-Aldrich, St Louis, MO). The protein content of the cell and tissue extracts was then measured using the RC DC Protein Assay (Bio-Rad Butein Laboratories, Hercules, CA) and the samples run on 12% SDS PAGE gels. Proteins were then transferred to PVDF membranes using iBlot Gel Transfer Device (Invitrogen, Carlsbad, CA). Membranes were then blocked using 5% non-fat dry milk in PBS-T (0.2% Tween 20) and then immunostained using antibodies to PPAR (Dilution 1:500). Membranes were then rinsed with PBS-T and then incubated with goat anti-rabbit IgG (H+L) HRP (Dilution 1:2500; Invitrogen), rinsed with PBS-T and incubated with SuperSignal? West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL). Immunostained bands were then detected using photographic film. All western blots were repeated CIT 3 times. F. Immunoprecipitation Cytoplasmic extracts from cultured meibocytes were first immunopreciptated using goat anti-PPAR Butein antibodies (sc-22020, Santa Cruz Biotechnology, Dallas, TX) linked to Dynabeads-Protein G (Novex, Life Technologies, Oslo, Norway). Proteins were eluted and then western blotted using rabbit anti-PPAR antibodies (ab27649, Abcam, Cambridge, MA) to identify presence of cytoplasmic 50 and 72 kDa PPAR. Blots were then stripped using a mild stripping protocol and then reacted with rabbit monoclonal antibody to SUMO1 (ab133352, Abcam). G. RNA Isolation and Analysis of Gene Expression Gene expression was evaluated by real time PCR using previously published methods.34, 35 Briefly, cells were directly lysed using RLT buffer and the RNA isolated over RNeasy spin columns as suggested by the manufacturer (Quiagen, Valencia CA). RNA yield and quality was evaluated with the aid of a Nanodrop spectrophotometer (Thermo Scientific, Wilmington,DE). 0.5 g RNA was reverse transcribed using oligo dT and random primers as supplied in the QuantiTect Reverese Transcription Kit (Quiagen). Real time PCR was.