Proteins uniquely associated with ARC-purified hmCD40 as determined by mass spectrometry analysis.

<p>*All protein assignments listed met or exceeded the Scaffold 95% confidence filter.</p><p>**TRAF1 was detected in gel slices containing material at or above the known molecular weight for TRAF1 in hmCD40 ARC samples, but was also detected in both hmCD40Δ67 and hmCD40 gel slices containing material of approximately 35 kD.</p

Western blot verification of mass spectrometry results.

<p>hmCD40 and hmCD40Δ67 ARC samples (3.3×10⁶ cell equivalents per lane) were fractionated by SDS-PAGE and transferred to PVDF membrane for Western blotting with the indicated antibodies. Lanes containing whole cell lysates (1.0×10⁵ cell equivalents per lane) were run in parallel. Each blot is representative of three or more experiments.</p

TRAF-dependent recruitment of proteins to CD40.

<p>Endogenous CD40 from CH12.LX cells (WT), TRAF2-deficient CH12.LX (T2-), or TRAF3 deficient CH12.LX cells (T3-) was isolated using ARC (lane loading as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011380#pone-0011380-g002" target="_blank">Fig. 2</a>). Western blotting was performed with the indicated antibodies. Each blot is representative of three or more experiments.</p

HOIPΔRBR inhibits IκBα phosphorylation and degradation.

<p>TRAF6-deficient A20.2J cells were stably transfected with FLAG2X-tagged HOIP or HOIPΔRBR in an IPTG-inducible expression vector. A, Expression of FLAG2X-tagged HOIP or HOIPΔRBR in stably transfected cell lines. Western blots of cell lysates from uninduced and IPTG-induced cell lines were probed with anti-FLAG (left) and anti-HOIP (right). Blots were reprobed for actin to verify equivalent lane loading. B, The two cell lines were stimulated with CD154 (CD40 ligand) for the times indicated and then processed for Western blotting. Unstimulated cells (first lane) and cells incubated for 5 minutes with insect cells lacking CD154 served as negative controls. Western blotting for phosphorylated IκBα was performed; blots were then stripped and reprobed for total IκBα and actin. C, Quantification of IκBα degradation (panel B) in TRAF6-deficient A20.2J cells stably transfected with Lac repressor only (◊;♦), Lac repressor plus full-length HOIP (□;▪), or Lac repressor plus HOIPΔRBR (Δ; ▴). Filled symbols indicate cultures pre-treated with IPTG. IκBα bands in each lane were normalized to the actin signal. The degradation index is the fraction of IκBα remaining at each time point relative to the amount of IκBα present in cells incubated for 5 minutes with insect cells lacking CD154. The results presented are the mean of four experiments. Error bars indicate standard error of the mean.*, p<0.05 (one-sided Student's t test).</p

Ursolic acid reduces diet-induced fatty liver disease.

<p>Mice were provided ad libitum access to high fat diet (HFD) lacking or containing ursolic acid (UA) for 6 weeks. UA concentrations were 0.27% (B–C and E) or 0.14% (A, D and F–G). Data are means ± SEM. *P<0.05 by t-test. (A) Liver weights. n≥12 mice per diet. (B) Liver H&E-stained sections. 20x magnification. (C) Liver osmium-stained sections, 10x magnification. (D) Hepatic triglyceride content. n = 5 mice per diet. (E) Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. n = 5 mice per diet. (F) Liver mRNA levels were determined using qPCR. Levels in UA-treated mice were normalized to the average levels in mice fed HFD lacking ursolic acid, which were set at 1. n = 10 mice per diet. (G) Livers were harvested and subjected to SDS-PAGE and immunoblot analysis with anti-ACC and anti-tubulin antibodies. Upper: representative immunoblots. Lower: ACC and tubulin levels were quantitated with densitometry. In each mouse, the ACC/tubulin ratio was normalized to the average ACC/tubulin ratio in mice fed HFD lacking ursolic acid. n = 6 mice per diet.</p

Ursolic acid increases exercise capacity, does not alter blood pressure, and reduces resting heart rate in high fat-fed mice.

<p>Mice were fed high fat diet (HFD) lacking or containing 0.27% ursolic acid (UA) for 17 weeks, and then exercise treadmill capacity was determined according to an established protocol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039332#pone.0039332-Arany1" target="_blank">[36]</a> (A) and resting blood pressure and heart rate were determined with tail cuff plethysmography (B and C). Data are means ± SEM from ≥7 mice per diet. P-values were determined with t-tests. *P<0.05.</p

Chronic, but not acute, ursolic acid treatment increases food intake and energy expenditure.

<p>Mice were fed high fat diet (HFD) lacking or containing 0.27% ursolic acid (UA) for either 3 days (acute treatment) or 6 weeks (chronic treatment), and then food intake (A) and energy expenditure (B) were determined using a comprehensive lab animal monitoring system (CLAMS). <i>Left panels</i>: hourly measurements. Data are means from 12 mice per diet. <i>Right panels</i>: cumulative measurements during the dark and light periods. Data are means ± SEM from 12 mice per diet. <i>P-</i>values were determined with unpaired t-tests. *<i>P</i><0.05.</p

Ursolic acid reduces diet-induced obesity and glucose intolerance.

<p>Mice were provided ad libitum access to high fat diet (HFD) lacking or containing 0.14% ursolic acid (UA) for 6 weeks. Data are means ± SEM. *P<0.05 by t-test. (A) Total body weight was measured at the indicated times. n≥12 mice per diet. (B) Weights of bilateral epididymal and retroperitoneal fat pads. n = 10 mice per diet. (C) Fasting blood glucose levels. Mice were fasted for 16 h prior to tail vein glucose measurements. n≥12 mice per diet. (D) Glucose tolerance tests. Following a 16 h fast, 1 g/kg glucose was administered by i.p. injection at time  = 0 min. Blood glucose was then measured via the tail vein at the indicated times. n = 10 mice per diet. Left, blood glucose values. Right, areas under the curves.</p

In mice fed a high fat diet, ursolic acid increases skeletal muscle Akt signaling, anabolic mRNA expression, grip strength, skeletal muscle mass, and fast and slow skeletal muscle fiber size.

<p>Mice were provided ad libitum access to high fat diet (HFD) lacking or containing 0.14% ursolic acid (UA) for 6 weeks. Data are means ± SEM. *<i>P</i><0.05 by t-test. (A) Triceps muscles here harvested and subjected to SDS-PAGE and immunoblot analysis with anti-phospho(Ser473)-Akt and anti-Akt antibodies. <i>Upper</i>: representative immunoblots. <i>Lower</i>: Phospho-Akt (P-Akt) and total Akt levels were quantitated with densitometry. In each mouse, the phospho-Akt/total Akt ratio was normalized to the average phospho-Akt/total Akt ratio in mice fed HFD lacking UA. n = 5 mice per diet. (B) Quadriceps mRNA levels were determined using qualitative real-time RT-PCR (qPCR). Levels in UA-treated mice were normalized to the average levels in mice fed HFD lacking ursolic acid, which were set at 1. n = 10 mice per diet. (C) Grip strength. n = 10 mice per diet. (D) Weights of bilateral quadriceps and triceps brachii (triceps). n≥12 mice per diet. (E) Slow and fast muscle fiber diameters. Sections of triceps muscle were subjected to immunohistochemical analysis with anti-slow myosin and anti-fast myosin antibodies, and then fiber diameter was measured. Slow fibers: n≥50 fibers/triceps from 5 mice per condition. Fast fibers: n≥100 fibers/triceps from 5 triceps per condition.</p