Materials The following materials were purchased from Sigma Chemical (St. Louis, MO): LPS (E.coli 0111:B4), ATP, UDP-glucose (microbial-derived; catalog #U4625, multiple vials of lot #074K7024 over a period of 15 months) and UDP-galactose (catalog #U4500). Synthetic UDP-glucose was purchased from MP Biomedicals (Solon, OH) and B. pertussis toxin (PTX) was obtained from Calbiochem (San Diego, CA).
Murine N9 microglia [9
] were cultured routinely in Dulbecco’s Modified Eagle’s Medium (DMEM; Cellgro, Herndon, VA) supplemented with 5% fetal bovine serum (Bio Whittaker, Walkersville, MD) and 100 U/ml penicillin/streptomycin (Cellgro) in 100-mm Sarstedt plates. Cells were grown to ~90% confluency and passaged every 2 days. For experimentation, cells were plated at densities of 1
cells per well in 24-well plates for nitrite measurements, or at 4
cells per well in 12-well plates for signaling studies and RT-PCR. The next day, the cells were treated as specified below.
Reverse-transcription polymerase chain reaction (RT-PCR)
RT-PCR was performed on 1 μg of total RNA from whole brain (positive control) and N9 microglia as previously reported [2
]. RT reactions were completed according to the manufacturer’s protocol with and without reverse transcriptase (+/-RT). The cDNA was then used for PCR using the GoTaq Green Master Mix (Promega, Madison, WI). The murine P2Y14 receptor gene was amplified using the following primers: 5’TAGAGGCCATAAACTGTGCTT and 5’AATTCTTCCTGGACTTGAGGT (expected amplimer size 742 bp). As a control, GAPDH (glyceraldehyde-3-phosphate dehydrogenase; expected amplimer size 325 bp) was amplified using the following primers: 5’CCATCACCATCTTCCAGGAG and 5’GATGGCATGGACTGTGGTC. PCR products were separated and visualized by ethidium bromide-stained 1% agarose gel electrophoresis.
Measurement of nitrite production
Cells were stimulated with LPS (1 μg/mL) either alone or together with UDPG for 18–22 h at the concentrations indicated. In some experiments, cells were treated with increasing concentrations of UDPG or UDP-gal alone (50–300 μM), or with UDPG that had been boiled at 105°C for 30–60 min. In other experiments, microglia were pretreated with PTX (100 ng/mL) for 18 h, followed by stimulation with UDPG. Nitric oxide (NO) levels were measured in the culture medium using the Greiss reagent to analyze nitrite production, a stable breakdown product of NO generation [7
]. The cells were collected for immunoblot analyses.
Whole-cell lysates were prepared and total protein content determined as previously described [4
]. Equal amounts of protein (~25 μg) were loaded per lane and separated by 10% SDS-PAGE [6
]. Proteins in the gels were transferred to Immobilon polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA), and the membranes were subjected to immunoblot analyses for iNOS, COX-2 and phospho-CREB as described previously [4
]. To confirm equal protein loading, membranes were probed with antibodies recognizing the cytosolic proteins Grb-2 or GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA). The data shown are representative of at least three independent experiments performed in triplicate.
Statistical analyses were performed using an ANOVA pre-hoc test and Tukey-Kramer, Dunnett or Bonferroni multiple comparison post-hoc analyses. Statistical significance was set at the 95% confidence limit (P
0.05). Quantitative data are expressed as the mean ± SD of three to six independent experiments.