Journal: The Journal of Biological Chemistry

Article Title: Nascent RNA Cleavage by Arrested RNA Polymerase II Does Not Require Upstream Translocation of the Elongation Complex on DNA *

doi:

Figure Lengend Snippet: A, time course of SII-dependent elongation from site Ia in the presence of all four nucleotides. Washed complexes (lane Ia) were split into 2 aliquots. One received bovine brain SII and 7 mM MgCl2 and was incubated at 28 °C for 1.5 or 15 min to generate the first (*) and second (**) cleavage intermediates, respectively. The second aliquot of washed complexes received bovine brain SII, MgCl2, and 800 μM each of all four NTPs. Portions of this reaction were stopped after the indicated times at 28 °C and analyzed by electrophoresis with the first and second cleavage intermediates. RO, runoff RNA. B, RNA elongation by an SII-independent elongation complex in the presence of SII. RNA in washed complexes was extended for 10 min at 28 °C to positions G218/G220 (indicated by dash at left, lane 0) in the presence of UTP, CTP, and GTP (800 μM each), bovine brain SII, and 7 mM MgCl2. The reaction was chilled to 4 °C, ATP (800 μM) was added, and samples were stopped at the indicated times after incubation at 28 °C. One sample (sar) was adjusted to 0.25% in Sarkosyl and another (α) to 1 μg/ml in α-amanitin before the addition of ATP and incubation at 28 °C. Arrowheads indicate the position of marker RNAs of 260, 380, 420, and 540 nucleotides (bottom to top). C, RNA elongation by a second SII-independent elongation complex in the presence of SII. Elongation complexes were assembled at site Ia (Ia) and moved to positions G218/G220 (dash to left of figure) as described in the legend to B. These complexes were washed free of nucleotides by centrifugation and resuspension and moved to position C230 (U) after an 8-min incubation at 28 °C in the presence of bovine brain SII, 7 mM MgCl2, and 800 μM each of ATP, GTP, and CTP. The reaction was incubated at 28 °C with UTP (800 μM) for the indicated times. One sample (sar) was made 0.25% in Sarkosyl before the addition of UTP and incubation at 28 °C.

Article Snippet: Fast protein liquid chromatography-purified NTPs, 4 ddNTPs, and 3′- O -methyl GTP were purchased from Pharmacia LKB Biotechnology Inc., 3′-dUTP was purchased from Boehringer Mannheim.

Techniques: Incubation, Electrophoresis, Marker, Centrifugation

Journal: The Journal of Biological Chemistry

Article Title: Decreased Expression of ARV1 Results in Cholesterol Retention in the Endoplasmic Reticulum and Abnormal Bile Acid Metabolism *

doi: 10.1074/jbc.M110.165761

Figure Lengend Snippet: Knockdown of hepatic ARV1 affects plasma lipid homeostasis. A and B, FPLC analysis of the plasma lipoproteins of pooled plasma samples derived from four to six mice injected with ASOs biweekly for 1 week (A) or 2 weeks (B). C, measurement of TG secretion rates in mice treated with control ASO or ARV1 ASO. Wild type mice treated with control ASO or ARV1 ASO for a week and mice were injected intraperitoneally with 400 μl of P-407 (1 mg/g) solution at zero time point. Rate of TG production was measured over varying time periods in P-407-treated mice. Two to three separate identical experiments were performed with four to six mice/group per treatment in all experimental groups. Values are expressed as means ± S.E. (error bars) of the mice in the representative experiment. Asterisks denote statistical significant differences from control for ASO-treated animals with p < 0.05 (one asterisk) and p < 0.001 (two asterisks) by unpaired t test. D, hepatic LDLR mRNA levels of mice injected with either control ASO or ARV1 ASO over the time course of 2 weeks.

Article Snippet: Pooled plasma samples (120 μl) from four to six mice were subjected to fast protein liquid chromatography (FPLC) gel filtration (Pharmacia LKB Biotechnology) on two Superose 6 columns at a flow rate of 0.5 ml/min (one run/sample), and fractions of 500 μl each were collected ( 12 ).

Techniques: Knockdown, Clinical Proteomics, Derivative Assay, Injection, Control