Journal: The Journal of Biological Chemistry

Article Title: Identification of RNF8 as a Ubiquitin Ligase Involved in Targeting the p12 Subunit of DNA Polymerase ? for Degradation in Response to DNA Damage * ^♦

doi: 10.1074/jbc.M112.423392

Figure Lengend Snippet: Effects of RNF8 depletion on p12 degradation by UV. A, four different shRNAs (shR1–shR4) and a control shRNA (C) were used to produce respective stable A549 cells (see ”Experimental Procedures“). Each sample was treated in duplicate with UVC (20 J/m2) and Western blotted for RNF8 and p12 after 4 h. GAPDH was used as a loading control. B, UV dose dependence of p12 degradation in RNF8-depleted A549 cells; shR2 was used in these and the following experiments. Cell cultures were treated with UVC (0, 6, 12, 18, and 24 J/m2) and analyzed 4 h later by Western blotting for p12, Chk1-p, γH2AX, and β-actin (loading control). C, the blots for p12 were quantified by densitometry and normalized against the loading control. The relative levels of p12 were then plotted against UVC dose. D, comparison of p12 levels in unperturbed RNF8 knockdown cells. The two left lanes show p12 levels in control shRNA-treated cells in the absence and presence of the proteasome inhibitor MG132 (10 μm), which was added 30 min prior to analysis. The two right lanes with the three rows of numbers show p12 levels in six individual shR2 RNF8 clonal isolates without MG132.

Article Snippet: HuSH 29-mer shRNA constructs against human RNF8 and control shRNA (noneffective shGFP) were synthesized by OriGene Technologies, Inc. (Rockville, MD).

Techniques: shRNA, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Identification of RNF8 as a Ubiquitin Ligase Involved in Targeting the p12 Subunit of DNA Polymerase ? for Degradation in Response to DNA Damage * ^♦

doi: 10.1074/jbc.M112.423392

Figure Lengend Snippet: Determination of the half-lives of p12 in control RNF8 knockdown cells. A, control and shR2 RNF8 cells were transferred to fresh medium containing 10 μg/ml cycloheximide (CHX). Cells were harvested at the indicated times (0–12 h) after treatment and analyzed by Western blotting for p12. B, control and shR2 RNF8 cells were treated with UVC (10 J/m2) and transferred to fresh medium containing 10 μg/ml cycloheximide. Cells were harvested at the indicated times (0–6 h) after treatment and analyzed by Western blotting for p12. β-Actin was used as the loading control. C, the relative amounts of p12 for the Western blots shown in A and B were determined by densitometry and were plotted on a semi-log plot against time. Data for control shRNA in the presence of cycloheximide or cycloheximide + UV are shown as open or closed squares, respectively; data for RNF8-shRNA cells in the presence of cycloheximide or cycloheximide + UV are shown as open or closed circles, respectively.

Article Snippet: HuSH 29-mer shRNA constructs against human RNF8 and control shRNA (noneffective shGFP) were synthesized by OriGene Technologies, Inc. (Rockville, MD).

Techniques: Western Blot, shRNA

Journal: PLoS Pathogens

Article Title: CD14 Signaling Restrains Chronic Inflammation through Induction of p38-MAPK/SOCS-Dependent Tolerance

doi: 10.1371/journal.ppat.1000687

Figure Lengend Snippet: A) MΦ isolated from CD14 +/+ and CD14 −/− mice were incubated with B. burgdorferi at a MOI of 10 for 24 h and TNF-α levels in culture supernatant were measured by CBA. B) Lentiviral transduction was used to knock down CD14 in MΦ as determined by a reduction in mean fluorescent intensity (MFI) and by Western blot analysis (inset). C) The lentivirus-treated MΦ were incubated with B. burgdorferi for 24 h and TNF-α levels were measured as for (A). D) MΦ isolated from CD14 +/+ and CD14 −/− mice were incubated with B. burgdorferi for 3, 6 and 24 h. Total RNA was isolated and used to perform qPCR for simultaneous interrogation of 84 genes associated with TLR signaling. The results presented are the ratio of fold change in HPRT-normalized gene activity in CD14 −/− versus CD14 +/+ MΦ and error bars represent SEM calculated on the basis of three independent experiments. E) Total RNA isolated from CD14 +/+ and CD14 −/− MΦ incubated with B. burgdorferi was analyzed by qPCR for socs1 , socs3 and cis . Results are presented as fold change over respective mock-infected control and were normalized with 18S rRNA transcript. F) Equivalent protein from lysed MΦ were separated by 12% SDS-PAGE, transferred to a PVDF membrane and probed with antibodies directed against SOCS1, SOCS3, CIS or β-actin. G) Total RNA isolated from CD14 +/+ and CD14 −/− MΦ incubated with B. burgdorferi was analyzed by qPCR for irf1 , irf7 and stat1 . Dotted lines denote the 2-fold over mock change considered significant for the purposes of interpretation. H) Equivalent protein from lysed MΦ were subjected to Western blot analysis using antibodies directed against phospho-specific STAT1 or STAT3, or β-actin. Results represent mean±SEM from three to four independent experiments. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Knock down of CD14 was achieved using a pRS-shGFP lentiviral expression vector (OriGene Technologies, Rockville, MD) to transduce either a CD14 shRNA or a scrambled control shRNA into CD14 +/+ MΦ.

Techniques: Isolation, Incubation, Transduction, Western Blot, Activity Assay, Infection, SDS Page

Journal: PLoS Pathogens

Article Title: CD14 Signaling Restrains Chronic Inflammation through Induction of p38-MAPK/SOCS-Dependent Tolerance

doi: 10.1371/journal.ppat.1000687

Figure Lengend Snippet: A) Equal protein from lysates of CD14 +/+ and CD14 −/− MΦ incubated with B. burgdorferi were separated by 10% SDS-PAGE, transferred to a nitrocellulose membrane and probed with phospho-p38 and β-actin antibodies. B) CD14 was knocked down using lentiviral transduction as in . MΦ were incubated with B. burgdorferi for 30 min and phospho-p38 and β-actin were detected as in . C) CD14 +/+ MΦ were treated with DMSO, SB202190 (0.5 µM) or arctigenin (1 µM) for 30 min prior to incubation with B. burgdorferi and total RNA was analyzed by qPCR for inos and socs3 . D) CD14 +/+ MΦ were treated as described in (C), and culture supernatants were analyzed for TNF-α by CBA. E) CD14 +/+ MΦ were treated with DMSO or increasing concentrations of arctigenin or SB202190 for 30 min prior to incubation with B. burgdorferi . Cell culture supernatants were collected 24 h p.i. and TNF-α was measured by CBA. F) CD14 +/+ MΦ were treated with DMSO or increasing concentrations of SB203580 for 30 min prior to incubation with live B. burgdorferi or B. burgdorferi lysate (10µg/ml). Cell culture supernatants were assayed for TNF-α by CBA. Results represent mean±SEM from two to five independent experiments. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Knock down of CD14 was achieved using a pRS-shGFP lentiviral expression vector (OriGene Technologies, Rockville, MD) to transduce either a CD14 shRNA or a scrambled control shRNA into CD14 +/+ MΦ.

Techniques: Incubation, SDS Page, Transduction, Cell Culture