Journal: Advances in biological chemistry

Article Title: Rad7 E3 Ubiquitin Ligase Attenuates Polyubiquitylation of Rpn10 and Dsk2 Following DNA Damage in Saccharomyces cerevisiae

doi: 10.4236/abc.2015.57021

Figure Lengend Snippet: (a) Merged RFP and DAPI fluorescent images are shown (left) of a Ub-RFP cell 15 min following induction with UV-C (20 J/m 2 ) followed by images containing only RFP and DAPI channels (middle) and a differential interference contrast (DIC) image (right). Bright cytosolic RFP foci are visible throughout the cell (green arrow indicates one focus). A nuclear-localized RFP focus is indicated (orange arrow); (b) Same as (a) except cell shown does not contain a nuclear-localized RFP focus. Bright cytosolic RFP foci are visible throughout the cell (green arrow indicates one focus); (c) Immunoblot of WCEs from the indicated yeast strains probed with an antibody to ubiquitin (Ub). Lower panel shows the same blot reprobed with an antibody to β-Actin. Lane 1: MagicMark ™ protein standard, lane 2: wild-type (W1588-4C), lane 3 Ub-RFP PF038-1D); (d) The Ub-RFP Rad10-YFP strain (PF038-1D) was incubated with BPDE (10 μM, 1 h) and imaged as a single focal plane. Merged, RFP, YFP and DIC images are shown. A white arrow indicates a nuclear-localized RFP focus; (e) Same as panel (c), except the strain was Ub-RFP Rad14-CFP (PF040-3A) and the DNA damaging agent was AAAF. A nuclear-localized RFP focus is indicated with an orange arrow. In both (d) and (e), green arrows indicate examples of RFP foci that are not nuclear or colocalized; (f) Graph of nuclear Ub-RFP foci counts from 11-slice Z-stacks (rather than single focal plane images) acquired during experiments described/depicted in panel (d). Data are shown for Ub-RFP Rad10-YFP strain (PF038-1D) samples induced with BPDE (10 μM, 1 h) or controls mock-induced with tetrahydrofuran (THF). Error bars represent standard error.

Article Snippet: Functional Ub-RFP was confirmed by immunoblotting of yeast WCEs using standard methods with a ubiquitin antibody (Ubiquitin (P4D1) Mouse mAb, Cat No 3936, Cell Signalling, Danvers, MA) and α - β -actin (Abcam ab8224, Cambridge, MA).

Techniques: Western Blot, Ubiquitin Proteomics, Incubation

Journal: Advances in biological chemistry

Article Title: Rad7 E3 Ubiquitin Ligase Attenuates Polyubiquitylation of Rpn10 and Dsk2 Following DNA Damage in Saccharomyces cerevisiae

doi: 10.4236/abc.2015.57021

Figure Lengend Snippet: Increased levels of ubiquitylated proteins are observed in rad7SOCS cells following UV and increased monoubiquitylated Rpn11 is observed in rad7SOCS without damage. (a) Cells from strains PF038-1D, (“wild-type”, lanes 1 and 2), PF084-7A, (“ rad7SOCS ”, lanes 3 and 4) and MGSC104, (“ rad7Δ ”, lanes 5 and 6), were cell cycle arrested at the G2/M boundary, released briefly back into cell cycle, treated with 100 J/m 2 UV-C (lanes 2, 4 or 6, “+”) for 3 minutes or mock treated (lanes 1, 3 or 5, “-”) and disrupted. Normalized quantities of protein from WCEs were analyzed on a NuPAGE Novex 4% – 12% Bis-Tris gel with MES SDS running buffer SDS-PAGE and immunoblotted with antibodies to Ub (upper panel) or GAPDH (lower panel) as a loading control. “M” indicates MagicMark ™ molecular weight marker. (b) Same as upper panel of (a) except that extracts were analyzed on a NuPAGE Novex 3% – 8% Tris-Acetate Gel with Tris-Acetate running buffer. Arrows indicate ubiquitin; brackets indicate putative ubiquitin-conjugates. (c) The Rpn11-TAP strain (YFR004W) and an isogenic Rpn11-TAP/rad7SOCS derivative (PF168) were transformed with YEplac195 CUP1::His7-Ub, cell cycle arrested, released briefly back into cell cycle and disrupted. Denatured protein extracts were subjected to Ni-NTA affinity capture experiments. Lane 1, MagicMark ™ molecular weight marker; lane 2, 10% input; lane 3, affinity capture in which Ni-NTA beads were omitted; lane 4, affinity capture from Rpn11-TAP , no UV; lane 5 affinity capture from Rpn11-TAP with UV; lane 6 affinity capture from Rpn11-TAP / rad7SOCS , no UV; lane 7 affinity capture from Rpn11-TAP with UV.

Article Snippet: Functional Ub-RFP was confirmed by immunoblotting of yeast WCEs using standard methods with a ubiquitin antibody (Ubiquitin (P4D1) Mouse mAb, Cat No 3936, Cell Signalling, Danvers, MA) and α - β -actin (Abcam ab8224, Cambridge, MA).

Techniques: SDS Page, Control, Molecular Weight, Marker, Ubiquitin Proteomics, Transformation Assay