Journal: PLoS ONE

Article Title: Identification of the ER-resident E3 ubiquitin ligase RNF145 as a novel LXR-regulated gene

doi: 10.1371/journal.pone.0172721

Figure Lengend Snippet: ( A ) Expression of Rnf145 was evaluated in the indicated mouse tissues by qPCR. Bars indicate mean ± SD (n = 3) ( B , C ) The indicated ( B ) human and ( C ) mouse cell lines and primary macrophages were cultured in lipoprotein-depletion medium for 16 hours and subsequently treated for 6 hours with 1μM GW3965 (GW). ( D ) THP1 cells were cultured in sterol depletion medium for 16 hrs and then treated with 1μM GW3965 (GW) and 100nM LG100268 (LG) for 6 hrs as indicated. Subsequently, ABCA1 and RNF145 expression was determined by qPCR and each bar and error represents the mean fold-change of ligand-treated cells over sterol-depleted cells ± SD (n≥3).

Article Snippet: Membranes were probed with the following antibodies: LDLR (Abcam, clone EP1553Y, 1:4000), tubulin (Sigma, clone DM1A, ascites fluid, 1:5000), ABCA1 (Novus Biologicals, NB400-105, 1:1000), FLAG (Sigma, clone M2, 1:1000), GFP (Santa Cruz sc-9996, 1:500), Myc (Santa Cruz 9E10, 1:3000), HA (Covance, clone 16B12, ascites fluid, 1:6000), HMGCR (rabbit polyserum was a kind gift from Dr. Peter Edwards, UCLA), SREBP2 (BD Biosciences, clone 1C6, 1:1000), SREBP1 (ThermoFisher, clone 2A4, 1:1000), actin (Merck Millipore, clone C4, 1:5000), V5 (Invitrogen, 46–0705, 1:3000), RNF145 (Abgent AP18281b, 1:8000), and ubiquitin (Enzo life Sciences, clone FK2, 1:1000).

Techniques: Expressing, Cell Culture

Journal: PLoS ONE

Article Title: Identification of the ER-resident E3 ubiquitin ligase RNF145 as a novel LXR-regulated gene

doi: 10.1371/journal.pone.0172721

Figure Lengend Snippet: ( A ) RAW264.7 macrophages were treated with 5μg/mL Actinomycin D (ActD) for the indicated time and expression of Abca1 and Rnf145 was determined by qPCR and plotted as mean ± SD relative to untreated cells (n = 3), ( B ) HepG2 and RAW264.7 cells were treated with 1μM GW3965 for 6 hours in the presence or absence of 5μg/ml actinomycinD for 4 hours, after which expression of the indicated genes was measured by qPCR. Bars indicate mean ± SD (n = 3) ( C , D ) THP1 macrophages were cultured in sterol-depletion medium for 16 hrs and then treated with ( C ) 1μM GW3965 (GW) for the indicated time, or ( D ) with the indicated concentration of GW3965 for 4 hrs. Subsequently, gene expression was evaluated qPCR and bars indicate mean ± SD (n = 3)

Article Snippet: Membranes were probed with the following antibodies: LDLR (Abcam, clone EP1553Y, 1:4000), tubulin (Sigma, clone DM1A, ascites fluid, 1:5000), ABCA1 (Novus Biologicals, NB400-105, 1:1000), FLAG (Sigma, clone M2, 1:1000), GFP (Santa Cruz sc-9996, 1:500), Myc (Santa Cruz 9E10, 1:3000), HA (Covance, clone 16B12, ascites fluid, 1:6000), HMGCR (rabbit polyserum was a kind gift from Dr. Peter Edwards, UCLA), SREBP2 (BD Biosciences, clone 1C6, 1:1000), SREBP1 (ThermoFisher, clone 2A4, 1:1000), actin (Merck Millipore, clone C4, 1:5000), V5 (Invitrogen, 46–0705, 1:3000), RNF145 (Abgent AP18281b, 1:8000), and ubiquitin (Enzo life Sciences, clone FK2, 1:1000).

Techniques: Expressing, Cell Culture, Concentration Assay, Gene Expression

Journal: PLoS ONE

Article Title: Identification of the ER-resident E3 ubiquitin ligase RNF145 as a novel LXR-regulated gene

doi: 10.1371/journal.pone.0172721

Figure Lengend Snippet: ( A ) LXR ChIP-seq experiments in human THP1 cells (GSE28319) and RAW macrophage-like cells (GSE50944) were analyzed and used to identify active LXREs within the Rnf145/Rnf145 loci, as graphically illustrated. ( B , C ) Genomic location of the identified LXREs. In bold, nucleotides that were mutated to disrupt LXR binding ( C ) A 1kb genomic region upstream of the transcriptional start site of hRNF145 was cloned into a pGL3basic. The putative LXRE was also mutated as indicated above. The empty, RNF145 WT , RNF145 MUT , and ABCA1 reporter plasmids were co-transfected with or without RXRα and LXRα expression plasmids in HEK 293T cells. 24 hours post-transfection the cells were treated with 1μM GW3965 (LXR) and 100nM LG100268 (RXR) for 24 hours and measured for luciferase signal (n≥3). ( D ) Cells were transfected with an empty or a tandem LXRE-containing pGL2 as in C . In all luciferase experiments the transfection efficiency was normalized to co-transfected Renilla luciferase. Bars report normalized chemiluminescence relative to untreated control ± SD (n = 3).

Article Snippet: Membranes were probed with the following antibodies: LDLR (Abcam, clone EP1553Y, 1:4000), tubulin (Sigma, clone DM1A, ascites fluid, 1:5000), ABCA1 (Novus Biologicals, NB400-105, 1:1000), FLAG (Sigma, clone M2, 1:1000), GFP (Santa Cruz sc-9996, 1:500), Myc (Santa Cruz 9E10, 1:3000), HA (Covance, clone 16B12, ascites fluid, 1:6000), HMGCR (rabbit polyserum was a kind gift from Dr. Peter Edwards, UCLA), SREBP2 (BD Biosciences, clone 1C6, 1:1000), SREBP1 (ThermoFisher, clone 2A4, 1:1000), actin (Merck Millipore, clone C4, 1:5000), V5 (Invitrogen, 46–0705, 1:3000), RNF145 (Abgent AP18281b, 1:8000), and ubiquitin (Enzo life Sciences, clone FK2, 1:1000).

Techniques: ChIP-sequencing, Binding Assay, Clone Assay, Transfection, Expressing, Luciferase, Control

Journal: eLife

Article Title: Patient-specific mutations impair BESTROPHIN1’s essential role in mediating Ca 2+ -dependent Cl - currents in human RPE

doi: 10.7554/eLife.29914

Figure Lengend Snippet:

Article Snippet: antibody , RPE65 , Novus Biologicals NB100-355 , RRID: AB_10002148 , 1:1000.

Techniques: Transfection, Construct, Mutagenesis, Generated, Recombinant, Virus, Produced, Sequencing, Purification, Size-exclusion Chromatography, Clone Assay, Software

Journal: bioRxiv

Article Title: Molecular architecture and domain arrangement of the placental malaria protein VAR2CSA suggests a model for receptor binding

doi: 10.1101/2020.04.16.045096

Figure Lengend Snippet: VAR2CSA and its deletion constructs expressed in HEK293-F cells and SDS-PAGE of the purified recombinant VAR2CSA proteins. (A) Schematic indicating the locations of DBL domains for constructs I-V. For each construct, the numbers at either end denote the beginning and ending amino acid sequence numbers. I, II and IV contain a non-cleavable C-terminal cMyc tag and hexahistidine tag, as coded in the commercial vector. For the III and V constructs, the C-terminal tag is replaced with a TEV-cleavable 3X-FLAG tag and a hexahistidine tag, as defined in Table S2, GBLOCK2. In addition, P1 contains a glycine and threonine residue inserted between native residues 58 and 59 as a cloning artefact. (B) Purified proteins (each 2 μg/lane), electrophoresed using 4-20 % gradient gels (1 mm thick) under reducing conditions and (C) non-reducing conditions are shown. In each gel, the lanes are: M, Molecular weight markers; I, NTS-DBL6ε (~310 kDa); II, DBL1x-ID2a (~125 kDa); III, ID2b-DBL6ε (~200 kDa); IV, DBL3x-DBL6ε (~180 kDa); V, DBL4ε-DBL6ε (~130 kDa). The molecular mass marker (kDa) standards are indicated in the left margin.

Article Snippet: cMyc mAb (Cat NB600-302, Novus Biologicals, CO) and NTS-DBL6ε or DBL1x-ID2a proteins were mixed at a ratio of 1:2 and incubated for 30 min on ice.

Techniques: Construct, SDS Page, Purification, Recombinant, Sequencing, Plasmid Preparation, FLAG-tag, Residue, Cloning, Molecular Weight, Marker

Journal: bioRxiv

Article Title: Molecular architecture and domain arrangement of the placental malaria protein VAR2CSA suggests a model for receptor binding

doi: 10.1101/2020.04.16.045096

Figure Lengend Snippet: VAR2CSA is an asymmetric molecule with the C-terminus located within the smaller lobe. (A) Micrograph of NTS-DBL6ε. The white bar is 50 nm. (B) Subset of 2D classes obtained during initial 2D classification of NTS-DBL6ε. (C) Single 2D class showing the layers (L1, L2, L3), separated by broken white lines, defined in the text. (D) Micrograph of NTS-DBL6ε bound to anti-cMyc antibody. (E) Representative 2D classes of unbound NTS-DBL6ε. (F) Representative 2D classes of unbound anti-cMyc antibody. (G) Representative 2D classes of NTS-DBL6ε:anti-cMyc-antibody complex. The letter A indicates the location of the cMyc antibody at the tip of the smaller lobe of NTS-DBL6ε.

Article Snippet: cMyc mAb (Cat NB600-302, Novus Biologicals, CO) and NTS-DBL6ε or DBL1x-ID2a proteins were mixed at a ratio of 1:2 and incubated for 30 min on ice.

Techniques:

Journal: The Journal of Pathology

Article Title: Hypoxia‐inducible factor 1‐alpha does not regulate osteoclastogenesis but enhances bone resorption activity via prolyl‐4‐hydroxylase 2

doi: 10.1002/path.4906

Figure Lengend Snippet: HIF induction during osteoclast differentiation. (A) Western blot analysis of HIF‐1α protein expression in CD14+ monocytes treated with 25 ng/ml M‐CSF for 2–24 h versus the untreated normoxic control (Nx). Hx = hypoxia (2% O 2 , 24 h). (B) RT‐qPCR comparing HIF1A , HIF2A, LDHA , and SLC2A1 ( GLUT1 ) mRNA on days 3, 5, 7 and 9 of osteoclast differentiation. * p < 0.05; ** p < 0.01. (C) Western blot analysis of HIF‐1α, GLUT1, and LDHA protein on days 3, 5, and 9 of osteoclast differentiation. (D) Normalized HRE‐driven luciferase reporter luminescence on days 3, 5, 7, and 9 of osteoclast differentiation. * p < 0.05; ** p < 0.01.

Article Snippet: Primary antibodies were against HIF‐1α (clone 54, 1:1000; BD Biosciences, Oxford, UK), GLUT1 (ab14683, 1:2500; Abcam, Cambridge, UK), LDHA (NBP1‐48336, 1:2000; Novus Biologicals, Cambridge, UK), and β‐tubulin (clone TUB2.1, 1:2500; Sigma‐Aldrich, Dorset, UK).

Techniques: Western Blot, Expressing, Control, Quantitative RT-PCR, Luciferase