Characterization of novel elongated Parvulin isoforms that are ubiquitously expressed in human tissues and originate from alternative transcription initiation-4

<p><b>Copyright information:</b></p><p>Taken from "Characterization of novel elongated Parvulin isoforms that are ubiquitously expressed in human tissues and originate from alternative transcription initiation"</p><p>BMC Molecular Biology 2006;7():9-9.</p><p>Published online 7 Mar 2006</p><p>PMCID:PMC1420321.</p><p></p>sequence two additional in-frame ATG codons (highlighted in blue). The only other 5' ATG codon is present in the sequence; it is out of frame and followed by a TAA stop codon (both underlined). All in-frame stop codons are marked in red. Only the human sequence displays an extended open reading frame, both as QR and RS isoform (SNPs highlighted in grey)

Characterization of novel elongated Parvulin isoforms that are ubiquitously expressed in human tissues and originate from alternative transcription initiation-2

<p><b>Copyright information:</b></p><p>Taken from "Characterization of novel elongated Parvulin isoforms that are ubiquitously expressed in human tissues and originate from alternative transcription initiation"</p><p>BMC Molecular Biology 2006;7():9-9.</p><p>Published online 7 Mar 2006</p><p>PMCID:PMC1420321.</p><p></p>. Reaction products were separated by 17.5% SDS-PAGE following autoradiography. . Equal amounts of 35S-methionine labeled Par17-QR, Par17-RS and Par14 were incubated with increasing concentrations of proteinaseK (PK) for 30 min at 10°C. Enzyme concentrations are given in μg/ml. Reactions were stopped by an excess of PMSF, separated by SDS-PAGE and subjected to autoradiography. . Western blot of HepG2 (lane 1 and 3) and HeLa (lane 2 and 4) cell lysates. Proteins were separated by SDS-PAGE with MES as running buffer and SeeBlue2 as protein standard, transferred to nitrocellulose membranes. Blots were incubated with pre-immune (lane 1 and 2) or anti-Par17 serum (lane 3 and 4; both at 500-fold dilution). . Coding sequences for Par17-RS and -QR were subcloned in the pET-28 vector with N-terminal His6 tag and expressed in . Lysates were subjected to reducing SDS-PAGE in MES buffer and SeeBlue2 as protein standard, transferred to nitrocellulose membranes and incubated with the anti-Par17 antibody. -, before IPTG induction; RS, Par17-RS lysates; QR, Par17-QR lysates. Coomassie stained gel of lysates to show equal loading. Par17 fusions with His6 tag and thrombin cleavage sites show apparent molecular weights of about 22 KDa in SDS-PAGE. The induction band in E. coli lysates and the corresponding band recognized by Ab-EXT are labeled with arrows. The lower migrating band may be caused by proteolytic degradation. . Par14 coding sequence was expressed as GFP fusion in HeLa cells (lane 3). Lane 1 and 2 are HeLa cell lysates not transfected with this construct. Lysates were subjected to reducing SDS-PAGE in Tris-glycine buffer with MagicMark as protein standard, transferred to nitrocellulose membranes and incubated with anti-Par17 and anti-GFP antibodies. Coomassie stained gel of lysates to show equal loading

Characterization of novel elongated Parvulin isoforms that are ubiquitously expressed in human tissues and originate from alternative transcription initiation-0

<p><b>Copyright information:</b></p><p>Taken from "Characterization of novel elongated Parvulin isoforms that are ubiquitously expressed in human tissues and originate from alternative transcription initiation"</p><p>BMC Molecular Biology 2006;7():9-9.</p><p>Published online 7 Mar 2006</p><p>PMCID:PMC1420321.</p><p></p>n mRNA contains a 92 bp extension at the 5' side depicted in white. Primers used for RT-PCR are indicated by arrows. Primers 246 and 247 are complementary to the sequences around the two start ATG codons, 248 binds at the sequence around the TAA stop codon. . RT-PCR products with mRNA from liver, kidney and Caco-2 cells after 30 amplification cycles. Primers 246 and 248 yield a 488 bp PCR product only on those Par14 mRNAs with 5' extension. Primers 247 and 248 give rise to a 385 bp DNA fragment with all Par14 mRNAs. All longer RT-PCR products were eluted from the gel and sequenced. . Two start ATG codons are indicated in bold capital letters. The sequence of Parvulin common to both originally described cDNAs by Uchida . [GenBank:] [3] and Rulten . [GenBank:] [9] begins at the caa codon depicted in bold. The peptide sequence used for antibody production is shaded in grey. Two SNPs are shown leading to amino acid substitutions Q16R and R18S

Instructed fear learning, extinction, and recall: additive effects of cognitive information on emotional learning of fear

<p>The effects of instruction on learning of fear and safety are rarely studied. We aimed to examine the effects of cognitive information and experience on fear learning. Fourty healthy participants, randomly assigned to three groups, went through fear conditioning, extinction learning, and extinction recall with two conditioned stimuli (CS+). Information was presented about the presence or absence of conditioned stimulus–unconditioned stimulus (CS–US) contingency at different stages of the experiment. Information about the CS–US contingency prior to fear conditioning enhanced fear response and reduced extinction recall. Information about the absence of CS–US contingency promoted extinction learning and recall, while omission of this information prior to recall resulted in fear renewal. These findings indicate that contingency information can facilitate fear expression during fear learning, and can facilitate extinction learning and recall. Information seems to function as an element of the larger context in which conditioning occurs.</p

LRRK2 constructs used in this study.

<p>ARM: armadillo repeats; ANK: ankyrin repeats; LRR: leucine rich repeats; ROC: Ras of complex; COR: C-terminal of ROC; Kin: kinase domain; WD40: WD40 domain; c/c: coiled coil motif (aa 319–348).</p

Co-localization analysis of LRRK2-GFP and DsRed-Monomer-Rab32.

<p>(A) NIH3T3 cells were co-transfected with plasmids encoding DsRed-Monomer-Rab32 wt and LRRK2-GFP. Living cells were imaged using a Zeiss LSM5 live microscope. The image shows a still frame from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111632#pone.0111632.s008" target="_blank">Movie S1</a>. Scale bar = 10 µm. (B) NIH3T3 cells were co-transfected with plasmids encoding DsRed-Monomer-Rab32 Q85L and LRRK2-GFP. After 48 hours the cells were fixed in 4% PFA and subsequently analyzed with a laser scanning microscope. Scale bar = 10 µm. (C) Co-transport and sorting of LRRK2-GFP by DsRed-Monomer-Rab32 wt. Image series are a detail view from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111632#pone.0111632.s009" target="_blank">Movie S2</a>. Both channels were recorded simultaneously every second. Scale bar = 2 µm. (D and E) NIH3T3 cells expressing either DsRed-Monomer-Rab32 wt or LRRK2-GFP were fixed and subsequently subjected to immunofluorescence labeling of β-tubulin (D) or the Golgi marker Rab6A (E). Scale bar = 10 µm.</p

Mapping of Rab32 binding motif within LRRK2 aminoterminus.

<p>After co-transformation of the yeast strain Y190 with the indicated plasmids with and without the hypothetical coiled-coil motif, cells were grown on synthetic media lacking histidine, supplemented with 30 mM 3 AT. Cell growth on these media and blue staining in subsequent β-gal-filter assays indicated direct interaction of the proteins.</p><p>no growth on selection media or staining in ß-galactosidase filter assay, + growth on selection media and blue staining in in ß-galactosidase filter assay. n = 3 independent experiments.</p><p>Mapping of Rab32 binding motif within LRRK2 aminoterminus.</p

Co-localization analysis of Rab32 wt and the constitutively active mutant Rab32 Q85L with different endosomal markers.

<p>(A) NIH3T3 cells were co-transfected with plasmids encoding the recycling endosome marker GFP-Rab11B and DsRed-Monomer-Rab32 wt or DsRed-Monomer-Rab32 Q85L, fixed and analyzed by fluorescence microscopy. Scale bar = 10 µm. (B) Cells were transfected with plasmids encoding for GFP-Rab32 wt or GFP-Rab32 Q85L followed by fixation and subsequent immunofluorescence staining of Rab7. Scale bar = 10 µm. (C–E) NIH3T3 cells expressing either GFP-Rab32 wt or GFP-Rab32 Q85L were fixed and stained for Rab7. (C) Microscopic analysis of GFP-Rab32 Q85L that co-localized with endogenous Rab7 (arrows) in the perinuclear area. Non co-localizing Rab7 was indicated by arrowheads. The perinuclear area was defined by the red (outlines nucleus) and the yellow line (outer border for perinuclear area). The image illustrates the cellular area used for the following analysis. (D) Rab7 perinuclear aggregates co-localizing with GFP-Rab32 wt or GFP-Rab32 Q85L and non co-localizing ones. ctrl. = control (untransfected IHKE-1 cells) (E) Quantification of perinuclear Rab7-positive structures. ctrl. (control): perinuclear Rab7 in untransfected cells; wt/Q85L: perinuclear Rab7 co-localizing with GFP-Rab32 constructs. control/Rab32 wt/Rab32 Q85L: n = 1660/18/167 structures in 67/18/60 cells, 1/1/3 independent experiments. Statistical significance was tested by a Student's T-test: p_control-wt = 0.02; p_control-Q85L<0.005; p_wt-Q85L = 0.02. Scale bar = 10 µm.</p

Binding of endogenous LRRK2 by the small GTPase Rab32.

<p>(A) GST-Rab32 wt, GST-Rab32 Q85L or GST as control was applied to glutathione agarose beads followed by incubation with NIH3T3 lysate overnight. Samples were analyzed by 6% SDS-PAGE and subsequent Western blot analysis to detect LRRK2. n≥3 independent experiments. (B) Lysates from IHKE-1 cells stably expressing GFP-Rab32 wt were incubated overnight with an anti-LRRK2 antibody (1E11). IP control = no antibody was added. Co-precipitated GFP-Rab32 wt was detected using an anti-Rab32 antibody. n = 3 independent experiments. (C) Lysates from IHKE-1 cells expressing GFP-Rab32 wt, GFP-Rab32 Q85L or GFP as control were subjected to immunoprecipitation by the GFP-Trap kit. Co-precipitated endogenous LRRK2 was detected using an anti LRRK2 antibody. n = 2 independent experiments.</p