Journal: Histopathology

Article Title: Differential expression of the semaphorin 3A pathway in prostatic cancer.

doi: 10.1111/j.1365-2559.2009.03406.x

Figure Lengend Snippet: Figure 1. Neuropilin 1 (NRP1) expression in the prostatic cancer cell line PC3 (A). Vascular endothelial growth factor is expressed in the cytoplasm of prostatic cancer cells (B). In clinically localized prostatic cancer, semaphorin 3A (Sema3A) (C) and NRP1 (D) expression is strongly associated. In contrast, most hormone-refractory prostatic cancers are characterized by absence of Sema3A immunoreactivity (E) and diffuse NRP1 expression at the membrane of cancer cells (F).

Article Snippet: For each case, three cores (0.6 mm diameter) of tumour and three cores of normal prostate (when available from the transurethral resection) were transferred from the selected areas to the recipient block. immunohistochemistry Immunohistochemistry was performed on tissue sections from the TMA blocks, using the following antibodies: Sema3A (R&D Systems, Minneapolis, MN, USA; dilution 1:200), NRP1 (Invitrogen, San Francisco, CA, USA; dilution 1:200) and VEGF (Novus Biologicals, Littleton, CO, USA; clone MM0009-2B86, dilution 1:50).

Techniques: Expressing, Membrane

Journal: Acta biochimica et biophysica Sinica

Article Title: RanBPM is an acetylcholinesterase-interacting protein that translocates into the nucleus during apoptosis.

doi: 10.1093/abbs/gmp082

Figure Lengend Snippet: Figure 1 AChE interacts with RanBPM (A) The self-expression assay of the bait. AH109/pGBKT7-AChEC-terminal cells were cultured on SD/-Trp plate or SD/-Trp/-His plates. (B) Library screening. AH109 clones transfected with pGBKT7-AChEC-terminal and human fetal brain library were cultured on SD/-Ade/-His/-Leu/-Trp/X-a-gal plates. The arrows indicate the positive clones (blue clones). (C) The scheme of the two positive clones (A and C6). The putative SPRY domain (amino acids 212–333) is indicated. (D) Clones A and C6 showed strong lacZ reporter activation. Pos., a clone consisting of BD-p53 and AD-SV40 T-antigen was used as the positive control. Neg., a combination of BD-laminC and AD-SV40 T-antigen was used as the negative control; b-galactosidase activity is expressed as a percentage of the positive control. The data are represented as the mean+ SD of three independent transformations. (E,F) HEK293T cells transfected with the indicated expression plasmid for 24 h; the whole-cell lysates were immunoprecipitated with anti-Myc antibody and analyzed by immunoblotting with anti-HA antibody (top panel). Separate aliquots of the lysates were immunoblotted with anti-HA (middle panel) or anti-Myc (bottom panel) to confirm the expression of HA-RanBPM or Myc-AChEC-terminal (or Myc-AChE), respectively. (G) Kidney tissues from the rat model of ischemia/reperfusion injury were immunoprecipitated using the anti-AChE antibody (E70), and analyzed by immunoblotting with the anti-RanBPM antibody (top panel). Separate aliquots of the lysates were immunoblotted with anti-RanBPM (middle panel) or anti-AChE (E70) (bottom panel) to confirm the expression of RanBPM or AChE, respectively, in the rat kidney tissues.

Article Snippet: The primary antibodies used were as follows: anti-RanBPM antibody (NB100-1281; Novus Biologicals) (1:1000), anti-AChE antibody (the antibody against AChE was prepared in the rabbit [37]) (1:1000), anti-Myc antibody (1:5000), anti-AChE antibody (1:1000), anti-GFP antibody (sc-9996; Santa Cruz) (1:1000), anti-HA antibody (sc-805; Santa Cruz) (1:500), anti-caspase 3 antibody (Cell Signalling, #9662) (1:500), anti-histone 3 antibody (ab1791; ABCAM) (1:3000), anti-tubulin antibody (T6074; Sigma) (1:5000), anti-actin antibody (A5441; Sigma) (1:5000).

Techniques: Expressing, Cell Culture, Library Screening, Clone Assay, Transfection, Activation Assay, Positive Control, Negative Control, Activity Assay, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Acta biochimica et biophysica Sinica

Article Title: RanBPM is an acetylcholinesterase-interacting protein that translocates into the nucleus during apoptosis.

doi: 10.1093/abbs/gmp082

Figure Lengend Snippet: Figure 3 Subcellular distribution of RanBPM and AChE (A,B) Fractionation of HEK293T cell extracts. HEK293T cells were transfected with the indicated expression plasmids. At 24 h after transfection, the cells were fractionated into nuclear (N) and cytoplasmic (C) fractions and analyzed directly by immunoblotting with the anti-HA (first panel) or anti-Myc antibody (second panel). The purity of the nuclear and cytoplasmic fractions was examined by immunoblotting with anti-histone 3 (fourth panel) and anti-a-tubulin antibody (third panel), respectively. (C) Determination of co-localization of AChE and RanBPM by immunofluorescence. HEK293T cells were transfected with Myc-RanBPM and GFP-AChE. At 24 h after transfection, the cells were fixed and incubated with anti-Myc (red), followed by incubation with Rhodamine-conjugated secondary antibodies. The cells were examined by fluorescence microscopy.

Article Snippet: The primary antibodies used were as follows: anti-RanBPM antibody (NB100-1281; Novus Biologicals) (1:1000), anti-AChE antibody (the antibody against AChE was prepared in the rabbit [37]) (1:1000), anti-Myc antibody (1:5000), anti-AChE antibody (1:1000), anti-GFP antibody (sc-9996; Santa Cruz) (1:1000), anti-HA antibody (sc-805; Santa Cruz) (1:500), anti-caspase 3 antibody (Cell Signalling, #9662) (1:500), anti-histone 3 antibody (ab1791; ABCAM) (1:3000), anti-tubulin antibody (T6074; Sigma) (1:5000), anti-actin antibody (A5441; Sigma) (1:5000).

Techniques: Fractionation, Transfection, Expressing, Western Blot, Incubation, Fluorescence, Microscopy

Journal: Acta biochimica et biophysica Sinica

Article Title: RanBPM is an acetylcholinesterase-interacting protein that translocates into the nucleus during apoptosis.

doi: 10.1093/abbs/gmp082

Figure Lengend Snippet: Figure 4 Subcellular distribution of RanBPM and AChE after cisplatin treatment (A) HEK293T cell viability was assessed by MTT after treatment with different concentrations of cisplatin for 24 h. The graph shows the mean+ SE of three independent experiments. (B) The transcription level of p73a gene detected by RT–PCR. HEK293T cells were treated with different concentrations of cisplatin for 24 h and then total mRNA was extracted for RT–PCR. (C) Cisplatin of 100 mM or higher induced DNA fragmentation. (D) The amount of RanBPM and AChE in the nuclear fractions increased after treatment with cisplatin. After cotransfection with pCMV-Myc-RanBPM and pEGFP-C1-AChE for 24 h, HEK293T cells were treated with the indicated concentrations of cisplatin for another 24 h. Then, the cells were fractionated into nuclear (N) and cytoplasmic (C) fractions. The cell fractions and the whole-cell lysates were immunoblotted with anti-Myc (left line) or anti-GFP (light line). The graph shows the densitometric values of the proteins in the cytoplasm versus those in the nuclei.

Article Snippet: The primary antibodies used were as follows: anti-RanBPM antibody (NB100-1281; Novus Biologicals) (1:1000), anti-AChE antibody (the antibody against AChE was prepared in the rabbit [37]) (1:1000), anti-Myc antibody (1:5000), anti-AChE antibody (1:1000), anti-GFP antibody (sc-9996; Santa Cruz) (1:1000), anti-HA antibody (sc-805; Santa Cruz) (1:500), anti-caspase 3 antibody (Cell Signalling, #9662) (1:500), anti-histone 3 antibody (ab1791; ABCAM) (1:3000), anti-tubulin antibody (T6074; Sigma) (1:5000), anti-actin antibody (A5441; Sigma) (1:5000).

Techniques: Reverse Transcription Polymerase Chain Reaction, Cotransfection

Journal: Acta biochimica et biophysica Sinica

Article Title: RanBPM is an acetylcholinesterase-interacting protein that translocates into the nucleus during apoptosis.

doi: 10.1093/abbs/gmp082

Figure Lengend Snippet: Figure 5 Nuclear distribution of endogenous RanBPM and AChE after long-term culture (A) Cleaved caspase 3 was detected in long-term cultured HEK293T cells. HEK293T cells were cultured without medium changed for 3 days (3D) or 7 days (7D), and then the whole-cell lysates were immunoblotted with anti-capspase 3, or anti-b-actin antibody. (B) The amount of endogenous RanBPM and AChE in the nuclear fractions was increased after long-term culture. HEK293T cells were cultured without medium changed for 2 days (2D), 5 days (5D) or 8 days (8D), and then the nuclear fractions were immunoblotted with anti-AChE (first panel) or anti-RanBPM antibody (second panel, NB 100–1281). The purity of the nuclear and cytoplasmic fractions was examined by immunoblotting with anti-a-tubulin (third panel) or anti-histone 3 antibody (fourth panel), respectively.

Article Snippet: The primary antibodies used were as follows: anti-RanBPM antibody (NB100-1281; Novus Biologicals) (1:1000), anti-AChE antibody (the antibody against AChE was prepared in the rabbit [37]) (1:1000), anti-Myc antibody (1:5000), anti-AChE antibody (1:1000), anti-GFP antibody (sc-9996; Santa Cruz) (1:1000), anti-HA antibody (sc-805; Santa Cruz) (1:500), anti-caspase 3 antibody (Cell Signalling, #9662) (1:500), anti-histone 3 antibody (ab1791; ABCAM) (1:3000), anti-tubulin antibody (T6074; Sigma) (1:5000), anti-actin antibody (A5441; Sigma) (1:5000).

Techniques: Cell Culture, Western Blot

Journal: Journal of neurochemistry

Article Title: The mitochondrial intramembrane protease PARL cleaves human Pink1 to regulate Pink1 trafficking.

doi: 10.1111/j.1471-4159.2011.07253.x

Figure Lengend Snippet: Fig. 1 PARL over-expression leads to increased processing of Pink1-66. (a) Schematic representation of human Pink1. The predicted matrix targeting sequence (MTS), the transmembrane domain (TMD), the kinase domain and the putative PARL processing site are indicated. Comparison of the TMDs of human (Hs) and D. melanog- aster (Dm) Pink1 using the EMBOSS pair- wise alignment algorithm reveals significant sequence conservation. The hydrophobicity plot of the relevant region is shown [using the scale of Kyte and Doolittle (1982), with a window size of 7] indicating the potential TMD boundaries. (b) Co-expression of hu- man Pink1 with PARL leads to an increased processing of the full-length 66 kDa form (open triangle). A catalytically inactive PARL mutant (SA) shows no activity. Subcellular fractionation reveals that Pink1-66 and the processed Pink-55 (filled triangle) are found in mitochondria (m) and also the non-mito- chondrial soluble fraction (c). IRES GFP and the cellular markers VDAC, AIF and actin were used as transfection and loading con- trol. t, total cell extract (10% loaded). (c) Quantification of Pink1 66/55 kDa ratio in the mitochondrial fraction upon PARL wt and SA over-expression (n = 3, means ± SEM). Significant changes versus mock are indi- cated (**p < 0.01; One-way ANOVA with Bonferroni’s post-test). (d) Titration of PARL cDNA significantly increases processing of Pink1-66 (n = 3, means ± SEM). Significant changes versus mock are indicated (*p < 0.05; One-way ANOVA with Bonferroni’s post-test).

Article Snippet: The following antibodies were used at dilutions recommended by the manufacturer: polyclonal rabbit Pink1 (BC100-494) (Novus Biologicals, Littelton, CO, USA), polyclonal rabbit voltage-dependent anion-selective channel protein (VDAC) (Pierce, Rockford, IL, USA), monoclonal mouse apoptosis-inducing factor (AIF; Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal mouse FLAG (M2) (Sigma, St Louis, MO, USA), polyclonal rabbit PARL and a-actin (Abcam, Cambridge, MA, USA).

Techniques: Over Expression, Sequencing, Comparison, Expressing, Mutagenesis, Activity Assay, Fractionation, Transfection, Titration

Journal: Journal of neurochemistry

Article Title: The mitochondrial intramembrane protease PARL cleaves human Pink1 to regulate Pink1 trafficking.

doi: 10.1111/j.1471-4159.2011.07253.x

Figure Lengend Snippet: Fig. 2 PARL is required for Pink1 processing. (a) Left panel: PARL knockdown by two non-overlapping siRNAs (PARL-1 and -2) blocks Pink1-66 processing and reduces the level of Pink1-55, when com- pared to a non-targeting siRNA (nt). Other proteins like VDAC, AIF, actin and IRES GFP are not affected. t, total cell extracts; m, mito- chondrial pellet and c, non-mitochondrial fraction. Right panel: Quan- tification of the Pink1 66/55 kDa ratio in the mitochondrial fractions upon knockdown of endogenous PARL by siRNAs (means ± SEM, n = 3). Significant changes versus mock are indicated (**p < 0.01; One-way ANOVA with Bonferroni’s post-test). Right bottom panel: PARL knockdown was monitored in the mitochondrial fraction. (b) Left panel: Doxycyclin-induced expression of PARL-specific shRNA in Hek293- shRNA cells for 6 days diminishes Pink1-66 processing. For PARL

Article Snippet: The following antibodies were used at dilutions recommended by the manufacturer: polyclonal rabbit Pink1 (BC100-494) (Novus Biologicals, Littelton, CO, USA), polyclonal rabbit voltage-dependent anion-selective channel protein (VDAC) (Pierce, Rockford, IL, USA), monoclonal mouse apoptosis-inducing factor (AIF; Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal mouse FLAG (M2) (Sigma, St Louis, MO, USA), polyclonal rabbit PARL and a-actin (Abcam, Cambridge, MA, USA).

Techniques: Knockdown, Expressing, shRNA

Journal: Journal of neurochemistry

Article Title: The mitochondrial intramembrane protease PARL cleaves human Pink1 to regulate Pink1 trafficking.

doi: 10.1111/j.1471-4159.2011.07253.x

Figure Lengend Snippet: Fig. 4 Both uncoupling of the mitochondrial membrane potential and PARL ablation lead to the accumulation of Pink1-66 at the outer mitochondrial membrane. (a) Upper panel: Uncoupling of the mitochondrial membrane potential in Hek293-shRNA cells (by CCCP) leads to a Pink1 66/55 kDa ratio as observed upon PARL knockdown (shRNA). Total cell extracts (t), mitochondrial fractions (m) and non-mitochondrial soluble fractions (c) were analyzed. Lower panel: Quantification of Pink1 66/55 kDa ratio of the indicated fractions is shown (means ± SEM, n = 3). Mitochondrial Pink1 66/ 55 kDa ratio is significantly lower after PARL shRNA and CCCP treatment than after treatment either with PARL shRNA or CCCP alone (*p < 0.05; **p < 0.01; paired t-test). (b) Upper panel: Outline of the Pink1 construct with a juxtamembrane factor Xa protease cleavage site (Pink1Xa). Lower panel: Mitochondria isolated from cells expressing Pink1Xa were subjected to digestion by factor Xa protease. Factor Xa cleaves Pink1-66 (open triangle) but not Pink1-55 (filled triangle) generating a 52 kDa form (grey triangle). In mitochondria isolated from cells expressing the PARL-specific shRNA or CCCP-treated cells, the factor Xa-sensitive outer membrane form of Pink1-66 increased. Note that upon PARL knockdown Pink1Xa was cleaved by an alternative protease within the N-terminal MTS leading to a 64 kDa form (labeled with an asterisk), which is not observed upon expression of Pink1 wild type.

Article Snippet: The following antibodies were used at dilutions recommended by the manufacturer: polyclonal rabbit Pink1 (BC100-494) (Novus Biologicals, Littelton, CO, USA), polyclonal rabbit voltage-dependent anion-selective channel protein (VDAC) (Pierce, Rockford, IL, USA), monoclonal mouse apoptosis-inducing factor (AIF; Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal mouse FLAG (M2) (Sigma, St Louis, MO, USA), polyclonal rabbit PARL and a-actin (Abcam, Cambridge, MA, USA).

Techniques: Membrane, shRNA, Knockdown, Construct, Isolation, Expressing, Labeling

Journal: Journal of neurochemistry

Article Title: The mitochondrial intramembrane protease PARL cleaves human Pink1 to regulate Pink1 trafficking.

doi: 10.1111/j.1471-4159.2011.07253.x

Figure Lengend Snippet: Fig. 5 Pink1 TMD mutations block PARL-catalyzed processing. (a) TMD sequences of Pink1 wild type (wt), Pink1G107L, Pink1G109L, Pink1R98W and Pink1I111S are shown. Mutated residues are highlighted in bold. (b) and (c) Western analysis of Pink1 mutants expressed in Hek293-shRNA cells after doxycyclin-induced knockdown of PARL (shRNA) and CCCP treatment (total cell extracts are shown). Notably, for Pink1G107L, Pink1G109L and Pink1R98W, the Pink1-66 precursor was modified by an unknown post-translational modification leading to a 68 kDa species (labeled by an asterisk). Quantification of the Pink1 66/55 kDa ratio in the indicated fractions is shown (means ± SEM, n = 3). Pink1-55 levels are lower for all probed mutants tested. Pink1G107L and Pink1R98W processing is insensitive to PARL ablation showing highly reduced processing of these mutants by PARL.

Article Snippet: The following antibodies were used at dilutions recommended by the manufacturer: polyclonal rabbit Pink1 (BC100-494) (Novus Biologicals, Littelton, CO, USA), polyclonal rabbit voltage-dependent anion-selective channel protein (VDAC) (Pierce, Rockford, IL, USA), monoclonal mouse apoptosis-inducing factor (AIF; Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal mouse FLAG (M2) (Sigma, St Louis, MO, USA), polyclonal rabbit PARL and a-actin (Abcam, Cambridge, MA, USA).

Techniques: Blocking Assay, Western Blot, shRNA, Knockdown, Labeling

Journal: Biochimica et biophysica acta

Article Title: Involvement of caspase-9 in autophagy-mediated cell survival pathway.

doi: 10.1016/j.bbamcr.2010.09.016

Figure Lengend Snippet: Fig. 3. FR122047 treatment induces autophagy in MCF-7 cells. (A) MCF-7 cells were treated with 30 μM FR122047 (FR) or DMSO alone for 18 h and processed for transmission electron microscopy. Arrows and arrowheads indicate autophagosomes and autophagolysosomes, respectively. Scale bars represent 2 μm (8000×) and 500 nm (12,000×). (B) Cells were treated with 30 μM FR for the indicated times, and cell lysates were analyzed by Western blotting for levels of LC3-I (18 kDa) and LC3-II (16 kDa). Representative blots from three independent experiments were shown. (C) Cells were treated with 30 μM FR for the indicated times, and the mRNA level of LC3B or ATG 7 was analyzed by RT-PCR. Expression levels relative to GAPDH are presented. The experiments were repeated independently at least three times. (D) Cells transfected with GFP-LC3 were treated for 24 h with 30 μM FR or DMSO alone and examined by confocal microscopy. Nuclei were stained with TOPRO-3 (blue). Scale bars represent 20 μm. Images were representative of the results of three independent experiments. (E) Cells were treated with 30 μM FR or DMSO alone for 24 h and labeled with MDC. Images were acquired using fluorescence microscopy. Scale bars represent 20 μm. Right, enlarged images of areas indicated by squares. Images representative of the results of three different experiments were presented. (F) Cells were treated with 30 μM FR or DMSO alone for 24 h, and formation of AVOs (red fluorescence) was examined under a fluorescence microscope.

Article Snippet: LC3 antibody was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Transmission Assay, Electron Microscopy, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Confocal Microscopy, Staining, Labeling, Microscopy

Journal: Biochimica et biophysica acta

Article Title: Involvement of caspase-9 in autophagy-mediated cell survival pathway.

doi: 10.1016/j.bbamcr.2010.09.016

Figure Lengend Snippet: Fig. 4. Treatment with caspase-9 inhibitor suppresses FR122047-induced autophagy. (A) Cells were pretreated with 20 nM rapamycin (RA) or 20 μM chloroquine (CQ) for 1 h before the addition of 30 μM FR122047 (FR). The cells were incubated for another 24 h and then analyzed by FACScan. Values represent the mean±SE from three independent experiments. *Pb0.05 versus FR alone. Bottom panel: cells were preincubated with 20 nM rapamycin (RA) for 1 h prior to treatment with 30 μM FR for 24 h and Western blot analysis was conducted with antibodies to LC3 and actin. (B) Cells were pretreated with 20 μM chloroquine (CQ) or 20 μM caspase-9 inhibitor, z-LEHD-FMK (Cas9-inh) for 1 h before the addition of 30 μM FR. Cells were incubated for another 24 h, and cell lysates were analyzed by Western blotting. Images representative of the results of three different experiments were presented. (C) Cells were pretreated with 100 nM bafilomycin A1 or cathepsin inhibitors (E64d+Pepstatin A methyl ester (E/P), 10 μg/ml for each) for 1 h before the addition of 30 μM FR. Cells were incubated for another 24 h, and cell lysates were analyzed by Western blotting. (D) Cells were pretreated with 20 μM chloroquine (CQ), 20 nM rapamycin (RA), 20 μM caspase-9 inhibitor, z-LEHD-FMK (Cas9-inh), or 100 nM bafilomycin A1 for 1 h before the addition of 30 μM FR. Cells were incubated for another 24 h, and formation of AVOs (red fluorescence) was examined under a fluorescence microscope. (E) Cathepsin B activity assay was performed after treatment for 12 h with 30 μM FR alone or in combination with 20 μM chloroquine or 20 μM caspase-9 inhibitor. Values represent the mean±SE of from three independent experiments. *Pb0.05 versus the control (DMSO), **Pb0.01 versus FR alone. (F) GFP-LC3-expressing cells were pretreated with 20 μM caspase-9 inhibitor, z-LEHD-FMK (Cas9-inh) for 1 h before the addition of 30 μM FR. Cells were incubated for another 24 h, stained with Lysotracker Red dye (Lysotracker), and observed under a confocal microscope. Scale bars represent 50 μm.

Article Snippet: LC3 antibody was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Incubation, Western Blot, Microscopy, Activity Assay, Control, Expressing, Staining

Journal: Biochimica et biophysica acta

Article Title: Involvement of caspase-9 in autophagy-mediated cell survival pathway.

doi: 10.1016/j.bbamcr.2010.09.016

Figure Lengend Snippet: Fig. 5. siRNA knockdown of caspase-9 expression sensitizes MCF-7 cells to FR122047-induced cell death. (A) Cells were treated with 100 μM sulforaphane (SFN), 400 μM etoposide (Etopo), or 10 μM doxorubicin (Doxo) for 24 h and levels of LC3-I and LC3-II in lysates were analyzed by Western blotting. Blots representative of the results of three independent experiments were presented. (B) MCF-7 cells were transfected with control (Con-si) or caspase-9 (Cas9-si) siRNA. Whole-cell lysates were harvested for Western blot analysis 24 and 48 h after transfection. Blots representative of the results of three independent experiments were presented. (C) At 24 h post-transfection, MCF-7 cells were treated with 30 μM FR122047 (FR) or 100 μM sulforaphane (SFN) for a further 24 h, and the proportion of subdiploid cells was analyzed by FACScan. Values represent the mean±SE from three independent experiments. *Pb0.05 versus FR or SFN alone. (D) Cathepsin B activity assay was performed after treatment for 12 h with 30 μM FR or 100 μM SFN at 24 h post- transfection. Values represent the mean±SE of from three independent experiments. *Pb0.05 versus the control (DMSO), **Pb0.05 versus FR or SFN alone.

Article Snippet: LC3 antibody was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Knockdown, Expressing, Western Blot, Transfection, Control, Activity Assay

Journal: Arthritis and rheumatism

Article Title: Defective DNA double-strand break repair in pediatric systemic lupus erythematosus.

doi: 10.1002/art.33334

Figure Lengend Snippet: Figure 4. Kinetics of phosphorylation of structural maintenance of chromosomes protein 1 (SMC1). A, Sixteen lymphoblastoid cell lines from patients with systemic lupus erythematosus (SLE) were irradiated with 10 Gy and harvested following incubation periods of 0 minutes, 15 minutes, 30 minutes, 1 hour, 4 hours, and 24 hours. Nuclear lysates were used in immunoblot analysis. WT (CHOC6, Paris1) control cell lines and RS (AT204LA, RS13) control cell lines were used in each experiment. Asterisks indicate SLE cell lines with slower resolution of SMC1 phosphorylation. Lower rows in each panel show loading controls of native SMC1. B, Shown is quantification of SMC1 phosphorylation. WT control cell lines 1 and 2 reach peak phosphorylation between 30 minutes and 1 hour postirradiation, with near-complete resolution of phosphorylation by 24 hours. Dots indicate where phosphorylation was aberrant. Lig 4 indicates DNA ligase IV (Lig 4)–deficient cell line (RS13). See Figure 1 for other definitions.

Article Snippet: For visualizing Fanconi protein D2 monoubiquitination products or SMC1 phosphorylation, a 7.5% SDSPAGE gel was electrophoresed; the immunoblot was incubated overnight with either FANCD2 antibody or SMC1 [p Ser966] antibody (both from Novus Biologicals); secondary antibody was detected with an HRP conjugate.

Techniques: Phospho-proteomics, Irradiation, Incubation, Western Blot, Control

Journal: Arthritis and rheumatism

Article Title: Defective DNA double-strand break repair in pediatric systemic lupus erythematosus.

doi: 10.1002/art.33334

Figure Lengend Snippet: Figure 5. A, Irradiation-induced 53BP1 foci formation. Retention of 53BP1 at double-strand break sites measures the integrity of the ubiquitin ligase cascade. Formation of 53BP1 foci was normal in all tested lymphoblastoid cell lines from patients with systemic lupus erythematosus (SLE) at 1 hour after irradiation with 3 Gy. WT (Paris1) and RS66 (RNF168-deficient) cell lines were used as positive and negative controls, respectively. H2AX foci (not shown) were used as irradiation controls and were positive for each cell line shown. Original magnification 40. B, Bromodeoxyuridine (BrdU) incorporation after irradiation with 10 Gy. All 8 SLE cell lines tested were within a normal range. Mean SD BrdU incorporation was 16.97 6.54% in a WT cell line (NAT9) and 79.26 14.52% in an A-T cell line (AT204LA). C, Monoubiquitination of Fanconi protein D2 (FANCD2). Both the ubiquitinated (FANCD2-L) and nonubiquitinated (FANCD2-S) forms were present in the protein extracts from all SLE cell lines. Nuclear extract from a Fanconi protein D2–deficient cell line (PD20.L) was used as a negative control; structural maintenance of chromosomes protein 1 (SMC1) was used as a loading control. D, Immunoblot showing ATM protein levels. ATM protein levels were within normal limits for the 8 SLE cell lines tested. The SMC1 loading control shows that SLE cell line 71 was slightly underloaded. See Figure 1 for other definitions.

Article Snippet: For visualizing Fanconi protein D2 monoubiquitination products or SMC1 phosphorylation, a 7.5% SDSPAGE gel was electrophoresed; the immunoblot was incubated overnight with either FANCD2 antibody or SMC1 [p Ser966] antibody (both from Novus Biologicals); secondary antibody was detected with an HRP conjugate.

Techniques: Irradiation, Ubiquitin Proteomics, BrdU Incorporation Assay, Negative Control, Control, Western Blot

Journal: Arthritis and rheumatism

Article Title: Defective DNA double-strand break repair in pediatric systemic lupus erythematosus.

doi: 10.1002/art.33334

Figure Lengend Snippet: Figure 6. Non-homologous DNA end joining (NHEJ) pathway. A, NHEJ assay. Ten micrograms of nuclear lysate from lymphoblastoid cell lines from patients with systemic lupus erythematosus (SLE) was added to 250 ng of Sma I–digested (SmaI[]) pUC19 DNA. Nuclear lysates from all SLE cell lines and a wild-type (WT) control cell line (Paris1) efficiently ligated Sma I–digested plasmid, forming higher-order multimers (upper right). Exclusion of MgCl2 (WT[]MgCl) served as a negative control (see Materials and Methods). Quantification of ligation efficiency was calculated using the dimer and a representative multimer band (asterisk). A linear form of the plasmid (Uncut pUC19) was used as the loading control. B, Immunoblot analysis of 7 core proteins involved in NHEJ. Fifty micrograms of whole cell extract isolated from each SLE lymphoblastoid cell line was used to immunoblot for DNA-dependent protein kinase, catalytic subunit (DNA-PKcs), Ku70, Ku80, Artemis, XLF/Cernunnos (XLF), DNA ligase IV (Lig 4), and XRCC4. Beta-actin or structural maintenance of chromosomes protein 1 (SMC1) was used as a loading control and is shown below the corresponding immunoblots. All 7 core proteins were present in the 16 SLE cell line extracts tested. SLE 64 and SLE 73 were retested for Ku70 and Ku80; SLE 68 was retested for XRCC4 and Lig 4. All cell lines were normal (not all data are shown).

Article Snippet: For visualizing Fanconi protein D2 monoubiquitination products or SMC1 phosphorylation, a 7.5% SDSPAGE gel was electrophoresed; the immunoblot was incubated overnight with either FANCD2 antibody or SMC1 [p Ser966] antibody (both from Novus Biologicals); secondary antibody was detected with an HRP conjugate.

Techniques: NHEJ Assay, Control, Plasmid Preparation, Negative Control, Ligation, Western Blot, Isolation