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

Journal: PLoS Pathogens

Article Title: The JAK-STAT Transcriptional Regulator, STAT-5, Activates the ATM DNA Damage Pathway to Induce HPV 31 Genome Amplification upon Epithelial Differentiation

doi: 10.1371/journal.ppat.1003295

Figure Lengend Snippet: HPV31-positive CIN612 cells were transduced as described in legend to . A) Western blot analysis of p-ATM, ATM, p-CHK2, CHK2, involucrin and GAPDH protein levels in uninfected and shRNA lentivirus infected CIN612 cells upon differentiation in high-calcium media for indicated times. B) Western blot analysis of p-CHK2, CHK2, STAT-5α, STAT-5β, and GAPDH protein levels in shRNA control and shRNA lentivirus infected CIN612 cells upon differentiation in high-calcium media for indicated times. C) Western blot analysis of BRCA-1. BRCA-2, SMC-1, p-SMC-1, RAD51 and GAPDH protein at total or phosphorylation levels in uninfected and shRNA lentivirus infected CIN612 cells upon differentiation for indicated times. The quantification of the band intensities is shown as bar graph figures in . All results are representative of observations from 3 independent experiments.

Article Snippet: The antibodies used in this study are as follows: anti- STAT-5α and STAT-5β (Sigma, St. Louis, MO); anti-Bcl-XL, anti-Involucrin, anti-GAPDH, and anti-PARP are from Santa Cruz, Santa Cruz, CA); anti-STAT-5, anti-p-STAT-5, anti-CHK2, anti-ATM, anti-p-CHK2 (Thr68), anti-p-ATM (Ser1981), anti-SMC-1, anti-p-SMC-1, anti-BRCA1, anti-p-BRCA1, anti-BRCA2, anti-PPARγ (Cell Signaling, Danvers, MA); anti-RAD51 (Millipore, Hayward, CA); and anti-keratin-10 (Abcam, Cambridge, MA).

Techniques: Western Blot, shRNA, Infection

Journal: Leukemia & lymphoma

Article Title: Synergistic cytotoxicity of busulfan, melphalan, gemcitabine, panobinostat, and bortezomib in lymphoma cells

doi: 10.3109/10428194.2016.1157871

Figure Lengend Snippet: List of primary antibodies, their sources, dilutions and molecular weights.

Article Snippet: p-SMC1 (Ser957) , Novus Biologicals/100–205 , pAb , 1500 , 140.

Techniques: Molecular Weight

Journal: Journal of Biological Chemistry

Article Title: ATM-dependent CHK2 Activation Induced by Anticancer Agent, Irofulven

doi: 10.1074/jbc.m400015200

Figure Lengend Snippet: FIG. 7. ATM and CHK2 target proteins activated by irofulven. A and B, human ovarian cancer cell lines A2780, CAOV3, SKOV3, and OVCAR3 were treated with 1 IC50 concentration of irofulven (A); and human normal fibroblast GM00637 and AT fibroblast GM05849 were treated with 8 M of irofulven (B) for 1 h followed by additional 24 h of incubation. Western blot analyses were performed with antibodies against phosphorylated NBS1 on Ser343, NBS1, phosphorylated SMC1 on Ser957, SMC1, and phosphorylated p53 on Ser15. The nonspecific band was indicated as n.s. C and D, A2780 and CAOV3 cells (C); and human colon cancer cell line HCT116 and its CHK2 knockout subline (D) were treated with 1 IC50 concentration of irofulven for 1 h followed by additional 24 h of incubation, Western blots were performed with antibodies against phosphorylated p53 on Ser20, p53, and actin.

Article Snippet: Polyclonal antibodies against phosphorylated NBS1 on Ser343 and phosphorylated SMC1 on Ser957 were purchased from Novus Biologicals (Littleton, CO).

Techniques: Concentration Assay, Incubation, Western Blot, Knock-Out