Journal: Cell

Article Title: Architectures of lipid transport systems for the bacterial outer membrane

doi: 10.1016/j.cell.2017.03.019

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Seleno-methionine , Chem-impex international , 662.

Techniques: Virus, Mutagenesis, Recombinant, Electron Microscopy, Cryo-EM Sample Prep, Staining, Plasmid Preparation, Expressing, Construct, Residue, Software, Tomography

Journal: Clinical Cancer Research

Article Title: Unique Cell Surface Expression of Receptor Tyrosine Kinase ROR1 in Human B-Cell Chronic Lymphocytic Leukemia

doi: 10.1158/1078-0432.ccr-07-1823

Figure Lengend Snippet: Fig. 1. ROR1 mRNA expression in B-CLL. A, ROR1 mRNA versus ZAP-70 mRNA expression. A cohort of 107 B-CLL patient samples (79 IgVH mutated and 28 IgVH unmutated) was analyzed for ROR1 versus ZAP-70 mRNA expression by gene expression profiling. ROR1 values on the y axis are given as fold expression in log2 over the background expression level in nine pooled lymphoma cell lines (5). ZAP-70 values on the x axis are centered to the median of all samples. Dashed line, scaled cutoff between ZAP-70 ^ negative (left) and ZAP-70 ^ positive (right) samples as described (5).The median ROR1 mRNA expression was 6.5-fold higher (2.7 log2) in the B-CLL patient samples compared with the nine pooled lymphoma cell lines. No significant difference in median ROR1 mRNA expression was found between ZAP-70 ^ negative and ZAP-70 ^ positive (P = 0.906) or between IgVH-mutated and IgVH-unmutated (P = 0.176) B-CLL patient samples. B, detection of theT518M polymorphism of ROR1by RT-PCR followed by DNA sequencing. Left, predicted domain structure of ROR1protein (12). Numbers, amino acid sequence of ROR1protein before signal peptide cleavage. Right, chromatograms from three representative mRNA samples prepared from B-CLL PBMC and amplified by RT-PCR.Top, uniform ACG (threonine) allele expression found in 8 of 15 B-CLL patient samples; center, uniform ATG (methionine) allele expression found in 1of 15 B-CLL patient samples; bottom, ACG/ATG allele expression mixture found in 6 of15 B-CLL patient samples.

Article Snippet: Affinity-purified polyclonal goat anti-human ROR1 (gahROR1 pAb), polyclonal goat anti-human ROR2 antibodies (gahROR2 pAb), and polyclonal goat anti-human CD5 antibodies (gahCD5 pAb) were used as primary antibodies (R&D Systems).

Techniques: Expressing, Gene Expression, Reverse Transcription Polymerase Chain Reaction, DNA Sequencing, Sequencing, Amplification

Journal: Clinical Cancer Research

Article Title: Unique Cell Surface Expression of Receptor Tyrosine Kinase ROR1 in Human B-Cell Chronic Lymphocytic Leukemia

doi: 10.1158/1078-0432.ccr-07-1823

Figure Lengend Snippet: Fig. 2. ROR1protein expression on the surface of B-CLL cells. A, flow cytometry profiles of PBMC from a representative B-CLL patient. Four-color flow cytometry was carried out with a mouse anti-human CD19 mAb conjugated to PE, a mouse anti-human CD5 mAb conjugated toAPC, gahROR1pAb or gahROR2 pAb, swine anti-goat-FITC conjugate, and propidium iodide.The histograms on the right represent staining for ROR1 (green) and ROR2 (pink) in CD5+ CD19+ B-CLL cells (top) and CD5+ CD19- Tcells (bottom).The background signal with normal goat immunoglobulin or secondary antibody alone is shownin black. B, biotinylated cell surface proteins from B-CLL cells or normal B cells were separated by SDS-PAGE. Subsequent Western blotting with gahROR1pAb and donkey anti-goat-HRP conjugate detected a single band of f120 kDa in B-CLL cells (left lane). No band was detected in normal B cells (right lane). C, B-CLL cells were incubated with gahROR1pAb for1h at 4jC. Subsequently, the cells were washed and either left at 4jC (green) or incubated for 1h at 37jC in the absence (red) or presence (blue) of 3 Amol/L phenylarsine oxide, followed by flow cytometry analysis with swine anti-goat-FITC conjugate.The background signal with secondary antibody alone is shown in black.

Article Snippet: Affinity-purified polyclonal goat anti-human ROR1 (gahROR1 pAb), polyclonal goat anti-human ROR2 antibodies (gahROR2 pAb), and polyclonal goat anti-human CD5 antibodies (gahCD5 pAb) were used as primary antibodies (R&D Systems).

Techniques: Expressing, Flow Cytometry, Staining, SDS Page, Western Blot, Incubation

Journal: Clinical Cancer Research

Article Title: Unique Cell Surface Expression of Receptor Tyrosine Kinase ROR1 in Human B-Cell Chronic Lymphocytic Leukemia

doi: 10.1158/1078-0432.ccr-07-1823

Figure Lengend Snippet: Fig. 5. Normal adult tissues do not express cell surface ROR1. Lysates from PBMC of B-CLL patients (5 Ag total protein per lane) were compared with lysates from 28 normal adult tissues (20 Ag total protein per lane) byWestern blotting using gahROR1 pAb followed by donkey anti-goat-HRP conjugate. Arrows, f120-kDa band that specifies cell surface ROR1 (Fig. 2B). Rabbit anti-human glyceraldehyde-3- phosphate dehydrogenase pAb followed by donkey anti-rabbit-HRP conjugate were used as positive control (bottom). Normal adult tissues: Brn, brain; Brt, breast;Tes, testis; Ova, ovary; Ute, uterus; Pro, prostate; Lyn, lymph node; Int, small intestine; Hrt, heart; Liv, liver; Lun, lung; Kid, kidney; Pan, pancreas; Adi, adipose; Skn, skin; Spl, spleen; Adr, adrenal gland; Amy, amygdala; Pit, pituitary gland; Pla, placenta; Bla, bladder;Ton, tonsil; Sto, stomach; Col, colon; Rec, rectum; Mus, skeletal muscle; Spn, spinal cord; Eye, eye.

Article Snippet: Affinity-purified polyclonal goat anti-human ROR1 (gahROR1 pAb), polyclonal goat anti-human ROR2 antibodies (gahROR2 pAb), and polyclonal goat anti-human CD5 antibodies (gahCD5 pAb) were used as primary antibodies (R&D Systems).

Techniques: Positive Control

Journal: PLoS ONE

Article Title: Silencing of Receptor Tyrosine Kinase ROR1 Inhibits Tumor-Cell Proliferation via PI3K/AKT/mTOR Signaling Pathway in Lung Adenocarcinoma

doi: 10.1371/journal.pone.0127092

Figure Lengend Snippet: (A) Flow cytometry analysis of ROR1 protein expression on the surface of NSCLC cell lines. The histograms on the right represent staining for ROR1 with polyclonal goat anti-ROR1 antibody. The background signal with normal goat IgG is shown in gray shadow. (B) Representative images of lung adenocarcinoma tissues (LA) detected by immunohistochemical analysis. Formalin-fixed, paraffin-embedded tissues were stained with polyclonal rabbit anti-ROR1 antibody or normal rabbit IgG. Tissue-bound ROR1 is shown in brown and the nucleus counterstained with hematoxylin is in blue (scale bar in the bottom right picture represents 50μm). A score of 0 indicates that none of the cells within the sample bound to the anti-ROR1 pAb; a score of 1 indicates low-level binding of the pAb on more than 25% of tumor cells; a score of 2 indicates low-level binding of the pAb on more than 50% of tumor cells or moderate-level staining on more than 25% of tumor cells; a score of 3 indicates moderate-level staining on more than 75% of tumor cells or high level staining on more than 50% of tumor cells. Controls include: Chronic lymphocytic leukemia lymph node (CLL LN) stained with normal rabbit IgG as negative control and with polyclonal rabbit anti-ROR1 antibody as positive control; Reactive hyperplasia of lymph node (RH LN) and tissues judged to be adjacent to cancer (TAC) stained with polyclonal rabbit anti-ROR1 antibody as negative controls. (C) A summary of immunohistochemical analysis for ROR1 staining in lung adenocarcinoma specimens. The proportion of lung tumor tissues found negative (Score 0) or having weak (Score 1), moderate (Score 2) or strong staining (Score 3) for ROR1 is indicated in the pie chart. (D-E) The proportion of lung adenocarcinoma tissues of different stages or sex found lacking staining (Score 0) or having weak (Score 1), moderate (Score 2) or strong staining (Score 3) for ROR1 is indicated in each bar. The number of different cases examined for each group is indicated in the parentheses. Statistical analyses was performed using Mann–Whitney U test. p = 0.048.

Article Snippet: Five μg/ml of polyclonal goat anti-ROR1 antibody (R&D system, Minneapolis, MN, USA) or chimeric rabbit/human anti-ROR1 monoclonal antibody R12 with HA tag which was developed by the correspondence author in Christoph Rader’s lab [ ], or normal goat IgG (Santa Cruz Biotechnology, Dallas, TX, USA) was added to the cells and incubated on ice for 30 min before washing twice with flow cytometry buffer.

Techniques: Flow Cytometry, Expressing, Staining, Immunohistochemical staining, Formalin-fixed Paraffin-Embedded, Binding Assay, Negative Control, Positive Control, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Silencing of Receptor Tyrosine Kinase ROR1 Inhibits Tumor-Cell Proliferation via PI3K/AKT/mTOR Signaling Pathway in Lung Adenocarcinoma

doi: 10.1371/journal.pone.0127092

Figure Lengend Snippet: (A-C) PC9, XLA-07, and NCI-H1975 were treated with ROR1 siRNA (siROR1) or control siRNA (siControl) for 72 h and examined for ROR1 protein expression with chimeric rabbit/human anti-ROR1 monoclonal antibody R12 by flow cytometry (A), growth-inhibition by MTS assay (B) and apoptosis induction by Annexin-V/PI staining (C). The height of each bar in the graph B provides the mean number of viable cells that are representative of more than three independent experiments. *** p<0.001, ** p<0.01 by Student’s t test.

Article Snippet: Five μg/ml of polyclonal goat anti-ROR1 antibody (R&D system, Minneapolis, MN, USA) or chimeric rabbit/human anti-ROR1 monoclonal antibody R12 with HA tag which was developed by the correspondence author in Christoph Rader’s lab [ ], or normal goat IgG (Santa Cruz Biotechnology, Dallas, TX, USA) was added to the cells and incubated on ice for 30 min before washing twice with flow cytometry buffer.

Techniques: Control, Expressing, Flow Cytometry, Inhibition, MTS Assay, Staining

Journal: PLoS ONE

Article Title: Silencing of Receptor Tyrosine Kinase ROR1 Inhibits Tumor-Cell Proliferation via PI3K/AKT/mTOR Signaling Pathway in Lung Adenocarcinoma

doi: 10.1371/journal.pone.0127092

Figure Lengend Snippet: PC9 and NCI-H1975 were treated with ROR1 siRNA (siROR1) or control siRNA (siControl) for 72 h and examined for phosphorylated AKT at Ser-473 (p-AKT), phosphorylated mTOR at Ser-2448, and phosphorylated PTEN at Ser-380/Thr-382/383 (p-PTEN) by immunoblot analysis.

Article Snippet: Five μg/ml of polyclonal goat anti-ROR1 antibody (R&D system, Minneapolis, MN, USA) or chimeric rabbit/human anti-ROR1 monoclonal antibody R12 with HA tag which was developed by the correspondence author in Christoph Rader’s lab [ ], or normal goat IgG (Santa Cruz Biotechnology, Dallas, TX, USA) was added to the cells and incubated on ice for 30 min before washing twice with flow cytometry buffer.

Techniques: Control, Western Blot

Journal: PLoS ONE

Article Title: Silencing of Receptor Tyrosine Kinase ROR1 Inhibits Tumor-Cell Proliferation via PI3K/AKT/mTOR Signaling Pathway in Lung Adenocarcinoma

doi: 10.1371/journal.pone.0127092

Figure Lengend Snippet: Activation of ROR1 significantly enhanced phosphorylation of both AKT and mTOR, and deactivated PTEN, a negative regulator of PI3K/AKT.

Article Snippet: Five μg/ml of polyclonal goat anti-ROR1 antibody (R&D system, Minneapolis, MN, USA) or chimeric rabbit/human anti-ROR1 monoclonal antibody R12 with HA tag which was developed by the correspondence author in Christoph Rader’s lab [ ], or normal goat IgG (Santa Cruz Biotechnology, Dallas, TX, USA) was added to the cells and incubated on ice for 30 min before washing twice with flow cytometry buffer.

Techniques: Activation Assay, Phospho-proteomics

Journal: Pharmaceutics

Article Title: Intracellular Delivery of Active Proteins by Polyphosphazene Polymers.

doi: 10.3390/pharmaceutics13020249

Figure Lengend Snippet: Figure 1. Schematics and characterization of polyphosphazenes (PZ)/protein complexes. (A) Chemi- cal structures of PZ-PYR and PZ-PEG polymers and their schematic presentations. (B) Representative AF4 profiles of FITC-avidin, PZ-PYR, and PZ-PYR/FITC-avidin as detected at 495 nm (PZ-PYR profile at 210 nm detection is shown for comparison purposes). (C) Dynamic light scattering profiles of FITC-avidin, PZ-PYR, and PZ-PYR/FITC-avidin (25 mM phosphate buffer, pH 7.4). (D) Efficiency of protein or peptide binding to PZ-PEG and PZ-PYR expressed as a percent of bound molecules of their total amount for FITC-avidin, Bax-BH3 peptide, and anti-F-actin antibody (25 mM phosphate buffer, pH 7.4).

Article Snippet: Asymmetric Flow Field Flow Fractionation (AF4) characterization was conducted using Postnova AF2000 MT series instrument (Postnova Analytics, Landsberg, Germany) equipped with UV-Vis detector (SPD-20A/20AV, Shimadzu Scientific Instruments, Columbia, MD, USA) and regenerated cellulose membrane (10 kDa molecular weight cutoff, Postnova Analytics, Landsberg, Germany).

Techniques: Avidin-Biotin Assay, Comparison, Binding Assay