Journal: PLoS ONE

Article Title: The Tyrosine Kinase Receptor ROR1 Is Constitutively Phosphorylated in Chronic Lymphocytic Leukemia (CLL) Cells

doi: 10.1371/journal.pone.0078339

Figure Lengend Snippet: Western blot analysis of CLL lysates from 3 CLL patients (5072, 5194, 5002) treated with a lambda phosphatase enzyme. - : enzyme-untreated sample, + : enzyme-treated sample. PVDF blot was stripped and reprobed with a goat anti-ROR1 polyclonal Ab.

Article Snippet: Filters were incubated with an anti-pROR1 mAb (against the intracellular tyrosine kinase domain of ROR1) (a gift from Monoclonal Antibody Research Center, Avicenna Research Institute, Tehran, Iran), anti-phospho-tyrosine mAb (clone 4G10), anti-phospho-serine mAb (clone 4A4) (Millipore) and a polyclonal goat anti-human ROR1 Ab (R&D) for 2 h at room temperature.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: The Tyrosine Kinase Receptor ROR1 Is Constitutively Phosphorylated in Chronic Lymphocytic Leukemia (CLL) Cells

doi: 10.1371/journal.pone.0078339

Figure Lengend Snippet: Representative experiments using non-immunoprecipitated CLL cell lysates from 5 CLL patients showing phosphorylated dimerized ROR1 (260 kDa), fully glycosylated (130 kDa) and non-glycosylated (105 kDa) ROR1 molecules (A). NP indicates non-progressive disease and P progressive disease. As controls, CLL cell lysates immunoprecipitated with a non-relevant mAb (mouse IgG1 isotype) were used. No bands could be seen. Furthermore, in PBMC of healthy donors, no bands were detected (data not shown). Representative experiments of PBMC using surface staining for ROR1 (left panel) and intracytoplasmic staining for pROR1 (right panel) in three CLL patients (B). Expression of ROR1 isoforms in the cytoplasm (C), nucleus (N) and total cell lysate (TCL) of leukemic CLL cells (C). Confirmation of protein localization was done using antibodies against α/β –tubulin, histone H3 and nucleolin before analysing the expression pattern of ROR1.

Article Snippet: Filters were incubated with an anti-pROR1 mAb (against the intracellular tyrosine kinase domain of ROR1) (a gift from Monoclonal Antibody Research Center, Avicenna Research Institute, Tehran, Iran), anti-phospho-tyrosine mAb (clone 4G10), anti-phospho-serine mAb (clone 4A4) (Millipore) and a polyclonal goat anti-human ROR1 Ab (R&D) for 2 h at room temperature.

Techniques: Immunoprecipitation, Staining, Expressing

Journal: PLoS ONE

Article Title: The Tyrosine Kinase Receptor ROR1 Is Constitutively Phosphorylated in Chronic Lymphocytic Leukemia (CLL) Cells

doi: 10.1371/journal.pone.0078339

Figure Lengend Snippet: pROR1/ROR1 intensity (mean ± SEM) of the 105, 130 and combined 105+130 kDa bands in progressive (■) (n=21) and non-progressive (☐) CLL patients (n=17). *** p= 0.001.

Article Snippet: Filters were incubated with an anti-pROR1 mAb (against the intracellular tyrosine kinase domain of ROR1) (a gift from Monoclonal Antibody Research Center, Avicenna Research Institute, Tehran, Iran), anti-phospho-tyrosine mAb (clone 4G10), anti-phospho-serine mAb (clone 4A4) (Millipore) and a polyclonal goat anti-human ROR1 Ab (R&D) for 2 h at room temperature.

Techniques:

Journal: PLoS ONE

Article Title: The Tyrosine Kinase Receptor ROR1 Is Constitutively Phosphorylated in Chronic Lymphocytic Leukemia (CLL) Cells

doi: 10.1371/journal.pone.0078339

Figure Lengend Snippet: Representative experiments of time-dependent ROR1 dephosphorylation and apoptosis in vitro incubated with an anti-KNG (4A7) (A and C) and anti-CRD (1D8) (B and D) anti-ROR1 mAb respectively. Representative experiments from two progressive (CLL 5102 and CLL 5116) CLL patients are shown. Leukemic cells were incubated for 20 min, 1, 4 hours with a non-relevant IgG1 isotype control mAb (-) and the ROR1 mAbs (+). Cells were harvested and lysed for western blot experiments (A and B). The non-relevant isotype control mAb did not induce dephosphorylation of ROR1 (130 kDa) while the anti-ROR1 mAb induced dephosphorylation already after 20 min, which increased by time. The intensity of pROR1 was measured by ImageJ software. The ratio of pROR1/ROR1 intensity of treated sample to pROR1/ROR1 intensity of untreated sample (relative intensity) is shown in a histogram. A value <1 indicates dephosphorylation of ROR1 after treatment with the anti-ROR1 mAb compared to the non-relevant isotype control mAb. Apoptosis of CLL cells (Annexin V + /PI + ) after 0, 12 and 18 h incubation with the anti ROR1 mAbs without the addition of immune effector cells or complement (C and D). An anti-CD20 mAb (ofatumumab) did not induce dephosphorylation of ROR1 after 4h incubation in two CLL patients. A representative experiment is shown for one of the CLL patients (E).

Article Snippet: Filters were incubated with an anti-pROR1 mAb (against the intracellular tyrosine kinase domain of ROR1) (a gift from Monoclonal Antibody Research Center, Avicenna Research Institute, Tehran, Iran), anti-phospho-tyrosine mAb (clone 4G10), anti-phospho-serine mAb (clone 4A4) (Millipore) and a polyclonal goat anti-human ROR1 Ab (R&D) for 2 h at room temperature.

Techniques: De-Phosphorylation Assay, In Vitro, Incubation, Western Blot, Software

Journal: Gastroenterology Report

Article Title: TRUB1 is a novel biomarker for promoting malignancy in colorectal cancer via NFκB signaling

doi: 10.1093/gastro/goaf027

Figure Lengend Snippet: Potential downstream targets of TRUB1 in CRC. (A) Scatter plot showing genes with significant changes in RNA-seq after TRUB1 knock-down; (B) GSEA analysis showing enrichment of TNFα signaling via NFκB in cells with high TRUB1 expression; (C) Western blot results for TRUB1, P65, and pP65 in control and TRUB1-knock-down HCT116 cells; (D) intersection analysis of gene sets between HCT116-sh-TRUB1-1 and HCT116-sh-TRUB1-2 (FC ≥ 1.5, FDR ≤ 0.05); (E) intersection analysis of TNFα signaling via NFκB gene sets between HCT116-sh-TRUB1-1 and HCT116-sh-TRUB1-2 ( P ≤ 0.05); (F) intersection analysis of gene sets between GSEA_NFκB signaling (61 genes), RNA-seq (261 genes), and apoptosis via NFκB (33 genes); (G) expression of TRUB1 and BIRC3 mRNA in control and TRUB1-knock-down HCT116 cells. GSEA = Gene Set Enrichment Analysis, pP65 = phosphorylated P65, BIRC3 = Baculoviral IAP repeat-containing protein 3.

Article Snippet: The antibodies that were used in the Western blot were TRUB1 (1:2,000; #PA536003; Thermo, Waltham, MA, USA); TRUB2 (1:2,000; #K110029P; Solarbio, Beijing, China); p65 (1:2,000; #a22331; ABclonal, Woburn, MA, USA); phosphorylated-P65 (pP65; 1:2,000; #af2006; Affinity, Nottingham, UK); GAPDH (1:1,000; #BM3876; BOSTER, Pleasanton, CA, USA); Cytochrome C (1:2,000; #A13430; ABclonal); Bcl 2 (1:2,000; #A0208; ABclonal); Caspase-8 (1:2,000; #A0215; ABclonal); Caspase-3 (1:2,000; #A02156; ABclonal); Caspase-9 (1:2,000; #A11910; ABclonal); cleaved Caspase-3 (1:2,000; #9661 T; CST, Danvers, MA, USA); and β-Actin (1:2,000; #AC038; ABclonal).

Techniques: RNA Sequencing, Knockdown, Expressing, Western Blot, Control