Journal: Experimental & Molecular Medicine

Article Title: Increased p190RhoGEF expression in activated B cells correlates with the induction of the plasma cell differentiation

doi: 10.3858/emm.2012.44.2.009

Figure Lengend Snippet: p190RhoGEF protein levels and changes after CD40 stimulation of B cells of various maturational stages. (A) Mouse splenic B cells (1 × 10 6 /ml) and the B cell lines WEHI 231, BAL17, CH12.LX (5 × 10 5 /ml) were stimulated with isotype control Ig or anti-CD40 at a concentration of 1 µg/ml for 48 h. (B) HEK 293T cells were transfected with plasmids carrying an empty vector (EV), WT, or DN p190RhoGEF (HA). (C) WEHI 231 B cells were stably transfected with MIGR1 (EV), WT, or DN p190RhoGEF. Lysates were prepared from these cells, subjected to a SDS-PAGE (6% or 8% gels), transferred to nitrocellulose, and probed with an Ab specific for p190RhoGEF (A-C) or HA (B). Equal loading of the protein samples was demonstrated by anti-β actin IB in (A). (D, E) Mouse splenic B cells (1 × 10 6 /ml) were stimulated with control Ig or anti-CD40 at a concentration of 1 µg/ml. After 48 h of stimulation, cells were fixed, permeabilized, and stained for endogenous p190RhoGEF, CD23, and IgD. The fluorescence intensities of cells stained with an isotype control Ab or antiserum were all corrected (data not shown). The results from flow cytometry are presented as a dot plot, showing IgD versus CD23 positive cells. For the cell populations in R1, R2, and R3 as indicated in (D), the population of cells (%) and the expression level of p190RhoGEF as mean fluorescence intensity (MFI) in the absence (control) and presence of CD40 stimulation is presented in (E). The results shown are representative of three independent experiments.

Article Snippet: The rat anti-mouse CD40 mAb (clone 1C10) from R&D Systems, Inc. (Minneapolis, MN) and the hamster anti-mouse CD40 mAb (HM40-3) from BD PharMingen (San Diego, CA) were used to stimulate B cells.

Techniques: Control, Concentration Assay, Transfection, Plasmid Preparation, Stable Transfection, SDS Page, Staining, Fluorescence, Flow Cytometry, Expressing

Journal: Experimental & Molecular Medicine

Article Title: Increased p190RhoGEF expression in activated B cells correlates with the induction of the plasma cell differentiation

doi: 10.3858/emm.2012.44.2.009

Figure Lengend Snippet: Expression of p190RhoGEF, CD138, and B220 in B cells at various maturational stages. (A, B) Mouse splenic B cells (1 × 10 6 /ml) were stimulated with control Ig or anti-CD40 at a concentration of 1 µg/ml. After 72 h of stimulation, cells were fixed, permeabilized, and stained for endogenous p190RhoGEF, B220, and CD138. The fluorescence intensities of cells stained with an isotype control Ab or antiserum were all corrected (data not shown). The results from flow cytometry are presented as a dot plot, showing the B220 positive cells versus the CD138 positive cells. The data shown are representative of three independent experiments. For the cell populations indicated in (A), expression of p190RhoGEF is presented as MFI in (B). Data are presented as the mean ( n = 4) ± SD. (C, D) The B cell lines, BAL17, CH12.LX, and NS-1 were fixed, permeabilized, and stained for endogenous p190RhoGEF, CD138, and B220. The results were obtained as described above. For the cell populations indicated in (C), expression of p190RhoGEF is presented as MFI in (D). Data are presented as the mean ( n = 3) ± SD.

Article Snippet: The rat anti-mouse CD40 mAb (clone 1C10) from R&D Systems, Inc. (Minneapolis, MN) and the hamster anti-mouse CD40 mAb (HM40-3) from BD PharMingen (San Diego, CA) were used to stimulate B cells.

Techniques: Expressing, Control, Concentration Assay, Staining, Fluorescence, Flow Cytometry

Journal: Experimental & Molecular Medicine

Article Title: Increased p190RhoGEF expression in activated B cells correlates with the induction of the plasma cell differentiation

doi: 10.3858/emm.2012.44.2.009

Figure Lengend Snippet: Expression changes of transcriptional regulators after CD40 stimulation and in response to p190RhoGEF and RhoA down-regulation in B cells at various maturational stages. (A) cDNA was prepared from B cells that were either untreated (control) or stimulated for 24 h with anti-CD40 (1 µg/mL). (B) cDNA was prepared from BAL17 and NS-1 cells that were stably transfected with MIGR1 (EV), p190RhoGEF (Y1003A) or RhoA (T19N). The PCR products for p190RhoGEF, RhoA, Blimp-1, XBP-1, IRF-4, and GAPDH were separated as described in "Methods." The data shown are representative of three independent experiments. Values indicate normalization for GAPDH.

Article Snippet: The rat anti-mouse CD40 mAb (clone 1C10) from R&D Systems, Inc. (Minneapolis, MN) and the hamster anti-mouse CD40 mAb (HM40-3) from BD PharMingen (San Diego, CA) were used to stimulate B cells.

Techniques: Expressing, Control, Stable Transfection, Transfection

Journal: Experimental & Molecular Medicine

Article Title: Increased p190RhoGEF expression in activated B cells correlates with the induction of the plasma cell differentiation

doi: 10.3858/emm.2012.44.2.009

Figure Lengend Snippet: Direct effects of manipulating the activity of p190RhoGEF and RhoA on B cell maturation and downstream transcriptional regulators. WEHI 231 B cells were stably transfected with MIGR1 (EV), WT p190RhoGEF, DN p190RhoGEF (Y1003A), CA RhoA (Q63L), DN RhoA (T19N), or a combination of WT p190RhoGEF and DN RhoA (T19N). (A) cDNA was prepared from these stably transfected cells. The PCR products for p190RhoGEF, RhoA, Blimp-1, XBP-1, IRF-4, Bcl-6, AID, and GAPDH were separated as described in "Methods." The data shown are representative of three independent experiments. Values indicate normalization for GAPDH. (B) These stably transfected cells were removed from the cultures and then washed, counted, and recultured in fresh medium for 24 h at 1 × 10 6 cells/ml. Some EV-transfected cells were also incubated with a medium alone (black bars) or with a combination of CD40 and IL4 as positive controls (white bars). Supernatants were harvested and serially diluted, and secreted Ig was measured by anti-IgM and anti-IgG ELISA as described in "Methods." Values of the optical densities at 490 nm in triplicate supernatant samples were collected in each independent experiment. Data are presented as the mean of three independent experiments ( n = 3) ± SD, * P < 0.05, ** P < 0.01, compared with cells transfected with EV. (C, D) These stably transfected cells were fixed, stained with anti-CD138 or anti-CXCR4, and analyzed by standard flow cytometry. The fluorescence intensities of cells stained with an isotype control Ab or antiserum were all corrected (data not shown).The bar graphs show the expression of CD138 (C) or CXCR4 (D), as indicated by MFI. Data are presented as the mean of three independent experiments ( n = 3) ± SEM, ** P < 0.01, *** P < 0.001, compared with cells transfected with EV.

Article Snippet: The rat anti-mouse CD40 mAb (clone 1C10) from R&D Systems, Inc. (Minneapolis, MN) and the hamster anti-mouse CD40 mAb (HM40-3) from BD PharMingen (San Diego, CA) were used to stimulate B cells.

Techniques: Activity Assay, Stable Transfection, Transfection, Incubation, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, Fluorescence, Control, Expressing

Journal: Experimental & Molecular Medicine

Article Title: Increased p190RhoGEF expression in activated B cells correlates with the induction of the plasma cell differentiation

doi: 10.3858/emm.2012.44.2.009

Figure Lengend Snippet: Effects of the overexpressed DN forms of p190RhoGEF or RhoA on the expression of CD138, CXCR4, and transcriptional regulators after CD40 stimulation. WEHI 231 B cells were stably transfected with either MIGR1 (EV), a plasmid encoding DN p190RhoGEF (Y1003A), or a plasmid encoding DN RhoA (T19N). (A, B) After CD40 stimulation for 48 h, cells were fixed, stained with anti-CD138 or anti-CXCR4, and measured by flow cytometry. The fluorescence intensities of cells stained with an isotype control Ab or antiserum were all corrected (data not shown). The results show the expression intensities of CD138 (A) or CXCR4 (B). Data are presented as the mean of three independent experiments ( n = 3) ± SD. (C) cDNA was prepared from these stably transfected cells, after being stimulated for 48 h with anti-CD40 Ab (1 µg/ml). The PCR products for p190RhoGEF, RhoA, Blimp-1, XBP-1, IRF-4, and GAPDH were separated as described in "Methods." The data shown are representative of three independent experiments. Values indicate normalization for GAPDH.

Article Snippet: The rat anti-mouse CD40 mAb (clone 1C10) from R&D Systems, Inc. (Minneapolis, MN) and the hamster anti-mouse CD40 mAb (HM40-3) from BD PharMingen (San Diego, CA) were used to stimulate B cells.

Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Staining, Flow Cytometry, Fluorescence, Control

Journal: Febs Letters

Article Title: Expression and functional characterization of the putative protein 8b of the severe acute respiratory syndrome‐associated coronavirus

doi: 10.1016/j.febslet.2006.05.051

Figure Lengend Snippet: Expression of recombinant SARS 8b‐EGFP protein in Vero E6 and CHO cells. Total cell proteins were extracted and 50–150 μg of protein were resolved by 12% SDS–PAGE. Western blots of these proteins were probed with anti‐GFP antibodies. Lane 1: 50 μg of proteins from Vero E6 cells transfected with pEGFP vectors. Lane 2: 150 μg of proteins from Vero E6 cells transfected with SARS 8b‐EGFP vectors. Lane 3: 50 μg of proteins from Vero E6 cells transfected with SARS 8b‐EGFP vectors. Lane 4: 30 μg of proteins from CHO cells transfected with pEGFP vectors. Lane 5: 30 μg of proteins from CHO cells transfected with SARS 8b‐EGFP vectors.

Article Snippet: Vero E6 (African green monkey kidney fibroblasts) and CHO (Chinese hamster ovary) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured at 37 °C in 5% CO 2 in Dulbecco modified eagle medium (Gibco BRL Life Technologies, Inc., Carlsbad, CA, USA) containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Expressing, Recombinant, SDS Page, Western Blot, Transfection

Journal: Febs Letters

Article Title: Expression and functional characterization of the putative protein 8b of the severe acute respiratory syndrome‐associated coronavirus

doi: 10.1016/j.febslet.2006.05.051

Figure Lengend Snippet: Subcellular localization of individually expressed recombinant SARS 8b‐EGFP and SARS 6‐RFP proteins. Images shown are Vero E6 cells transfected with vectors encoding (A) EGFP, (B) SARS 8b‐EGFP, and (C) SARS 6‐RFP; and CHO cells transfected with vectors encoding (D) EGFP, (E) SARS 8b‐EGFP, and (F) SARS 6‐RFP.

Article Snippet: Vero E6 (African green monkey kidney fibroblasts) and CHO (Chinese hamster ovary) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured at 37 °C in 5% CO 2 in Dulbecco modified eagle medium (Gibco BRL Life Technologies, Inc., Carlsbad, CA, USA) containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Recombinant, Transfection

Journal: Febs Letters

Article Title: Expression and functional characterization of the putative protein 8b of the severe acute respiratory syndrome‐associated coronavirus

doi: 10.1016/j.febslet.2006.05.051

Figure Lengend Snippet: Subcellular localization of co‐expressed SARS 8b‐EGFP and SARS 6‐RFP proteins. Localization of cotransfected SARS 8b‐EGFP and SARS 6‐RFP in Vero E6 cells: (A) SARS 8b‐EGFP, (B) SARS 6‐RFP, (C) overlay image of (A) and (B). Localization of cotransfected SARS 8b‐EGFP and SARS 6‐RFP in CHO cells: (D) SARS 8b‐EGFP, (E) SARS 6‐RFP, (F) overlay image of (D) and (E).

Article Snippet: Vero E6 (African green monkey kidney fibroblasts) and CHO (Chinese hamster ovary) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured at 37 °C in 5% CO 2 in Dulbecco modified eagle medium (Gibco BRL Life Technologies, Inc., Carlsbad, CA, USA) containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques:

Journal: Febs Letters

Article Title: Expression and functional characterization of the putative protein 8b of the severe acute respiratory syndrome‐associated coronavirus

doi: 10.1016/j.febslet.2006.05.051

Figure Lengend Snippet: Expression of untagged SARS 6 and SARS 8b mRNAs in Vero E6 cells. RT‐PCR was performed with β‐actin, SARS 6, and/ or SARS 8b primers on mRNAs extracted from control cells and cells transiently transfected with SARS 6 and/or SARS 8b vectors. PCR products for SARS 6, SARS 8b, β‐actin were 192, 255, and 481 bp, respectively.

Article Snippet: Vero E6 (African green monkey kidney fibroblasts) and CHO (Chinese hamster ovary) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured at 37 °C in 5% CO 2 in Dulbecco modified eagle medium (Gibco BRL Life Technologies, Inc., Carlsbad, CA, USA) containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Transfection

Journal: Febs Letters

Article Title: Expression and functional characterization of the putative protein 8b of the severe acute respiratory syndrome‐associated coronavirus

doi: 10.1016/j.febslet.2006.05.051

Figure Lengend Snippet: Stimulation of DNA synthesis by untagged SARS 8b expression. [ 3 H]‐thymidine incorporation assays were performed on (A) Vero E6 cells and (B) CHO cells transiently transfected with vector control and SARS 8b vectors. Each bar represents the means ± S.D. of three experiments in four‐ to six‐replicate setup. ∗ Significant difference between the control and SARS 8b‐expressing cells detected by Student's t test ( p < 0.05).

Article Snippet: Vero E6 (African green monkey kidney fibroblasts) and CHO (Chinese hamster ovary) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured at 37 °C in 5% CO 2 in Dulbecco modified eagle medium (Gibco BRL Life Technologies, Inc., Carlsbad, CA, USA) containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: DNA Synthesis, Expressing, Transfection, Plasmid Preparation, Control

Journal: Febs Letters

Article Title: Expression and functional characterization of the putative protein 8b of the severe acute respiratory syndrome‐associated coronavirus

doi: 10.1016/j.febslet.2006.05.051

Figure Lengend Snippet: Stimulation of DNA synthesis by untagged SARS 6 and SARS 8b expression. [ 3 H]‐thymidine incorporation assays were performed on (A) Vero E6 cells and (B) CHO cells transiently transfected with vector controls, SARS 6 and/or SARS 8b, respectively. Each bar represents the means ± S.D. of three to five experiments in four‐ to six‐replicate setup. ∗ Significant differences among the controls and SARS proteins‐expressing cells detected by Kruskal–Wallis ANOVA on Ranks ( p < 0.05).

Article Snippet: Vero E6 (African green monkey kidney fibroblasts) and CHO (Chinese hamster ovary) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured at 37 °C in 5% CO 2 in Dulbecco modified eagle medium (Gibco BRL Life Technologies, Inc., Carlsbad, CA, USA) containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: DNA Synthesis, Expressing, Transfection, Plasmid Preparation

Journal: The Lancet. Microbe

Article Title: Comparative tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 and SARS-CoV with implications for clinical manifestations, transmissibility, and laboratory studies of COVID-19: an observational study

doi: 10.1016/S2666-5247(20)30004-5

Figure Lengend Snippet: Human and non-human cell lines used in the study

Article Snippet: Hamster kidney fibroblast , BHK21 , ATCC CCL-10.

Techniques: