Journal:

Article Title: Helper-Free Foamy Virus Vectors

doi: 10.1089/hum.1998.9.17-2517

Figure Lengend Snippet: Time course of vector production by transient transfection of 293T cells. Three independent transfections were performed with plasmids pCGPES and pCGPMAPΔBel, and at 24, 36, 48, 56, 72, and 120 hr 100 μ l of vector-containing medium was removed from each well and frozen at −80°C until assayed. Samples were then thawed together at room temperature and titered on FAB cells. Controls of cells transfected with vector plasmid alone were negative (<1 AP FFU/ml). Values shown are titers (AP FFU/ml), with each symbol type representing a different transfection.

Article Snippet: Cell culture and virus production The cell lines used included human embryonic kidney 293 cells (ATCC CRL-1573), simian virus 40 (SV40) T antigen-transformed 293T cells ( DuBridge et al ., 1987 ), NIH 3T3 TK − mouse fibroblasts ( Wei et al ., 1981 ), human fibrosarcoma HT-1080 cells ( Rasheed et al ., 1974 ), SV40-transformed African green monkey kidney COS-1 cells (ATCC CRL-1650), baby hamster kidney BHK-21 cells ( Macpherson and Stoker, 1962 ), normal human foreskin fibroblasts ( Palmer et al ., 1987 ), and FAB hamster HFV β -galactosidase ( β -Gal) indicator cells ( Yu and Linial, 1993 ).

Techniques: Plasmid Preparation, Transfection

Journal:

Article Title: Helper-Free Foamy Virus Vectors

doi: 10.1089/hum.1998.9.17-2517

Figure Lengend Snippet: Comparison of vector harvest methods. 293T cells were transfected with both the helper construct pCGPES and vector construct pCGPMAPΔBel, and 72 hr later supernatants (vector-containing medium) or suspensions of cells and supernatants were harvested as follows and titered on FAB cells. Harvest conditions included supernatant filtered without freezing (SUP); filtered supernatant that was frozen at −80°C for 30 min, then thawed at room temperature (SUP 1×F/T); filtered supernatant with glycerol added to 40% (v/v) that was frozen at −80°C for 30 min, then thawed at room temperature (SUP 1×F/T GLYC); supernatant combined with a suspension of the transfected cells that was frozen at −80°C for 30 min, thawed at room temperature, then filtered (SUSP 1×F/T); or supernatant combined with a suspension of the transfected cells that was frozen in an ethanol–dry ice bath and thawed at 37°C three times, then filtered (SUSP 3×F/T). Controls of untransfected cells or cells transfected with vector plasmid pCGPMAPΔBel alone produced no AP FFU (<1/ml). Supernatant from a control well transfected with the same transfection mixture used for all treatments was tested by FAB assay and did not contain Bel-dependent RCR (<1 BFFU/ml). Mean titer values (AP FFU/ml) with standard errors from three independent measurements are plotted. Values in parentheses indicate the fold reduction as compared with unfrozen supernatants.

Article Snippet: Cell culture and virus production The cell lines used included human embryonic kidney 293 cells (ATCC CRL-1573), simian virus 40 (SV40) T antigen-transformed 293T cells ( DuBridge et al ., 1987 ), NIH 3T3 TK − mouse fibroblasts ( Wei et al ., 1981 ), human fibrosarcoma HT-1080 cells ( Rasheed et al ., 1974 ), SV40-transformed African green monkey kidney COS-1 cells (ATCC CRL-1650), baby hamster kidney BHK-21 cells ( Macpherson and Stoker, 1962 ), normal human foreskin fibroblasts ( Palmer et al ., 1987 ), and FAB hamster HFV β -galactosidase ( β -Gal) indicator cells ( Yu and Linial, 1993 ).

Techniques: Comparison, Plasmid Preparation, Transfection, Construct, Suspension, Produced, Control

Journal: Neuron

Article Title: Targeted proteoform mapping uncovers specific Neurexin-3 variants required for dendritic inhibition

doi: 10.1016/j.neuron.2022.04.017

Figure Lengend Snippet:

Article Snippet: COS-7 monkey kidney cells , ATCC , #CRL-1651.

Techniques: Virus, Recombinant, Reporter Assay, RNAscope, Multiplex Assay, Plasmid Preparation, Software, Targeted Proteomics, Microscopy

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: Frontiers in Immunology

Article Title: Uncoupling Splicing From Transcription Using Antisense Oligonucleotides Reveals a Dual Role for I Exon Donor Splice Sites in Antibody Class Switching

doi: 10.3389/fimmu.2020.00780

Figure Lengend Snippet: Specific IgG1 class switching inhibition in B cells treated by Iγ1 exon dss ASO. Splenic B cells were isolated from C57BL / 6 mice, stimulated with anti-CD40 + IL4 and treated with 2 μM Iγ1 dss ASO (ASO) or an irrelevant ASO (control) for 2 days (B,E) or 4 days (C,D,F,G) . (A) Schematic representation of unspliced ε GLT. Iε, ε I exon; Sε, ε switch region; CH1ε, ε constant exon 1; dss, donor splice site. (B,E) Unspliced γ1 (B) and ε (E) GLT expression monitored as described in . Iε-for-Q and SεU-rev-Q primers, described in schema A , were used for ε GLT expression determination. (C,F) Post-switch Iμ-Cγ1 and Iμ-Cε mRNA expressions monitored as described in . (D,G) Quantification of IgG1 and IgE in culture supernatants by ELISA. (B–G) Data are means ± SEM of two independent experiments, n = 3–4 for each group. Unpaired two-tailed Student's t test was used to determine significance. ns: non-significant, ** P <0.01, *** P <0.001.

Article Snippet: Splenic B cells were stimulated with either 1 or 5 μg/ml lipopolysaccharide (LPS) (LPS-EB Ultrapure, InvivoGen), 1 μg/ml LPS + 20 ng/ml interleukin 4 (IL4) (recombinant murine IL-4, PeproTech), or 5 μg/ml anti-CD40 [mouse CD40/TNFRSF5 MAb (Clone 1C10), Bio-Techne, USA] + 40 ng/ml IL4.

Techniques: Inhibition, Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test