Structured Review

TaKaRa n terminal gfp tag
CyDye fluorescent Western blots. a HeLa cell protein lysates present in unpurified and unbound lysate fractions probed with primary antibodies <t>anti-GFP</t> and anti-GAPDH and with secondary antibodies goat-anti-rabbit IgG Cy5 and goat-anti-mouse IgG Cy3. b Purified GFP-tagged <t>DNMT1</t> protein (DNMT1 225 kDa) and GFP (27 KDa) and DNMT1 variants generated by site-directed mutagenesis. The GFP bands in the fusion protein lanes in ( b ) likely represent cleavage products of the purified fusion protein
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1) Product Images from "Genetic variation affecting DNA methylation and the human imprinting disorder, Beckwith-Wiedemann syndrome"

Article Title: Genetic variation affecting DNA methylation and the human imprinting disorder, Beckwith-Wiedemann syndrome

Journal: Clinical Epigenetics

doi: 10.1186/s13148-018-0546-4

CyDye fluorescent Western blots. a HeLa cell protein lysates present in unpurified and unbound lysate fractions probed with primary antibodies anti-GFP and anti-GAPDH and with secondary antibodies goat-anti-rabbit IgG Cy5 and goat-anti-mouse IgG Cy3. b Purified GFP-tagged DNMT1 protein (DNMT1 225 kDa) and GFP (27 KDa) and DNMT1 variants generated by site-directed mutagenesis. The GFP bands in the fusion protein lanes in ( b ) likely represent cleavage products of the purified fusion protein
Figure Legend Snippet: CyDye fluorescent Western blots. a HeLa cell protein lysates present in unpurified and unbound lysate fractions probed with primary antibodies anti-GFP and anti-GAPDH and with secondary antibodies goat-anti-rabbit IgG Cy5 and goat-anti-mouse IgG Cy3. b Purified GFP-tagged DNMT1 protein (DNMT1 225 kDa) and GFP (27 KDa) and DNMT1 variants generated by site-directed mutagenesis. The GFP bands in the fusion protein lanes in ( b ) likely represent cleavage products of the purified fusion protein

Techniques Used: Western Blot, Purification, Generated, Mutagenesis

2) Product Images from "GSK3β-mediated Ser156 phosphorylation modulates a BH3-like domain in BCL2L12 during TMZ-induced apoptosis and autophagy in glioma cells"

Article Title: GSK3β-mediated Ser156 phosphorylation modulates a BH3-like domain in BCL2L12 during TMZ-induced apoptosis and autophagy in glioma cells

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2018.3672

Proposed mechanism of Ser156 phosphorylation as an allosteric site to modulate a BH3-like domain on BCL2L12 in glioma cells. When BCL2/BCL-XL interacts with the Beclin-1 BH3 domain, autophagy is inhibited. Overexpression of BCL2L12 may displace Beclin-1 in integrating with BCL2/BCL-XL via its BH3-like domain, leading to release of Beclin-1 and initiation of the autophagy process. In addition, since BCL2L12 occupies the hydrophobic groove of BCL2/BCL-XL, BH3 only BCL2 activator or sensitizer is unable to gain access, and the gross result of anti-apoptosis is observed. The BH3 domain mimetic agent, ABT-737, also binds to BCL2/BCL-XL, and hence competes and disrupts the interaction between BCL2/BCL-XL and BCL2L12, making tumor cells more vulnerable to apoptosis. Of note, GSK3β-mediated BCL2L12 S156 phosphorylation may affect BH3 domain function in glioma cells. BCL2L12, BCL2-like 12; BCL2, BCL2 apoptosis regulator; BCL-XL, BCL-extra large; GSK3β, glycogen synthase kinase 3β.
Figure Legend Snippet: Proposed mechanism of Ser156 phosphorylation as an allosteric site to modulate a BH3-like domain on BCL2L12 in glioma cells. When BCL2/BCL-XL interacts with the Beclin-1 BH3 domain, autophagy is inhibited. Overexpression of BCL2L12 may displace Beclin-1 in integrating with BCL2/BCL-XL via its BH3-like domain, leading to release of Beclin-1 and initiation of the autophagy process. In addition, since BCL2L12 occupies the hydrophobic groove of BCL2/BCL-XL, BH3 only BCL2 activator or sensitizer is unable to gain access, and the gross result of anti-apoptosis is observed. The BH3 domain mimetic agent, ABT-737, also binds to BCL2/BCL-XL, and hence competes and disrupts the interaction between BCL2/BCL-XL and BCL2L12, making tumor cells more vulnerable to apoptosis. Of note, GSK3β-mediated BCL2L12 S156 phosphorylation may affect BH3 domain function in glioma cells. BCL2L12, BCL2-like 12; BCL2, BCL2 apoptosis regulator; BCL-XL, BCL-extra large; GSK3β, glycogen synthase kinase 3β.

Techniques Used: Over Expression

3) Product Images from "Cell‐based high‐throughput screen for small molecule inhibitors of Bax translocation, et al. Cell‐based high‐throughput screen for small molecule inhibitors of Bax translocation"

Article Title: Cell‐based high‐throughput screen for small molecule inhibitors of Bax translocation, et al. Cell‐based high‐throughput screen for small molecule inhibitors of Bax translocation

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.14076

Enhanced green fluorescent protein (EGFP)‐Bax exhibits similar spatial and temporal redistribution as endogenous Bax. (A) Temporal pattern of Bax translocation for EGFP‐Bax and Bax following staurosporine‐mediated induction of programmed cell death. Relative Bax translocation at 0, 3, 6 and 9 h following ultraviolet C (UVC) treatment. Data shown are means ± SEM for experiments performed in triplicates in which ≥100 cells per test condition were examined for each group. No significant differences were observed between endogenous Bax and EGFP‐Bax. (B) In order to determine the fidelity of translocation for EGFP‐Bax, the subcellular localization of EGFP‐Bax was compared in cells to that seen for mitochondrial marker Tom20 as determined by immunofluorescence; scale bar: 5 μm
Figure Legend Snippet: Enhanced green fluorescent protein (EGFP)‐Bax exhibits similar spatial and temporal redistribution as endogenous Bax. (A) Temporal pattern of Bax translocation for EGFP‐Bax and Bax following staurosporine‐mediated induction of programmed cell death. Relative Bax translocation at 0, 3, 6 and 9 h following ultraviolet C (UVC) treatment. Data shown are means ± SEM for experiments performed in triplicates in which ≥100 cells per test condition were examined for each group. No significant differences were observed between endogenous Bax and EGFP‐Bax. (B) In order to determine the fidelity of translocation for EGFP‐Bax, the subcellular localization of EGFP‐Bax was compared in cells to that seen for mitochondrial marker Tom20 as determined by immunofluorescence; scale bar: 5 μm

Techniques Used: Translocation Assay, Marker, Immunofluorescence

Time‐dependent and Bcl‐w‐suppressible translocation of EGFP‐Bax following programmed cell death (PCD) initiation in Certified Chinese Hamster Ovary (CHO) and human embryonic kidney (HEK) 293T cells. (A‐F) Following transfection with enhanced green fluorescent protein (EGFP)‐Bax (48 h), CHO and HEK 293T cells were stimulated with 2 μM staurosporine or UVC irradiation (not shown) to initiate PCD. Subcellular localization of EGFP‐Bax was then examined at 0, 4 and 8 h following treatment. EGFP‐Bax was initially distributed homogeneously throughout the cell cytoplasm prior to PCD stimulation in CHO (A) and HEK 293T (D) cells. As observed at 4 (B, E) and 8 h (C, F) following staurosporine treatment, both CHO and HEK 293T cells exhibited punctate re‐localization of EGFP‐Bax to perinuclear regions of the cell. (G‐J) The ability of Bcl‐2 family proteins to functionally suppress EGFP‐Bax translocation was examined using Bcl‐w. Forty‐eight hours following transfection in HEK 293T cells, 2 μM staurosporine was used to initiate PCD. No difference in initial cytoplasmic distribution of EGFP‐Bax was observed prior to staurosporine treatment regardless of Bcl‐w status (G, I). At 8 h following staurosporine treatment, substantially fewer cells co‐transfected with Bcl‐w exhibited a punctate redistribution of EGFP‐Bax compared to cells transfected with EGFP‐Bax alone (H, J); scale bar: 5 μm
Figure Legend Snippet: Time‐dependent and Bcl‐w‐suppressible translocation of EGFP‐Bax following programmed cell death (PCD) initiation in Certified Chinese Hamster Ovary (CHO) and human embryonic kidney (HEK) 293T cells. (A‐F) Following transfection with enhanced green fluorescent protein (EGFP)‐Bax (48 h), CHO and HEK 293T cells were stimulated with 2 μM staurosporine or UVC irradiation (not shown) to initiate PCD. Subcellular localization of EGFP‐Bax was then examined at 0, 4 and 8 h following treatment. EGFP‐Bax was initially distributed homogeneously throughout the cell cytoplasm prior to PCD stimulation in CHO (A) and HEK 293T (D) cells. As observed at 4 (B, E) and 8 h (C, F) following staurosporine treatment, both CHO and HEK 293T cells exhibited punctate re‐localization of EGFP‐Bax to perinuclear regions of the cell. (G‐J) The ability of Bcl‐2 family proteins to functionally suppress EGFP‐Bax translocation was examined using Bcl‐w. Forty‐eight hours following transfection in HEK 293T cells, 2 μM staurosporine was used to initiate PCD. No difference in initial cytoplasmic distribution of EGFP‐Bax was observed prior to staurosporine treatment regardless of Bcl‐w status (G, I). At 8 h following staurosporine treatment, substantially fewer cells co‐transfected with Bcl‐w exhibited a punctate redistribution of EGFP‐Bax compared to cells transfected with EGFP‐Bax alone (H, J); scale bar: 5 μm

Techniques Used: Translocation Assay, Transfection, Irradiation

Intra‐ and inter‐plate variability of 384‐well assay for automated enhanced green fluorescent protein (EGFP)‐Bax translocation and identification of small molecule inhibitors of Bax translocation. Assay variability was assessed by examining cisplatin‐induced EGFP‐Bax translocation in six 384‐well plates (2304 wells) following cisplatin treatment. For each well, n ≥ 250 cells were examined. (A) Data points represent the level of observed EGFP‐Bax translocation as determined in single wells. Data collected from each plate are represented by different colours normalized to 100% mean. Red and blue and dotted lines represent mean ± 1 and 3 SDs, respectively. (B) Frequency distribution of control data presented in (A) demonstrating normalized distribution data. For control cisplatin data, 99.78% of values fall within 3σ of the normalized mean. Dotted lines represent successive ±SDs away from the normalized mean. Graph line represents idealized normal distribution for this dataset. (C) High‐throughput screening of four chemical libraries (6246 GRAS compounds) identified eight targets who response differed by > 3σ from the cisplatin controls dataset. These were selected for secondary screening together with 10 compounds whose B score suggested them as possible hits in comparison to controls. Control data from (A) are shown for reference. For each well, n ≥ 250 cells were examined. Blue dotted lines represent the mean ± 3 SD
Figure Legend Snippet: Intra‐ and inter‐plate variability of 384‐well assay for automated enhanced green fluorescent protein (EGFP)‐Bax translocation and identification of small molecule inhibitors of Bax translocation. Assay variability was assessed by examining cisplatin‐induced EGFP‐Bax translocation in six 384‐well plates (2304 wells) following cisplatin treatment. For each well, n ≥ 250 cells were examined. (A) Data points represent the level of observed EGFP‐Bax translocation as determined in single wells. Data collected from each plate are represented by different colours normalized to 100% mean. Red and blue and dotted lines represent mean ± 1 and 3 SDs, respectively. (B) Frequency distribution of control data presented in (A) demonstrating normalized distribution data. For control cisplatin data, 99.78% of values fall within 3σ of the normalized mean. Dotted lines represent successive ±SDs away from the normalized mean. Graph line represents idealized normal distribution for this dataset. (C) High‐throughput screening of four chemical libraries (6246 GRAS compounds) identified eight targets who response differed by > 3σ from the cisplatin controls dataset. These were selected for secondary screening together with 10 compounds whose B score suggested them as possible hits in comparison to controls. Control data from (A) are shown for reference. For each well, n ≥ 250 cells were examined. Blue dotted lines represent the mean ± 3 SD

Techniques Used: Translocation Assay, High Throughput Screening Assay

4) Product Images from "The Stress-Inducible Protein DRR1 Exerts Distinct Effects on Actin Dynamics"

Article Title: The Stress-Inducible Protein DRR1 Exerts Distinct Effects on Actin Dynamics

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19123993

DRR1 enhances actin bundling via its two actin binding regions and potentially through homo-dimerization. ( A ) DRR1 enhances bundling of F-actin in a concentration-dependent manner. Z-stacks from actin networks polymerized at room temperature (RT) for > 2 h in the presence of DRR1 and visualized with phalloidin-488. Scale bar denotes 50 µm. In all panels, “R” refers to the molar ratio of recombinant protein: actin protein; ( B ) Z-stacks from actin networks polymerized at RT for > 2 h in the presence of DRR1 proteins (R = 0.5) and visualized with phalloidin-488. Control = MBP added, because the DRR1 proteins are MBP-tagged. Scale bar denotes 20 µm.
Figure Legend Snippet: DRR1 enhances actin bundling via its two actin binding regions and potentially through homo-dimerization. ( A ) DRR1 enhances bundling of F-actin in a concentration-dependent manner. Z-stacks from actin networks polymerized at room temperature (RT) for > 2 h in the presence of DRR1 and visualized with phalloidin-488. Scale bar denotes 50 µm. In all panels, “R” refers to the molar ratio of recombinant protein: actin protein; ( B ) Z-stacks from actin networks polymerized at RT for > 2 h in the presence of DRR1 proteins (R = 0.5) and visualized with phalloidin-488. Control = MBP added, because the DRR1 proteins are MBP-tagged. Scale bar denotes 20 µm.

Techniques Used: Binding Assay, Concentration Assay, Recombinant

DRR1 bundling effect diminishes cellular actin treadmilling. DRR1 wt and the mutant dN—but none of the other mutants–slow down actin treadmilling in HeLa cells. Fluorescence recovery after photobleaching (FRAP) in HeLa cells co-transfected with plasmids expressing GFP-actin and untagged DRR1 wt, dN, dC, dM, and M was recorded. Representative cells are shown. Quantification was performed in ImageJ (25–30 cells from 2–3 independent experiments). Scale bar denotes 20 µm. Movies of FRAP experiments are available on request.
Figure Legend Snippet: DRR1 bundling effect diminishes cellular actin treadmilling. DRR1 wt and the mutant dN—but none of the other mutants–slow down actin treadmilling in HeLa cells. Fluorescence recovery after photobleaching (FRAP) in HeLa cells co-transfected with plasmids expressing GFP-actin and untagged DRR1 wt, dN, dC, dM, and M was recorded. Representative cells are shown. Quantification was performed in ImageJ (25–30 cells from 2–3 independent experiments). Scale bar denotes 20 µm. Movies of FRAP experiments are available on request.

Techniques Used: Mutagenesis, Fluorescence, Transfection, Expressing

DRR1 reduces actin filament elongation but increases nucleation. ( A , B ) DRR1 and the mutant dM exert an inhibitory effect on in vitro polymerization of pyrene-actin. 20% pyrene-labeled actin (4 µM) was polymerized in the presence of wt ( A , B ) and mutant ( B ) DRR1 proteins (purified via the MBP-tag) as indicated. Increase in fluorescence of pyrene-actin during polymerization was monitored in 5 s intervals for 90 min; ( C ) Single filament elongation of actin is strongly reduced by DRR1 and the mutant dM. Actin (c = 0.5 µM, 10% labeled with ATTO-488) was polymerized in the presence of DRR1 proteins or MBP as control (R = 0.5) and visualized by TIRF microscopy for 10 min with 3 s intervals starting 2 min after the beginning of the reaction. An endpoint image was taken at 2 h of polymerization. Scale bar denotes 10 µm for all images. Bars indicating the filament elongation rate and the nucleation rate represent means + SEM of three independent experiments. */ # p
Figure Legend Snippet: DRR1 reduces actin filament elongation but increases nucleation. ( A , B ) DRR1 and the mutant dM exert an inhibitory effect on in vitro polymerization of pyrene-actin. 20% pyrene-labeled actin (4 µM) was polymerized in the presence of wt ( A , B ) and mutant ( B ) DRR1 proteins (purified via the MBP-tag) as indicated. Increase in fluorescence of pyrene-actin during polymerization was monitored in 5 s intervals for 90 min; ( C ) Single filament elongation of actin is strongly reduced by DRR1 and the mutant dM. Actin (c = 0.5 µM, 10% labeled with ATTO-488) was polymerized in the presence of DRR1 proteins or MBP as control (R = 0.5) and visualized by TIRF microscopy for 10 min with 3 s intervals starting 2 min after the beginning of the reaction. An endpoint image was taken at 2 h of polymerization. Scale bar denotes 10 µm for all images. Bars indicating the filament elongation rate and the nucleation rate represent means + SEM of three independent experiments. */ # p

Techniques Used: Mutagenesis, In Vitro, Labeling, Purification, Fluorescence, Microscopy

In the presence of profilin, DRR1 and the mutants dM and dC block elongation more effectively, suggesting DRR1 as a novel barbed end capping factor. ( A , B ) Pyrene-actin polymerization is blocked by DRR1 and the mutant dM at R = 0.5 in the presence of profilin (12 µM). 20% pyrene-labeled actin (4 µM) was polymerized in the presence of wt ( A , B ) and mutant ( B ) DRR1 proteins (purified via the MBP tag) as indicated. An increase in fluorescence of pyrene-actin during polymerization was monitored in 5 s intervals for 60 min; ( C ) Visualization of actin in vitro polymerization by TIRF microscopy (c = 0.5 µM, 10% labeled with ATTO-488) in the presence of profilin (1.5 µM). Actin was polymerized in the presence of DRR1 proteins for 10 min with 3 s intervals imaging starting 2 min after the beginning of the reaction. An endpoint image was taken at 2 h of polymerization. Scale bar denotes 10 µm for all images. Bars indicating the filament elongation rate represent means + SEM of three independent experiments. */ # p
Figure Legend Snippet: In the presence of profilin, DRR1 and the mutants dM and dC block elongation more effectively, suggesting DRR1 as a novel barbed end capping factor. ( A , B ) Pyrene-actin polymerization is blocked by DRR1 and the mutant dM at R = 0.5 in the presence of profilin (12 µM). 20% pyrene-labeled actin (4 µM) was polymerized in the presence of wt ( A , B ) and mutant ( B ) DRR1 proteins (purified via the MBP tag) as indicated. An increase in fluorescence of pyrene-actin during polymerization was monitored in 5 s intervals for 60 min; ( C ) Visualization of actin in vitro polymerization by TIRF microscopy (c = 0.5 µM, 10% labeled with ATTO-488) in the presence of profilin (1.5 µM). Actin was polymerized in the presence of DRR1 proteins for 10 min with 3 s intervals imaging starting 2 min after the beginning of the reaction. An endpoint image was taken at 2 h of polymerization. Scale bar denotes 10 µm for all images. Bars indicating the filament elongation rate represent means + SEM of three independent experiments. */ # p

Techniques Used: Blocking Assay, Mutagenesis, Labeling, Purification, Fluorescence, In Vitro, Microscopy, Imaging

Downregulated in renal cell carcinoma 1 (DRR1) features an actin binding site at each terminus. ( A ) Domain structure of DRR1 wt and mutants. DRR1 harbors a conserved domain of unknown function from amino acid 16–133. Secondary structure prediction was performed with the “Predict Protein Server” (dark: helix, light: loop; https://www.predictprotein.org/ , accessed on 31 July 2012). Coiled coil prediction performed with “Coils” ( http://embnet.vital-it.ch/software/COILS_form.html ; accessed no 10 December 2018); ( B ) Co-immunoprecipitation of actin with DRR1 wt and mutants fused to Enhanced Green Fluorescent Protein EGFP overexpressed in Human embryonic kidney 293 cells (HEK)-293 cells using Green Fluorescent Protein (GFP)-Trap ® beads. Control was performed with EGFP alone. Lysate and eluate samples were analyzed by SDS-PAGE and Western blot. A representative Western blot is shown; ( C ) Quantification of Co-immunoprecipitation ( n = 8, dN and M n = 7); ( D ) Co-sedimentation of recombinant wt and mutant DRR1 protein with preformed F-actin by ultracentrifugation. Coomassie-stained sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis (PAGE) with total (T), supernatant (S) and pellet (P) fractions are shown; ( E ) Quantification of co-sedimented protein ( n = 3). Bars represent means + SEM. ** /## p
Figure Legend Snippet: Downregulated in renal cell carcinoma 1 (DRR1) features an actin binding site at each terminus. ( A ) Domain structure of DRR1 wt and mutants. DRR1 harbors a conserved domain of unknown function from amino acid 16–133. Secondary structure prediction was performed with the “Predict Protein Server” (dark: helix, light: loop; https://www.predictprotein.org/ , accessed on 31 July 2012). Coiled coil prediction performed with “Coils” ( http://embnet.vital-it.ch/software/COILS_form.html ; accessed no 10 December 2018); ( B ) Co-immunoprecipitation of actin with DRR1 wt and mutants fused to Enhanced Green Fluorescent Protein EGFP overexpressed in Human embryonic kidney 293 cells (HEK)-293 cells using Green Fluorescent Protein (GFP)-Trap ® beads. Control was performed with EGFP alone. Lysate and eluate samples were analyzed by SDS-PAGE and Western blot. A representative Western blot is shown; ( C ) Quantification of Co-immunoprecipitation ( n = 8, dN and M n = 7); ( D ) Co-sedimentation of recombinant wt and mutant DRR1 protein with preformed F-actin by ultracentrifugation. Coomassie-stained sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis (PAGE) with total (T), supernatant (S) and pellet (P) fractions are shown; ( E ) Quantification of co-sedimented protein ( n = 3). Bars represent means + SEM. ** /## p

Techniques Used: Binding Assay, Software, Immunoprecipitation, SDS Page, Western Blot, Sedimentation, Recombinant, Mutagenesis, Staining, Polyacrylamide Gel Electrophoresis

DRR1 modulates actin-dependent processes in cells. ( A ) DRR1 wt and the mutants dN, dC, and dM inhibit spreading of HeLa cells. Cells were transfected with constructs expressing EGFP-DRR1 wt or mutants (control: EGFP), cultivated for 24 h and re-plated on fibronectin-coated coverslips. After 30 min, cells were fixed and F-actin was stained with phalloidin. Representative cells are displayed (green: EGFP or EGFP-DRR1; red: F-actin). Scale bar denotes 20 µm. Bars represent mean cell sizes + SEM of four independent experiments (50–200 cells in each experiment). * p
Figure Legend Snippet: DRR1 modulates actin-dependent processes in cells. ( A ) DRR1 wt and the mutants dN, dC, and dM inhibit spreading of HeLa cells. Cells were transfected with constructs expressing EGFP-DRR1 wt or mutants (control: EGFP), cultivated for 24 h and re-plated on fibronectin-coated coverslips. After 30 min, cells were fixed and F-actin was stained with phalloidin. Representative cells are displayed (green: EGFP or EGFP-DRR1; red: F-actin). Scale bar denotes 20 µm. Bars represent mean cell sizes + SEM of four independent experiments (50–200 cells in each experiment). * p

Techniques Used: Transfection, Construct, Expressing, Staining

5) Product Images from "BZLF1 interacts with chromatin remodelers promoting escape from latent infections with EBV"

Article Title: BZLF1 interacts with chromatin remodelers promoting escape from latent infections with EBV

Journal: Life Science Alliance

doi: 10.26508/lsa.201800108

ATAC-seq coverage at the 66 BZLF1-binding sites in Raji EBV chromatin in non-induced and induced AD-truncated Raji p5694 cells. The two heatmaps summarize the individual ATAC-seq coverage at the identified 66 BZLF1 ChIP-seq peaks in Raji EBV chromatin arranged in hierarchical order. The left and right panels illustrate the situation in non-induced AD-truncated Raji p5694 cells (truncated, non-induced) and cells induced for 15 h (truncated, induced), respectively. The data show the mean of three independent ATAC-seq experiments.
Figure Legend Snippet: ATAC-seq coverage at the 66 BZLF1-binding sites in Raji EBV chromatin in non-induced and induced AD-truncated Raji p5694 cells. The two heatmaps summarize the individual ATAC-seq coverage at the identified 66 BZLF1 ChIP-seq peaks in Raji EBV chromatin arranged in hierarchical order. The left and right panels illustrate the situation in non-induced AD-truncated Raji p5694 cells (truncated, non-induced) and cells induced for 15 h (truncated, induced), respectively. The data show the mean of three independent ATAC-seq experiments.

Techniques Used: Binding Assay, Chromatin Immunoprecipitation

Conditional expression of BZLF1 in Raji cells. (A) The conditional expression plasmid p4816 encodes the unmodified full-length BZLF1 (aa 1–245) protein. The conditional expression plasmids p5693 and p5694 encode FLAG- and tandem Strep-tagged full-length BZLF1 (aa 1–245) and the activation domain (AD) truncated BZLF1 (aa 175–236) protein also termed bZIP, respectively. The bicistronic coding sequences of the tetracycline-controlled repressor rTS and transactivator rtTA2S-M2 are shown. The addition of doxycycline induces bidirectional transcription and concomitant expression of three transgenes: enhanced GFP (eGFP), the human truncated NGF-receptor (tNGF-R), and BZLF1. An internal ribosomal entry site (IRES) separates tNGF-R and eGFP. β-lactamase (amp) and puromycin N-acetyl-transferase (puro) serve as resistance genes in bacteria and Raji cells, respectively. DNA replication in E . coli initiates at the prokaryotic origin of replication (ori). Epstein–Barr nuclear antigen 1 (EBNA1) binds to the origin of plasmid replication (oriP) and supports extrachromosomal DNA replication of the plasmids in Raji cells. (B) The conditional expression plasmids (p4816, p5693, and p5694) were stably introduced into Raji cells, and single-cell clones were selected by limiting dilution, expanded, and analyzed further. GFP expression in Raji cells was a measure of the induced expression of BZLF1. Upon addition of 100 ng/ml doxycycline to the cells for 15 h, GFP expression was analyzed by flow cytometry.
Figure Legend Snippet: Conditional expression of BZLF1 in Raji cells. (A) The conditional expression plasmid p4816 encodes the unmodified full-length BZLF1 (aa 1–245) protein. The conditional expression plasmids p5693 and p5694 encode FLAG- and tandem Strep-tagged full-length BZLF1 (aa 1–245) and the activation domain (AD) truncated BZLF1 (aa 175–236) protein also termed bZIP, respectively. The bicistronic coding sequences of the tetracycline-controlled repressor rTS and transactivator rtTA2S-M2 are shown. The addition of doxycycline induces bidirectional transcription and concomitant expression of three transgenes: enhanced GFP (eGFP), the human truncated NGF-receptor (tNGF-R), and BZLF1. An internal ribosomal entry site (IRES) separates tNGF-R and eGFP. β-lactamase (amp) and puromycin N-acetyl-transferase (puro) serve as resistance genes in bacteria and Raji cells, respectively. DNA replication in E . coli initiates at the prokaryotic origin of replication (ori). Epstein–Barr nuclear antigen 1 (EBNA1) binds to the origin of plasmid replication (oriP) and supports extrachromosomal DNA replication of the plasmids in Raji cells. (B) The conditional expression plasmids (p4816, p5693, and p5694) were stably introduced into Raji cells, and single-cell clones were selected by limiting dilution, expanded, and analyzed further. GFP expression in Raji cells was a measure of the induced expression of BZLF1. Upon addition of 100 ng/ml doxycycline to the cells for 15 h, GFP expression was analyzed by flow cytometry.

Techniques Used: Expressing, Plasmid Preparation, Activation Assay, Stable Transfection, Clone Assay, Flow Cytometry, Cytometry

6) Product Images from "Src activation by Chk1 promotes actin patch formation and prevents chromatin bridge breakage in cytokinesis"

Article Title: Src activation by Chk1 promotes actin patch formation and prevents chromatin bridge breakage in cytokinesis

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201802102

Expression of dominant-negative Vps4-K173Q does not prevent chromatin breakage in Src or Chk1-deficient cells. (A and C) Examples of cells with DNA bridges exhibiting broken or intact intercellular canals after labeling with a lipophilic dye. Cells were transfected with negative siRNA (control), siSrc, or siChk1, or treated with TG003 for 5 h and stained with FM 1-43FX. (B) Frequency of cells with broken DNA bridges exhibiting intact intercellular canals. Error bars show the SD from the mean from three independent experiments. 15 cells with broken chromatin bridges were analyzed per experiment. (D) Cells expressing GFP or GFP–Vps4-K173Q were transfected with Aurora B siRNA (siAurora B; siAurB) or treated as in A–C. Broken DNA bridges or intercellular canals are indicated by dotted arrows. Bars, 5 µm. (E) Percentage of DNA bridges that appear broken in GFP-positive cells. Error bars show the SD from the mean from three independent experiments. A minimum of 30 cells with chromatin bridges was analyzed per experiment. ***, P
Figure Legend Snippet: Expression of dominant-negative Vps4-K173Q does not prevent chromatin breakage in Src or Chk1-deficient cells. (A and C) Examples of cells with DNA bridges exhibiting broken or intact intercellular canals after labeling with a lipophilic dye. Cells were transfected with negative siRNA (control), siSrc, or siChk1, or treated with TG003 for 5 h and stained with FM 1-43FX. (B) Frequency of cells with broken DNA bridges exhibiting intact intercellular canals. Error bars show the SD from the mean from three independent experiments. 15 cells with broken chromatin bridges were analyzed per experiment. (D) Cells expressing GFP or GFP–Vps4-K173Q were transfected with Aurora B siRNA (siAurora B; siAurB) or treated as in A–C. Broken DNA bridges or intercellular canals are indicated by dotted arrows. Bars, 5 µm. (E) Percentage of DNA bridges that appear broken in GFP-positive cells. Error bars show the SD from the mean from three independent experiments. A minimum of 30 cells with chromatin bridges was analyzed per experiment. ***, P

Techniques Used: Expressing, Dominant Negative Mutation, Labeling, Transfection, Staining

7) Product Images from "NSs Protein of Sandfly Fever Sicilian Phlebovirus Counteracts Interferon (IFN) Induction by Masking the DNA-Binding Domain of IFN Regulatory Factor 3"

Article Title: NSs Protein of Sandfly Fever Sicilian Phlebovirus Counteracts Interferon (IFN) Induction by Masking the DNA-Binding Domain of IFN Regulatory Factor 3

Journal: Journal of Virology

doi: 10.1128/JVI.01202-18

IFN-β promoter binding assay. HEK293 cells were cotransfected with plasmids encoding eGFP-IRF3 or eGFP or MAVS, as well as with increasing amounts of plasmids encoding 3×FLAG-tagged SFSV NSs, or the 3×FLAG-tagged control protein ΔMx, as indicated. Cell lysates were then incubated with an unlabeled, double-stranded DNA oligonucleotide comprising the IFN-β promoter or with a scrambled control oligonucleotide or were left untreated. Next, streptavidin-coated magnetic beads covered with biotinylated IFN-β promoter oligonucleotide were used to pull down activated IRF3. Bound proteins were eluted by boiling in Laemmli buffer and analyzed by immunoblotting.
Figure Legend Snippet: IFN-β promoter binding assay. HEK293 cells were cotransfected with plasmids encoding eGFP-IRF3 or eGFP or MAVS, as well as with increasing amounts of plasmids encoding 3×FLAG-tagged SFSV NSs, or the 3×FLAG-tagged control protein ΔMx, as indicated. Cell lysates were then incubated with an unlabeled, double-stranded DNA oligonucleotide comprising the IFN-β promoter or with a scrambled control oligonucleotide or were left untreated. Next, streptavidin-coated magnetic beads covered with biotinylated IFN-β promoter oligonucleotide were used to pull down activated IRF3. Bound proteins were eluted by boiling in Laemmli buffer and analyzed by immunoblotting.

Techniques Used: Binding Assay, Incubation, Magnetic Beads

8) Product Images from "Organelle-specific Subunit Interactions of the Vertebrate Two-pore Channel Family *"

Article Title: Organelle-specific Subunit Interactions of the Vertebrate Two-pore Channel Family *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.610493

Subcellular localization of hTPC1 stably expressed in HEK293 cells. A–F , stable cell lines were made in HEK293 cells using mCherry-tagged hTPC1 ( red ) and GFP-tagged organelle markers ( green ). The upper panels of each image show a GFP-labeled organelle ( left ) and mCherry-labeled hTPC1 ( right ). G , a stable HEK293 cell line that expressed GFP-hTPC1 ( green ; left ) and mCherry-hTPC2 ( red ; right ). The lower panels for all images show a merged image (colocalization in yellow ; left ) and a bright-field image for the cell (differential interference contrast; right ). Scale bars = 10 μm. H , Pearson coefficients for cells exemplified in A–G . Data are means ± S.E. of three cells for each pair. TfR , transferrin receptor.
Figure Legend Snippet: Subcellular localization of hTPC1 stably expressed in HEK293 cells. A–F , stable cell lines were made in HEK293 cells using mCherry-tagged hTPC1 ( red ) and GFP-tagged organelle markers ( green ). The upper panels of each image show a GFP-labeled organelle ( left ) and mCherry-labeled hTPC1 ( right ). G , a stable HEK293 cell line that expressed GFP-hTPC1 ( green ; left ) and mCherry-hTPC2 ( red ; right ). The lower panels for all images show a merged image (colocalization in yellow ; left ) and a bright-field image for the cell (differential interference contrast; right ). Scale bars = 10 μm. H , Pearson coefficients for cells exemplified in A–G . Data are means ± S.E. of three cells for each pair. TfR , transferrin receptor.

Techniques Used: Stable Transfection, Labeling

Subcellular localization of hTPC2 stably expressed in HEK293 cells. A–G , stable cell lines were made in HEK293 cells using mCherry-tagged hTPC2 ( red ) and GFP-tagged organelle markers ( green ). The upper panels of each image show a GFP-labeled organelle ( left ) and mCherry-labeled hTPC2 ( right ). The lower panels show a merged image (colocalization in yellow ; left ) and a bright-field image for the cell ( right ). Scale bars = 10 μm. H , Pearson coefficients for cells exemplified in A–G . Data are means ± S.E. of three cells for each pair. TfR , transferrin receptor.
Figure Legend Snippet: Subcellular localization of hTPC2 stably expressed in HEK293 cells. A–G , stable cell lines were made in HEK293 cells using mCherry-tagged hTPC2 ( red ) and GFP-tagged organelle markers ( green ). The upper panels of each image show a GFP-labeled organelle ( left ) and mCherry-labeled hTPC2 ( right ). The lower panels show a merged image (colocalization in yellow ; left ) and a bright-field image for the cell ( right ). Scale bars = 10 μm. H , Pearson coefficients for cells exemplified in A–G . Data are means ± S.E. of three cells for each pair. TfR , transferrin receptor.

Techniques Used: Stable Transfection, Labeling

Interaction between hTPC2 and other TPC isoforms when coexpressed in HEK293 cells. A , expression levels detected by immunoblotting ( IB ) of HA-hTPC2 ( upper panel ), mCherry ( middle left panel ), and mCherry-tagged TPC isoforms ( middle right panel ) in stable HA-hTPC2 cells transiently transfected with the cDNA for mCherry ( vec ) and N-terminal mCherry-tagged hTPC1 ( h1 ), hTPC2 ( h2 ), rTPC3 ( r3 ), or cTPC3 ( c3 ). Actin was used as a loading control ( lower panel ). B , co-IP of HA-hTPC2 by the anti-mCherry antibody. The immunoprecipitants were left untreated ( left panel ) or treated with PNGase F ( right panel ). Samples from mCherry-hTPC2-transfected cells ( h2 ) were loaded as 1/10 ( h2/10 ) and equivalent (h2) amounts compared with the other samples for immunoblotting. Open triangles indicate possible dimers. The filled arrowhead indicates reduced size of possible dimers after deglycosylation by PNGase F. Open arrows indicate the ∼85-kDa band mostly unaffected by PNGase F except for the mCherry-hTPC2-transfected cell samples. The filled arrow indicates the reduced size from the ∼85-kDa band. C , FRET efficiency between GFP and mCherry in HEK293 cells that coexpressed GFP-tagged hTPC2 (either N- or C-terminal tag) and N-terminal mCherry-tagged TPC isoforms as indicated. Data are means ± S.E. for the number of cells indicated in parentheses . ***, p
Figure Legend Snippet: Interaction between hTPC2 and other TPC isoforms when coexpressed in HEK293 cells. A , expression levels detected by immunoblotting ( IB ) of HA-hTPC2 ( upper panel ), mCherry ( middle left panel ), and mCherry-tagged TPC isoforms ( middle right panel ) in stable HA-hTPC2 cells transiently transfected with the cDNA for mCherry ( vec ) and N-terminal mCherry-tagged hTPC1 ( h1 ), hTPC2 ( h2 ), rTPC3 ( r3 ), or cTPC3 ( c3 ). Actin was used as a loading control ( lower panel ). B , co-IP of HA-hTPC2 by the anti-mCherry antibody. The immunoprecipitants were left untreated ( left panel ) or treated with PNGase F ( right panel ). Samples from mCherry-hTPC2-transfected cells ( h2 ) were loaded as 1/10 ( h2/10 ) and equivalent (h2) amounts compared with the other samples for immunoblotting. Open triangles indicate possible dimers. The filled arrowhead indicates reduced size of possible dimers after deglycosylation by PNGase F. Open arrows indicate the ∼85-kDa band mostly unaffected by PNGase F except for the mCherry-hTPC2-transfected cell samples. The filled arrow indicates the reduced size from the ∼85-kDa band. C , FRET efficiency between GFP and mCherry in HEK293 cells that coexpressed GFP-tagged hTPC2 (either N- or C-terminal tag) and N-terminal mCherry-tagged TPC isoforms as indicated. Data are means ± S.E. for the number of cells indicated in parentheses . ***, p

Techniques Used: Expressing, Transfection, Co-Immunoprecipitation Assay

9) Product Images from "hSnm1 Colocalizes and Physically Associates with 53BP1 before and after DNA Damage"

Article Title: hSnm1 Colocalizes and Physically Associates with 53BP1 before and after DNA Damage

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.22.24.8635-8647.2002

hSnm1 focus formation occurs normally in Nijmegen breakage syndrome cells. GM7166 primary fibroblasts were fixed 9 h after mock treatment or after exposure to 15 Gray of ionizing radiation (IR) and subjected to indirect immunofluorescence with anti-hSnm1. (a) Mock-treated cells; (b) after ionizing radiation exposure.
Figure Legend Snippet: hSnm1 focus formation occurs normally in Nijmegen breakage syndrome cells. GM7166 primary fibroblasts were fixed 9 h after mock treatment or after exposure to 15 Gray of ionizing radiation (IR) and subjected to indirect immunofluorescence with anti-hSnm1. (a) Mock-treated cells; (b) after ionizing radiation exposure.

Techniques Used: Immunofluorescence

Colocalization of hSnm1 foci with hMre11 and BRCA1 but not hRad51, as determined by indirect immunofluorescence. HT-1080 cells were irradiated with 10 Gray and stained 2 h later with (a) anti-hSnm1 and (b) anti-hRad51; (c) merged fields after DAPI staining. HT-1080 cells were irradiated with 15 Gray and stained 9 h later with (d) anti-hSnm1 and (e) anti-hMre11; (f) merged fields after DAPI staining. Partial colocalization with BRCA1 ionizing radiation-induced foci was observed 5 h after 10 Gray, as shown by (g) anti-hSnm1 and (h) anti-BRCA1; (i) both fields merged with DAPI staining.
Figure Legend Snippet: Colocalization of hSnm1 foci with hMre11 and BRCA1 but not hRad51, as determined by indirect immunofluorescence. HT-1080 cells were irradiated with 10 Gray and stained 2 h later with (a) anti-hSnm1 and (b) anti-hRad51; (c) merged fields after DAPI staining. HT-1080 cells were irradiated with 15 Gray and stained 9 h later with (d) anti-hSnm1 and (e) anti-hMre11; (f) merged fields after DAPI staining. Partial colocalization with BRCA1 ionizing radiation-induced foci was observed 5 h after 10 Gray, as shown by (g) anti-hSnm1 and (h) anti-BRCA1; (i) both fields merged with DAPI staining.

Techniques Used: Immunofluorescence, Irradiation, Staining

hSnm1 is localized to the nucleus. Indirect immunofluorescence of MCF-7 cells probed with hSnm1 affinity-purified polyclonal antibodies displaying (a) diffuse nuclear staining and hSnm1 bodies (as indicated by white arrow) or (b) multiple nuclear foci. Epifluorescence of MCF-7 cells expressing EGFP-hSnm1 fusion protein showing localization to (c) a nuclear body and (d) foci (transfected cell indicated by white arrow). An undamaged HT-1080 cell transiently expressing the Flag-hSnm1 fusion construct was stained with (e) anti-hSnm1 antibodies and (f) anti-Flag M2 monoclonal antibodies. (g) The two images are shown merged after DAPI staining.
Figure Legend Snippet: hSnm1 is localized to the nucleus. Indirect immunofluorescence of MCF-7 cells probed with hSnm1 affinity-purified polyclonal antibodies displaying (a) diffuse nuclear staining and hSnm1 bodies (as indicated by white arrow) or (b) multiple nuclear foci. Epifluorescence of MCF-7 cells expressing EGFP-hSnm1 fusion protein showing localization to (c) a nuclear body and (d) foci (transfected cell indicated by white arrow). An undamaged HT-1080 cell transiently expressing the Flag-hSnm1 fusion construct was stained with (e) anti-hSnm1 antibodies and (f) anti-Flag M2 monoclonal antibodies. (g) The two images are shown merged after DAPI staining.

Techniques Used: Immunofluorescence, Affinity Purification, Staining, Expressing, Transfection, Construct

Colocalization of EGFP-hSnm1 protein with 53BP1. MCF-7 cells were transfected with pEGFP-hSnm1 followed by staining with anti-53BP1 and Toto-3. Localization of (a) EGFP-hSnm1 and (b) 53BP1 to a nuclear body in the same cell; (c) the two fields merged with Toto-3.
Figure Legend Snippet: Colocalization of EGFP-hSnm1 protein with 53BP1. MCF-7 cells were transfected with pEGFP-hSnm1 followed by staining with anti-53BP1 and Toto-3. Localization of (a) EGFP-hSnm1 and (b) 53BP1 to a nuclear body in the same cell; (c) the two fields merged with Toto-3.

Techniques Used: Transfection, Staining

Coimmunoprecipitation of 53BP1 and hSnm1. (A) HeLa cell extracts were incubated with beads only, preimmune serum, or affinity-purified anti-hSnm1 antibodies. Polyclonal anti-53BP1 antibodies were used to detect 53BP1 by immunoblotting. (B) HEK293 nuclear extracts were prepared with or without infection with EGFP-hSnm1-expressing adenovirus and immunoblotted with monoclonal antibodies against EGFP or polyclonal antibodies against 53BP1. Immunoprecipitations (IP) were performed with these extracts with anti-hSnm1 and anti-53BP1 antibodies as shown.
Figure Legend Snippet: Coimmunoprecipitation of 53BP1 and hSnm1. (A) HeLa cell extracts were incubated with beads only, preimmune serum, or affinity-purified anti-hSnm1 antibodies. Polyclonal anti-53BP1 antibodies were used to detect 53BP1 by immunoblotting. (B) HEK293 nuclear extracts were prepared with or without infection with EGFP-hSnm1-expressing adenovirus and immunoblotted with monoclonal antibodies against EGFP or polyclonal antibodies against 53BP1. Immunoprecipitations (IP) were performed with these extracts with anti-hSnm1 and anti-53BP1 antibodies as shown.

Techniques Used: Incubation, Affinity Purification, Infection, Expressing

Deletion analysis of hSNM1. (A) Various truncations and in-frame deletions were constructed in the hSNM1 segment of the EGFP-hSNM1 fusion gene. The solid black region indicates EGFP, and vertical lines indicate the conserved metallo-β-lactamase domain. Each construct was transfected into HT-1080 cells, and the location of the EGFP signal was determined 24 h later. (B) Expression of EGFP-hSnm1 deletion mutants in HT-1080 cells.
Figure Legend Snippet: Deletion analysis of hSNM1. (A) Various truncations and in-frame deletions were constructed in the hSNM1 segment of the EGFP-hSNM1 fusion gene. The solid black region indicates EGFP, and vertical lines indicate the conserved metallo-β-lactamase domain. Each construct was transfected into HT-1080 cells, and the location of the EGFP signal was determined 24 h later. (B) Expression of EGFP-hSnm1 deletion mutants in HT-1080 cells.

Techniques Used: Construct, Transfection, Expressing

(A) Northern blot of mRNAs from various tissues was probed with hSnm1 cDNA. A probe for β-actin was used to control for loading. (B) In vitro-translated Flag-hSnm1 protein labeled with [ 35 S]methionine was precipitated by anti-hSnm1-protein A-agarose beads, protein A beads only, or preimmune-protein A beads. B and SN indicate beads and supernatant, respectively.
Figure Legend Snippet: (A) Northern blot of mRNAs from various tissues was probed with hSnm1 cDNA. A probe for β-actin was used to control for loading. (B) In vitro-translated Flag-hSnm1 protein labeled with [ 35 S]methionine was precipitated by anti-hSnm1-protein A-agarose beads, protein A beads only, or preimmune-protein A beads. B and SN indicate beads and supernatant, respectively.

Techniques Used: Northern Blot, In Vitro, Labeling

Quantitative analysis of hSnm1 focus induction after DNA damage in MCF-7 cells by indirect immunofluorescence. hSnm1 antibodies were used to probe cells at various times after treatment with either ionizing radiation (IR) or 4HC. The percentages of cells displaying diffuse nuclear staining with fewer than 10 foci (open bars), hSnm1 bodies (hatched bars), and more than 10 foci (black bars) were calculated after scoring at least 100 nuclei for each time point. Reported here are the averages and standard deviations of three data sets.
Figure Legend Snippet: Quantitative analysis of hSnm1 focus induction after DNA damage in MCF-7 cells by indirect immunofluorescence. hSnm1 antibodies were used to probe cells at various times after treatment with either ionizing radiation (IR) or 4HC. The percentages of cells displaying diffuse nuclear staining with fewer than 10 foci (open bars), hSnm1 bodies (hatched bars), and more than 10 foci (black bars) were calculated after scoring at least 100 nuclei for each time point. Reported here are the averages and standard deviations of three data sets.

Techniques Used: Immunofluorescence, Staining

Quantitative analysis of hSnm1 nuclear staining during the cell cycle. (A) Histogram displaying the DNA content of asynchronous untreated MCF-7 cells analyzed by laser scanning cytometry and the observed frequencies of the different hSnm1 staining patterns corresponding to the G 1 , S, and G 2 subpopulations. PI, propidium iodide. (B) Similar data obtained from MCF-7 cells taken 5 h after treatment with 10 Gray of ionizing radiation. The percentages of cells displaying diffuse nuclear staining with fewer than 10 foci (white bars), hSnm1 bodies (hatched bars), and more than 10 foci (black bars) were calculated after scoring at least 150 nuclei for each time point.
Figure Legend Snippet: Quantitative analysis of hSnm1 nuclear staining during the cell cycle. (A) Histogram displaying the DNA content of asynchronous untreated MCF-7 cells analyzed by laser scanning cytometry and the observed frequencies of the different hSnm1 staining patterns corresponding to the G 1 , S, and G 2 subpopulations. PI, propidium iodide. (B) Similar data obtained from MCF-7 cells taken 5 h after treatment with 10 Gray of ionizing radiation. The percentages of cells displaying diffuse nuclear staining with fewer than 10 foci (white bars), hSnm1 bodies (hatched bars), and more than 10 foci (black bars) were calculated after scoring at least 150 nuclei for each time point.

Techniques Used: Staining, Cytometry

Colocalization of hSnm1 and 53BP1 in foci as detected by indirect immunofluorescence. MCF-7 cells were mock treated (a, b, and c) or treated with 10 Gray of ionizing radiation and fixed after 30 min (d, e, and f), after 90 min (g, h, and i), or after 5 h (j, k, and l). (a, d, g, and j) Polyclonal anti-hSnm1 staining with fluorescein isothiocyanate; (b, e, h, and k) monoclonal anti-53BP1 staining with tetramethyl rhodamine isocyanate; (c, f, i, and l) merged fields plus DAPI staining.
Figure Legend Snippet: Colocalization of hSnm1 and 53BP1 in foci as detected by indirect immunofluorescence. MCF-7 cells were mock treated (a, b, and c) or treated with 10 Gray of ionizing radiation and fixed after 30 min (d, e, and f), after 90 min (g, h, and i), or after 5 h (j, k, and l). (a, d, g, and j) Polyclonal anti-hSnm1 staining with fluorescein isothiocyanate; (b, e, h, and k) monoclonal anti-53BP1 staining with tetramethyl rhodamine isocyanate; (c, f, i, and l) merged fields plus DAPI staining.

Techniques Used: Immunofluorescence, Staining

10) Product Images from "Replicating hepatitis delta virus RNA is edited in the nucleus by the small form of ADAR1"

Article Title: Replicating hepatitis delta virus RNA is edited in the nucleus by the small form of ADAR1

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.232416799

Overexpression of HA-tagged ADAR1-L and ADAR1-S during HDV replication. Western analyses were performed on total cell lysates expressing replicating-HDV RNA and either HA-tagged ADAR1-L or ADAR1-S. Posttransfection (3, 5, and 7 days), the samples were blotted with either anti-HA monoclonal antibody ( Upper ) to monitor protein expression or anti-delta antigen polyclonal antiserum ( Lower ) to monitor editing. The relative expression of the HA-tagged ADARs was obtained by comparison with a HA-tagged ADAR1 standard. An equal aliquot of this standard was loaded on all of the anti-HA Westerns, and the signal obtained for this standard was given a value of 10 (27).
Figure Legend Snippet: Overexpression of HA-tagged ADAR1-L and ADAR1-S during HDV replication. Western analyses were performed on total cell lysates expressing replicating-HDV RNA and either HA-tagged ADAR1-L or ADAR1-S. Posttransfection (3, 5, and 7 days), the samples were blotted with either anti-HA monoclonal antibody ( Upper ) to monitor protein expression or anti-delta antigen polyclonal antiserum ( Lower ) to monitor editing. The relative expression of the HA-tagged ADARs was obtained by comparison with a HA-tagged ADAR1 standard. An equal aliquot of this standard was loaded on all of the anti-HA Westerns, and the signal obtained for this standard was given a value of 10 (27).

Techniques Used: Over Expression, Western Blot, Expressing

( A ) RNA editing of the antigenome during HDV replication enables the virus to express two proteins from one coding sequence. The genome and antigenome are represented as rods, and the open boxes within the rods represent the sequences corresponding to the ORF of the HDAg-S. The black region of the box is the additional 19 amino acids of the HDAg-L. ( B ). The diagram shows the three splice variants, with exons (Ex) 1A, 1B, and 1C spliced to exon 2. The promoters are shown as shaded regions. Exon 1A arises from an IFN-inducible promoter (Pi-1A) and contains the first methionine (M1) of the hADAR1-L. Exons 1B and 1C are constitutively expressed from promoters Pc-1B and Pc-1C, respectively, and lack an in-frame AUG. Only transcripts with exon 1A encode ADAR1-L, whereas the others encode ADAR1-S. A fourth transcript arising from a constitutive promoter at exon 2 (Pc-2) also encodes ADAR1-S. The black squares denote the positions of the siRNAs targeted against only ADAR1-L (siL) or both forms of ADAR1 (siL+S). Sequences targeted by siL+S are duplicated naturally.
Figure Legend Snippet: ( A ) RNA editing of the antigenome during HDV replication enables the virus to express two proteins from one coding sequence. The genome and antigenome are represented as rods, and the open boxes within the rods represent the sequences corresponding to the ORF of the HDAg-S. The black region of the box is the additional 19 amino acids of the HDAg-L. ( B ). The diagram shows the three splice variants, with exons (Ex) 1A, 1B, and 1C spliced to exon 2. The promoters are shown as shaded regions. Exon 1A arises from an IFN-inducible promoter (Pi-1A) and contains the first methionine (M1) of the hADAR1-L. Exons 1B and 1C are constitutively expressed from promoters Pc-1B and Pc-1C, respectively, and lack an in-frame AUG. Only transcripts with exon 1A encode ADAR1-L, whereas the others encode ADAR1-S. A fourth transcript arising from a constitutive promoter at exon 2 (Pc-2) also encodes ADAR1-S. The black squares denote the positions of the siRNAs targeted against only ADAR1-L (siL) or both forms of ADAR1 (siL+S). Sequences targeted by siL+S are duplicated naturally.

Techniques Used: Sequencing

Expression of endogenous ADAR1 and ADAR2 during HDV replication in Huh7 cells. ( A ) Western analyses were performed on total cell lysates expressing replicating-HDV RNA (HDV+) or control vector (HDV−) 4–10 days posttransfection and immunoblotted with either anti-ADAR1 ( Upper ) or anti-ADAR2 ( Lower ) polyclonal antisera. std, standard. ( B ) Efficient editing was occurring during the course of replication. ( Upper ) A Western analysis performed with anti-delta antigen polyclonal antiserum showing the edited (HDAg-L) and unedited (HDAg-S) products. ( Lower ) A Northern blot probed to detect the HDV genome.
Figure Legend Snippet: Expression of endogenous ADAR1 and ADAR2 during HDV replication in Huh7 cells. ( A ) Western analyses were performed on total cell lysates expressing replicating-HDV RNA (HDV+) or control vector (HDV−) 4–10 days posttransfection and immunoblotted with either anti-ADAR1 ( Upper ) or anti-ADAR2 ( Lower ) polyclonal antisera. std, standard. ( B ) Efficient editing was occurring during the course of replication. ( Upper ) A Western analysis performed with anti-delta antigen polyclonal antiserum showing the edited (HDAg-L) and unedited (HDAg-S) products. ( Lower ) A Northern blot probed to detect the HDV genome.

Techniques Used: Expressing, Western Blot, Plasmid Preparation, Northern Blot

11) Product Images from "Activation of Mammalian Unfolded Protein Response Is Compatible with the Quality Control System Operating in the Endoplasmic Reticulum"

Article Title: Activation of Mammalian Unfolded Protein Response Is Compatible with the Quality Control System Operating in the Endoplasmic Reticulum

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E03-09-0693

Effects of using a full-length or truncated CMV promoter on the expression, localization and ER stress-induced processing of GFP-ATF6α. (A) CHO cells were transfected with pCMVfull-EGFP-ATF6α (Full) or pCMVshort-EGFP-ATF6α (Short). Eight or 24 h after transfection, cell lysates were prepared and analyzed by immunoblotting using anti-ATF6α or anti-ATF6β antibodies. The migration positions of GFP-ATF6α(P), endogenous pATF6α(P), endogenous pATF6α(N), and endogenous pATF6β(P) are marked. The positions of full-range rainbow molecular weight markers (Amersham Biosciences) are indicated on the left. (B) Eight hours after transfection with pCMVfull-EGFP-ATF6α (Full) or pCMVshort-EGFP-ATF6α (Short), CHO cells were fixed, stained with anti-KDEL or anti-GM130 antibodies, and analyzed by fluorescence microscopy. GFP-ATF6α was visualized by its own fluorescence (shown in green), whereas ER chaperones having the KDEL sequence at their C-termini and GM130 were visualized using rhodamine-conjugated secondary antibody (shown in red). An outline of the nucleus is indicated by a white line in each cell. (C) Eight hours after transfection with pCMVfull-EGFP-ATF6α (Full) or pCMVshort-EGFP-ATF6α (Short), CHO cells were untreated (-) or treated (+)with 1 mM DTT for 30 min before the preparation of cell lysates, which were then analyzed by immunoblotting using anti-ATF6α antibody. The migration positions of GFP-ATF6α(P), endogenous pATF6α(P), GFP-ATF6α(N), and endogenous pATF6α(N) are marked. (D) Twelve hours after mock transfection (-) or transfection of CHO cells with pCMVfull-EGFP (Full) or pCMVshort-EGFP (Short), total RNA was prepared and analyzed by Northern blot hybridization using a probe specific to GFP. The level of 28S rRNA in each lane is shown as a loading control. Similar experiments were carried out two more times. Chemiluminescence intensity of each band was determined using an LAS-1000plus Luminoimage analyzer and the averages are presented with standard deviations (error bars). (E) CHO cells were transfected with pGL3-Basic vector (Empty), pGL3-CMVfull (Full), or pGL3-CMVshort (Short) together with reference plasmid pRL-SV40. The relative luciferase activity was determined after overnight incubation of transfected cells. The averages from duplicate determination of three independent experiments are presented with standard deviations (error bars) after normalization to the value obtained for Empty.
Figure Legend Snippet: Effects of using a full-length or truncated CMV promoter on the expression, localization and ER stress-induced processing of GFP-ATF6α. (A) CHO cells were transfected with pCMVfull-EGFP-ATF6α (Full) or pCMVshort-EGFP-ATF6α (Short). Eight or 24 h after transfection, cell lysates were prepared and analyzed by immunoblotting using anti-ATF6α or anti-ATF6β antibodies. The migration positions of GFP-ATF6α(P), endogenous pATF6α(P), endogenous pATF6α(N), and endogenous pATF6β(P) are marked. The positions of full-range rainbow molecular weight markers (Amersham Biosciences) are indicated on the left. (B) Eight hours after transfection with pCMVfull-EGFP-ATF6α (Full) or pCMVshort-EGFP-ATF6α (Short), CHO cells were fixed, stained with anti-KDEL or anti-GM130 antibodies, and analyzed by fluorescence microscopy. GFP-ATF6α was visualized by its own fluorescence (shown in green), whereas ER chaperones having the KDEL sequence at their C-termini and GM130 were visualized using rhodamine-conjugated secondary antibody (shown in red). An outline of the nucleus is indicated by a white line in each cell. (C) Eight hours after transfection with pCMVfull-EGFP-ATF6α (Full) or pCMVshort-EGFP-ATF6α (Short), CHO cells were untreated (-) or treated (+)with 1 mM DTT for 30 min before the preparation of cell lysates, which were then analyzed by immunoblotting using anti-ATF6α antibody. The migration positions of GFP-ATF6α(P), endogenous pATF6α(P), GFP-ATF6α(N), and endogenous pATF6α(N) are marked. (D) Twelve hours after mock transfection (-) or transfection of CHO cells with pCMVfull-EGFP (Full) or pCMVshort-EGFP (Short), total RNA was prepared and analyzed by Northern blot hybridization using a probe specific to GFP. The level of 28S rRNA in each lane is shown as a loading control. Similar experiments were carried out two more times. Chemiluminescence intensity of each band was determined using an LAS-1000plus Luminoimage analyzer and the averages are presented with standard deviations (error bars). (E) CHO cells were transfected with pGL3-Basic vector (Empty), pGL3-CMVfull (Full), or pGL3-CMVshort (Short) together with reference plasmid pRL-SV40. The relative luciferase activity was determined after overnight incubation of transfected cells. The averages from duplicate determination of three independent experiments are presented with standard deviations (error bars) after normalization to the value obtained for Empty.

Techniques Used: Expressing, Transfection, Migration, Molecular Weight, Staining, Fluorescence, Microscopy, Sequencing, Northern Blot, Hybridization, Plasmid Preparation, Luciferase, Activity Assay, Incubation

12) Product Images from "Aberrant Assembly of RNA Recognition Motif 1 Links to Pathogenic Conversion of TAR DNA-binding Protein of 43 kDa (TDP-43) *"

Article Title: Aberrant Assembly of RNA Recognition Motif 1 Links to Pathogenic Conversion of TAR DNA-binding Protein of 43 kDa (TDP-43) *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.451849

RRM1-C/S mutations unravel the pathogenic properties of TDP-43. A , RRM1-C/S substitution enhances the aggregation propensity of TDP-43 with familial ALS-linked mutations in the C-terminal domain. EGFP-fused WT or A315T and Q331K mutants of TDP-43, either with ( d–f ) or without ( a–c ) the C175S mutation, were transiently expressed in HEK293A cells. B , quantification of cells with aggregates. Data represent the mean percentages of cells harboring aggregates in total transfected cells ± S.E. ( n = 8 images); 20–120 cells were counted in each image. *, p
Figure Legend Snippet: RRM1-C/S mutations unravel the pathogenic properties of TDP-43. A , RRM1-C/S substitution enhances the aggregation propensity of TDP-43 with familial ALS-linked mutations in the C-terminal domain. EGFP-fused WT or A315T and Q331K mutants of TDP-43, either with ( d–f ) or without ( a–c ) the C175S mutation, were transiently expressed in HEK293A cells. B , quantification of cells with aggregates. Data represent the mean percentages of cells harboring aggregates in total transfected cells ± S.E. ( n = 8 images); 20–120 cells were counted in each image. *, p

Techniques Used: Mutagenesis, Transfection

RRM1-C/S substitutions induced cytoplasmic and nuclear inclusions of TDP-43. Shown are confocal analyses of HEK293A cells transiently transfected with EGFP-fused TDP-43. A and B , RRM1-C/S mutations in TDP-43 induced aggregates in the nucleus and cytosol. Cells were transiently transfected with full-length WT or RRM1-C/S of TDP-43-EGFP ( green ) with WT ( A ) or modified ( B ) NLS. Scale bar , 10 μm. C , the RRM1 deletion mutant of TDP-43 formed marked nuclear inclusions, but RRM1-deleted TDP-43 with modified NLS did not. HEK293A cells were transiently transfected with EGFP-fused TDP-43 lacking RRM1 with WT ( a and b ) or modified ( c and d ) NLS. Scale bar , 10 μm. D , RRM2-C/S had no impact on the TDP-43 aggregation. Cells were transfected with full-length WT or RRM2-C/S double mutant ( RRM2 DCS ) TDP-43-EGFP ( green ). Scale bars , 10 μm ( a and b ) and 30 μm ( c and d ). E , disulfide bonding in RRM1 and RRM2 is not inevitable for TDP-43 aggregation. Cells were transfected with RRM1 single mutants and the RRM2-C/S double mutant (RRM2 DCS) of TDP-43-EGFP ( green ). Double C/S substitutions at Cys-198/Cys-244 ( RRM2 DCS ) did not affect nuclear or cytosolic TDP-43 inclusions caused by C/S mutations at RRM1. Scale bar , 10 μm. F , C/A mutants in Cys-173 and/or C175S showed the same aggregation as C/S mutants. Scale bar , 10 μm. Nuclei were stained by DAPI ( blue ).
Figure Legend Snippet: RRM1-C/S substitutions induced cytoplasmic and nuclear inclusions of TDP-43. Shown are confocal analyses of HEK293A cells transiently transfected with EGFP-fused TDP-43. A and B , RRM1-C/S mutations in TDP-43 induced aggregates in the nucleus and cytosol. Cells were transiently transfected with full-length WT or RRM1-C/S of TDP-43-EGFP ( green ) with WT ( A ) or modified ( B ) NLS. Scale bar , 10 μm. C , the RRM1 deletion mutant of TDP-43 formed marked nuclear inclusions, but RRM1-deleted TDP-43 with modified NLS did not. HEK293A cells were transiently transfected with EGFP-fused TDP-43 lacking RRM1 with WT ( a and b ) or modified ( c and d ) NLS. Scale bar , 10 μm. D , RRM2-C/S had no impact on the TDP-43 aggregation. Cells were transfected with full-length WT or RRM2-C/S double mutant ( RRM2 DCS ) TDP-43-EGFP ( green ). Scale bars , 10 μm ( a and b ) and 30 μm ( c and d ). E , disulfide bonding in RRM1 and RRM2 is not inevitable for TDP-43 aggregation. Cells were transfected with RRM1 single mutants and the RRM2-C/S double mutant (RRM2 DCS) of TDP-43-EGFP ( green ). Double C/S substitutions at Cys-198/Cys-244 ( RRM2 DCS ) did not affect nuclear or cytosolic TDP-43 inclusions caused by C/S mutations at RRM1. Scale bar , 10 μm. F , C/A mutants in Cys-173 and/or C175S showed the same aggregation as C/S mutants. Scale bar , 10 μm. Nuclei were stained by DAPI ( blue ).

Techniques Used: Transfection, Modification, Mutagenesis, Staining

Hypothetical roles of misfolding-relevant cores in RRM1. A , ribbon structures showing the locations of misfolding-relevant RRM1 core-a, -b, and -c. B , hypothetical scheme of TDP-43 proteinopathy development. For clarity, RRM1 is indicated by a shaded area ; core-a, -b, and -c are also indicated. These cores may not be exposed under physiological conditions. Various cellular stresses, such as oxidative stress or regional condensation of TDP-43, may alter the β-sheet assembly, which may induce transient disulfide bonds, ultimately resulting in pathological TDP-43 proteinopathies with the participation of the C terminus.
Figure Legend Snippet: Hypothetical roles of misfolding-relevant cores in RRM1. A , ribbon structures showing the locations of misfolding-relevant RRM1 core-a, -b, and -c. B , hypothetical scheme of TDP-43 proteinopathy development. For clarity, RRM1 is indicated by a shaded area ; core-a, -b, and -c are also indicated. These cores may not be exposed under physiological conditions. Various cellular stresses, such as oxidative stress or regional condensation of TDP-43, may alter the β-sheet assembly, which may induce transient disulfide bonds, ultimately resulting in pathological TDP-43 proteinopathies with the participation of the C terminus.

Techniques Used:

TDP-43 inclusions with RRM1-C/S mutation were phosphorylated, ubiquitinated, and disulfide-free. A and B , confocal analysis of SHSY-5Y cells transfected with full-length WT or RRM1-C/S mutants of TDP-43-EGFP ( green ). Cells were immunostained with antibodies ( red ) targeting phospho-TDP-43 at Ser-409/Ser-410 ( a–i ) or Lys-48-linked ubiquitin ( j–o ). In B , all constructs contained mNLS. Nuclei were stained by DAPI ( blue ). Phosphorylated or ubiquitinated inclusions in the cytoplasm are indicated by arrowheads . Note that cytoplasmic inclusions ( green ) are strongly immunoreactive to both antibodies compared with nuclear aggregates. Scale bar , 10 μm. C and D , Western blots showing that disulfide-irrelevant oligomers of TDP-43 harboring RRM1-C/S substitutions under the proteasome inhibitor lactacystin are more prominent in the cytosol ( D ) than in the nucleus ( C ). TDP-43-FLAG was overexpressed in HEK293A cells. Cell lysates were analyzed by SDS-PAGE and incubated with antibodies targeting FLAG and GAPDH. Note that DTT marginally reduced oligomerization (indicated by an asterisk ) and increased dimer formation in C173S and C175S mutants. E , Western blot of TDP-43 with RRM1-C/S substitutions using an antibody targeting TDP-43 phosphorylated at Ser-409/Ser-410. TDP-43 with mNLS is phosphorylated to a greater extent than WT, which is enhanced by lactacystin ( right , lanes 3 and 4 ). Conversely, TDP-43 harboring the RRM1-C/S substitution is more strongly phosphorylated, and modification of NLS or lactacystin treatment augmented the phosphorylated TDP-43 (both left and right , lanes 5–10 ).
Figure Legend Snippet: TDP-43 inclusions with RRM1-C/S mutation were phosphorylated, ubiquitinated, and disulfide-free. A and B , confocal analysis of SHSY-5Y cells transfected with full-length WT or RRM1-C/S mutants of TDP-43-EGFP ( green ). Cells were immunostained with antibodies ( red ) targeting phospho-TDP-43 at Ser-409/Ser-410 ( a–i ) or Lys-48-linked ubiquitin ( j–o ). In B , all constructs contained mNLS. Nuclei were stained by DAPI ( blue ). Phosphorylated or ubiquitinated inclusions in the cytoplasm are indicated by arrowheads . Note that cytoplasmic inclusions ( green ) are strongly immunoreactive to both antibodies compared with nuclear aggregates. Scale bar , 10 μm. C and D , Western blots showing that disulfide-irrelevant oligomers of TDP-43 harboring RRM1-C/S substitutions under the proteasome inhibitor lactacystin are more prominent in the cytosol ( D ) than in the nucleus ( C ). TDP-43-FLAG was overexpressed in HEK293A cells. Cell lysates were analyzed by SDS-PAGE and incubated with antibodies targeting FLAG and GAPDH. Note that DTT marginally reduced oligomerization (indicated by an asterisk ) and increased dimer formation in C173S and C175S mutants. E , Western blot of TDP-43 with RRM1-C/S substitutions using an antibody targeting TDP-43 phosphorylated at Ser-409/Ser-410. TDP-43 with mNLS is phosphorylated to a greater extent than WT, which is enhanced by lactacystin ( right , lanes 3 and 4 ). Conversely, TDP-43 harboring the RRM1-C/S substitution is more strongly phosphorylated, and modification of NLS or lactacystin treatment augmented the phosphorylated TDP-43 (both left and right , lanes 5–10 ).

Techniques Used: Mutagenesis, Transfection, Construct, Staining, Western Blot, SDS Page, Incubation, Modification

Evaluation of polyclonal antibody for the detection of aggregation-relevant cores. A , schematic illustration showing the antigenic sequences in RRM1 used to generate the polyclonal antibodies raised against core-a (aa 108–116), core-b (aa 134–142), and core-c (aa 163–173), which are designated as pAb RRM1-a, -b, and -c, respectively. Asterisks indicate the residues that showed irreversible chemical shifts on NMR. B , Western blot analysis using antibody against three aggregation-relevant domains (Ab1, Ab2, and Ab3) in the presence (DTT (+)) and absence (DTT (−)) of DTT. The antibody 1 (Ab1; against aa 108–116) and the antibody 3 (Ab3; against aa 167–172) recognize RRM1 and full-length recombinant TDP-43 but not RRM2 or the RRM1 deletion mutant of TDP-43. C , ELISA study showing the immunoreactivity of pAb RRM1-a ( a ) and pAb RRM1-c ( b ) against recombinant human TDP-43 proteins of various types.
Figure Legend Snippet: Evaluation of polyclonal antibody for the detection of aggregation-relevant cores. A , schematic illustration showing the antigenic sequences in RRM1 used to generate the polyclonal antibodies raised against core-a (aa 108–116), core-b (aa 134–142), and core-c (aa 163–173), which are designated as pAb RRM1-a, -b, and -c, respectively. Asterisks indicate the residues that showed irreversible chemical shifts on NMR. B , Western blot analysis using antibody against three aggregation-relevant domains (Ab1, Ab2, and Ab3) in the presence (DTT (+)) and absence (DTT (−)) of DTT. The antibody 1 (Ab1; against aa 108–116) and the antibody 3 (Ab3; against aa 167–172) recognize RRM1 and full-length recombinant TDP-43 but not RRM2 or the RRM1 deletion mutant of TDP-43. C , ELISA study showing the immunoreactivity of pAb RRM1-a ( a ) and pAb RRM1-c ( b ) against recombinant human TDP-43 proteins of various types.

Techniques Used: Nuclear Magnetic Resonance, Western Blot, Recombinant, Mutagenesis, Enzyme-linked Immunosorbent Assay

Misfolding-relevant core in RRM1 is an immunogenic marker of TDP-43 inclusions. A , confocal micrographs showing SHSY-5Y cells transiently expressing EGFP-fused WT or C/S mutant TDP-43. Cells were immunostained with the antibody targeting core-a ( a–i ; pAb RRM1-a) or core-c ( j–r ; pAb RRM1-c) or a commercially available anti-TDP-43 antibody ( s–x ; Proteintech). The bottom panels show overlaid images of the EGFP ( green ) and antibody ( red ) signals. Nuclei were stained with DAPI ( blue ). Scale bar , 10 μm. B , quantification of the aggregate-specific reactivity of the pAb RRM1-a and pAb RRM1-c antibodies, compared with that of a commercially available anti-TDP-43 antibody (Proteintech). The fluorescence of EGFP and the antibody was measured in each nucleus expressing WT or C173S/C175S mutant (DCS) TDP-43. The ratio of antibody to EGFP fluorescence was determined; WT data are normalized to the DCS data, which are expressed as Ab reactivity to nuclear TDP-43. Data represent the mean ± S.E. ( n = 15–117 nuclei). *, p
Figure Legend Snippet: Misfolding-relevant core in RRM1 is an immunogenic marker of TDP-43 inclusions. A , confocal micrographs showing SHSY-5Y cells transiently expressing EGFP-fused WT or C/S mutant TDP-43. Cells were immunostained with the antibody targeting core-a ( a–i ; pAb RRM1-a) or core-c ( j–r ; pAb RRM1-c) or a commercially available anti-TDP-43 antibody ( s–x ; Proteintech). The bottom panels show overlaid images of the EGFP ( green ) and antibody ( red ) signals. Nuclei were stained with DAPI ( blue ). Scale bar , 10 μm. B , quantification of the aggregate-specific reactivity of the pAb RRM1-a and pAb RRM1-c antibodies, compared with that of a commercially available anti-TDP-43 antibody (Proteintech). The fluorescence of EGFP and the antibody was measured in each nucleus expressing WT or C173S/C175S mutant (DCS) TDP-43. The ratio of antibody to EGFP fluorescence was determined; WT data are normalized to the DCS data, which are expressed as Ab reactivity to nuclear TDP-43. Data represent the mean ± S.E. ( n = 15–117 nuclei). *, p

Techniques Used: Marker, Expressing, Mutagenesis, Staining, Fluorescence

Crucial roles of cysteines in RRM1 and C-terminal domain in the formation of cytosolic inclusions of TDP-43. A , sporadic ALS mutation D169G mutation in RRM1 domain caused no TDP-43 aggregation. Confocal analysis of HEK293A cells overexpressing nuclear ( A ) or mislocalized ( mNLS ) ( B ) TDP-43-EGFP ( green ), with C175S ( c and d ) or D169G ( e and f ) mutations. Scale bar , 10 mm ( a , c , and e ) or 50 μm ( b , d , and f ). B , C terminus mediates RRM1-C/S-induced aggregation of TDP-43. Confocal analysis of HEK293A cells transiently transfected with nuclear ( a–c , WT NLS) or cytosolic ( d–f , mNLS) TDP-43-EGFP carrying the double C173S/C175S mutation ( DCS ), either with ( c and f ) or without ( b and e ) the C terminus (aa 266–414, Δ Ct ). WT *, WT NLS. g , quantification of the effect of the C-terminal tail of TDP-43 on nuclear or cytosolic aggregates caused by RRM1-C/S mutation. The percentage of aggregate holding cells in the total transfected cells was obtained by counting. *, p
Figure Legend Snippet: Crucial roles of cysteines in RRM1 and C-terminal domain in the formation of cytosolic inclusions of TDP-43. A , sporadic ALS mutation D169G mutation in RRM1 domain caused no TDP-43 aggregation. Confocal analysis of HEK293A cells overexpressing nuclear ( A ) or mislocalized ( mNLS ) ( B ) TDP-43-EGFP ( green ), with C175S ( c and d ) or D169G ( e and f ) mutations. Scale bar , 10 mm ( a , c , and e ) or 50 μm ( b , d , and f ). B , C terminus mediates RRM1-C/S-induced aggregation of TDP-43. Confocal analysis of HEK293A cells transiently transfected with nuclear ( a–c , WT NLS) or cytosolic ( d–f , mNLS) TDP-43-EGFP carrying the double C173S/C175S mutation ( DCS ), either with ( c and f ) or without ( b and e ) the C terminus (aa 266–414, Δ Ct ). WT *, WT NLS. g , quantification of the effect of the C-terminal tail of TDP-43 on nuclear or cytosolic aggregates caused by RRM1-C/S mutation. The percentage of aggregate holding cells in the total transfected cells was obtained by counting. *, p

Techniques Used: Mutagenesis, Transfection

13) Product Images from "Encephalomyocarditis Virus Disrupts Stress Granules, the Critical Platform for Triggering Antiviral Innate Immune Responses"

Article Title: Encephalomyocarditis Virus Disrupts Stress Granules, the Critical Platform for Triggering Antiviral Innate Immune Responses

Journal: Journal of Virology

doi: 10.1128/JVI.03248-12

EMCV infection results in the cleavage of G3BP1. (A) Immunoblotting (IB) showing the kinetics of G3BP1 cleavage in EMCV-infected HeLa/G-G3BP1 cells. N.C., negative control. (B) HeLa cells stably expressing FLAG-G3BP1 Q325E protein were infected with EMCV, and the G3BP1 Q325E protein level was monitored by immunoblotting. (C) Western blot analysis of HeLa/G-G3BP1 cells infected with EMCV. Lysates were prepared at the indicated time points after infection and subjected to immunoblotting with the indicated antibodies. FL, full-length; n.s., not significant. (D, left) HeLa cells were transiently transfected with an empty vector or the expression vector for leader or 3C and analyzed for endogenous G3BP1 by Western blotting. (Right) HeLa/G-G3BP1 and HeLa/G-G3BP1Q325E cells were transiently transfected with an empty vector or the expression vector for leader or 3C and analyzed by Western blotting using anti-GFP. MW, molecular weight (in thousands); p.h.i., hour postinfection; cp, cleavage fragment.
Figure Legend Snippet: EMCV infection results in the cleavage of G3BP1. (A) Immunoblotting (IB) showing the kinetics of G3BP1 cleavage in EMCV-infected HeLa/G-G3BP1 cells. N.C., negative control. (B) HeLa cells stably expressing FLAG-G3BP1 Q325E protein were infected with EMCV, and the G3BP1 Q325E protein level was monitored by immunoblotting. (C) Western blot analysis of HeLa/G-G3BP1 cells infected with EMCV. Lysates were prepared at the indicated time points after infection and subjected to immunoblotting with the indicated antibodies. FL, full-length; n.s., not significant. (D, left) HeLa cells were transiently transfected with an empty vector or the expression vector for leader or 3C and analyzed for endogenous G3BP1 by Western blotting. (Right) HeLa/G-G3BP1 and HeLa/G-G3BP1Q325E cells were transiently transfected with an empty vector or the expression vector for leader or 3C and analyzed by Western blotting using anti-GFP. MW, molecular weight (in thousands); p.h.i., hour postinfection; cp, cleavage fragment.

Techniques Used: Infection, Negative Control, Stable Transfection, Expressing, Western Blot, Transfection, Plasmid Preparation, Molecular Weight

14) Product Images from "Structure-Function Analyses of the Small GTPase Rab35 and Its Effector Protein Centaurin-β2/ACAP2 during Neurite Outgrowth of PC12 Cells *"

Article Title: Structure-Function Analyses of the Small GTPase Rab35 and Its Effector Protein Centaurin-β2/ACAP2 during Neurite Outgrowth of PC12 Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.611301

Effect of the N610A/N691A mutation of centaurin-β2 on NGF-induced neurite outgrowth of PC12 cells. A , typical images of PC12 cells (merged bright field images and EGFP fluorescence images) transiently expressing shControl or shCentβ2 together with pEGFP-C1 ( top row ) or pEGFP-C1-Centβ2 SR (WT, N610A/N691A, or ΔANKR) ( bottom row ). The cells were fixed after NGF stimulation for 36 h and examined under a confocal microscope. Note that knockdown of centaurin-β2 in PC12 cells inhibited neurite outgrowth ( top right panel ), whereas re-expression of Centβ2 SR (WT) ( bottom left panel ), but not of Centβ2 SR (N610A/N691A) ( bottom middle panel ) or Centβ2 SR (ΔANKR) ( bottom right panel ), in shCentβ2-treated PC12 cells restored total neurite length. Under our experimental conditions, manipulation of centaurin-β2 had no significant effect on the number of neurites (shControl, 2.2 ± 0.05; shCentβ2, 2.0 ± 0.04; shCentβ2 + Centβ2 SR (WT), 2.1 ± 0.04; shCentβ2 + Centβ2 SR (N610A/N691A), 2.0 ± 0.04; and shCentβ2 + Centβ2 SR (ΔANKR), 2.0 ± 0.05 (mean ± S.E.)), suggesting that centaurin-β2 is involved in neurite extension rather than in neuritogenesis, the same as Rab35 is. Scale bars , 20 μm. B , quantification of total neurite length shown in A (sum of the lengths of the broken white lines in each PC12 cell). The bars represent the means and S.E. of data from three independent experiments ( n = 100 cells; more than 30 cells were analyzed in each experiment). ***, p
Figure Legend Snippet: Effect of the N610A/N691A mutation of centaurin-β2 on NGF-induced neurite outgrowth of PC12 cells. A , typical images of PC12 cells (merged bright field images and EGFP fluorescence images) transiently expressing shControl or shCentβ2 together with pEGFP-C1 ( top row ) or pEGFP-C1-Centβ2 SR (WT, N610A/N691A, or ΔANKR) ( bottom row ). The cells were fixed after NGF stimulation for 36 h and examined under a confocal microscope. Note that knockdown of centaurin-β2 in PC12 cells inhibited neurite outgrowth ( top right panel ), whereas re-expression of Centβ2 SR (WT) ( bottom left panel ), but not of Centβ2 SR (N610A/N691A) ( bottom middle panel ) or Centβ2 SR (ΔANKR) ( bottom right panel ), in shCentβ2-treated PC12 cells restored total neurite length. Under our experimental conditions, manipulation of centaurin-β2 had no significant effect on the number of neurites (shControl, 2.2 ± 0.05; shCentβ2, 2.0 ± 0.04; shCentβ2 + Centβ2 SR (WT), 2.1 ± 0.04; shCentβ2 + Centβ2 SR (N610A/N691A), 2.0 ± 0.04; and shCentβ2 + Centβ2 SR (ΔANKR), 2.0 ± 0.05 (mean ± S.E.)), suggesting that centaurin-β2 is involved in neurite extension rather than in neuritogenesis, the same as Rab35 is. Scale bars , 20 μm. B , quantification of total neurite length shown in A (sum of the lengths of the broken white lines in each PC12 cell). The bars represent the means and S.E. of data from three independent experiments ( n = 100 cells; more than 30 cells were analyzed in each experiment). ***, p

Techniques Used: Mutagenesis, Fluorescence, Expressing, Microscopy

Identification of critical residues responsible for the specific binding of centaurin-β2 in the switch II region of Rab35 by site-directed mutagenesis. A , sequence alignment of the switch II regions of mouse Rab1A/B, Rab8A/B, Rab10, Rab13, Rab15, and Rab35. Amino acid residues in the sequences that are conserved in more than four switch II regions and that are similar are shown against a black background and a shaded background , respectively. Only two amino acids ( arrowheads ) in the switch II region of Rab1A/B and Rab35 are different, and we replaced the Thr-76 and Thr-81 of Rab35 with Ser and Ala, respectively, by site-directed mutagenesis ( i.e. produced a T76S/T81A mutant, which mimics the switch II region of Rab1A). B ). C ) were streaked on SC-LW ( top panels ) and SC-AHLW (selection medium; bottom panels ) and incubated at 30 °C for 1 day and 1 week, respectively. D , two Thr residues of Rab35, Thr-76 and Thr-81, are critical for binding centaurin-β2 but not for binding other Rab35BPs. Yeast cells containing the pAct2 (or pGAD) plasmid expressing Rab35BP and pGBD plasmid expressing the constitutively active form (Rab35(QL)) of Rab35(WT) or Rab35(T76S/T81A) mutant were streaked on SC-LW ( left panels ) and SC-AHLW ( right panels ) and incubated at 30 °C for 1 day and 1 week, respectively. Note that the Rab35 containing the T76S/T81A mutations specifically abrogated binding activity toward centaurin-β2 ( bottom right panel ). E ). Co-immunoprecipitated T7-tagged Facsin1 ( middle panel ) and immunoprecipitated FLAG-tagged Rab35(WT) or Rab35(T76S/T81A) mutant ( bottom panel ) were detected with HRP-conjugated anti-T7 tag antibody and HRP-conjugated anti-FLAG tag antibody, respectively. Input , 1/50 of the volume of the reaction mixture used for immunoprecipitation ( IP ) ( top panel ). The positions of the molecular mass markers (in kilodaltons) are shown on the left. F , the T76S/T81A mutation dramatically decreased the centaurin-β2 binding activity of Rab35, a finding that was consistent with the results of the yeast two-hybrid assays shown in D . Co-immunoprecipitation assays were performed as described in E .
Figure Legend Snippet: Identification of critical residues responsible for the specific binding of centaurin-β2 in the switch II region of Rab35 by site-directed mutagenesis. A , sequence alignment of the switch II regions of mouse Rab1A/B, Rab8A/B, Rab10, Rab13, Rab15, and Rab35. Amino acid residues in the sequences that are conserved in more than four switch II regions and that are similar are shown against a black background and a shaded background , respectively. Only two amino acids ( arrowheads ) in the switch II region of Rab1A/B and Rab35 are different, and we replaced the Thr-76 and Thr-81 of Rab35 with Ser and Ala, respectively, by site-directed mutagenesis ( i.e. produced a T76S/T81A mutant, which mimics the switch II region of Rab1A). B ). C ) were streaked on SC-LW ( top panels ) and SC-AHLW (selection medium; bottom panels ) and incubated at 30 °C for 1 day and 1 week, respectively. D , two Thr residues of Rab35, Thr-76 and Thr-81, are critical for binding centaurin-β2 but not for binding other Rab35BPs. Yeast cells containing the pAct2 (or pGAD) plasmid expressing Rab35BP and pGBD plasmid expressing the constitutively active form (Rab35(QL)) of Rab35(WT) or Rab35(T76S/T81A) mutant were streaked on SC-LW ( left panels ) and SC-AHLW ( right panels ) and incubated at 30 °C for 1 day and 1 week, respectively. Note that the Rab35 containing the T76S/T81A mutations specifically abrogated binding activity toward centaurin-β2 ( bottom right panel ). E ). Co-immunoprecipitated T7-tagged Facsin1 ( middle panel ) and immunoprecipitated FLAG-tagged Rab35(WT) or Rab35(T76S/T81A) mutant ( bottom panel ) were detected with HRP-conjugated anti-T7 tag antibody and HRP-conjugated anti-FLAG tag antibody, respectively. Input , 1/50 of the volume of the reaction mixture used for immunoprecipitation ( IP ) ( top panel ). The positions of the molecular mass markers (in kilodaltons) are shown on the left. F , the T76S/T81A mutation dramatically decreased the centaurin-β2 binding activity of Rab35, a finding that was consistent with the results of the yeast two-hybrid assays shown in D . Co-immunoprecipitation assays were performed as described in E .

Techniques Used: Binding Assay, Mutagenesis, Sequencing, Produced, Selection, Incubation, Plasmid Preparation, Expressing, Activity Assay, Immunoprecipitation, FLAG-tag

15) Product Images from "Structure-Function Analyses of the Small GTPase Rab35 and Its Effector Protein Centaurin-β2/ACAP2 during Neurite Outgrowth of PC12 Cells *"

Article Title: Structure-Function Analyses of the Small GTPase Rab35 and Its Effector Protein Centaurin-β2/ACAP2 during Neurite Outgrowth of PC12 Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.611301

Identification of critical residues responsible for the specific binding of centaurin-β2 in the switch II region of Rab35 by site-directed mutagenesis. A , sequence alignment of the switch II regions of mouse Rab1A/B, Rab8A/B, Rab10, Rab13, Rab15, and Rab35. Amino acid residues in the sequences that are conserved in more than four switch II regions and that are similar are shown against a black background and a shaded background , respectively. Only two amino acids ( arrowheads ) in the switch II region of Rab1A/B and Rab35 are different, and we replaced the Thr-76 and Thr-81 of Rab35 with Ser and Ala, respectively, by site-directed mutagenesis ( i.e. produced a T76S/T81A mutant, which mimics the switch II region of Rab1A). B ). C ) were streaked on SC-LW ( top panels ) and SC-AHLW (selection medium; bottom panels ) and incubated at 30 °C for 1 day and 1 week, respectively. D , two Thr residues of Rab35, Thr-76 and Thr-81, are critical for binding centaurin-β2 but not for binding other Rab35BPs. Yeast cells containing the pAct2 (or pGAD) plasmid expressing Rab35BP and pGBD plasmid expressing the constitutively active form (Rab35(QL)) of Rab35(WT) or Rab35(T76S/T81A) mutant were streaked on SC-LW ( left panels ) and SC-AHLW ( right panels ) and incubated at 30 °C for 1 day and 1 week, respectively. Note that the Rab35 containing the T76S/T81A mutations specifically abrogated binding activity toward centaurin-β2 ( bottom right panel ). E ). Co-immunoprecipitated T7-tagged Facsin1 ( middle panel ) and immunoprecipitated FLAG-tagged Rab35(WT) or Rab35(T76S/T81A) mutant ( bottom panel ) were detected with HRP-conjugated anti-T7 tag antibody and HRP-conjugated anti-FLAG tag antibody, respectively. Input , 1/50 of the volume of the reaction mixture used for immunoprecipitation ( IP ) ( top panel ). The positions of the molecular mass markers (in kilodaltons) are shown on the left. F , the T76S/T81A mutation dramatically decreased the centaurin-β2 binding activity of Rab35, a finding that was consistent with the results of the yeast two-hybrid assays shown in D . Co-immunoprecipitation assays were performed as described in E .
Figure Legend Snippet: Identification of critical residues responsible for the specific binding of centaurin-β2 in the switch II region of Rab35 by site-directed mutagenesis. A , sequence alignment of the switch II regions of mouse Rab1A/B, Rab8A/B, Rab10, Rab13, Rab15, and Rab35. Amino acid residues in the sequences that are conserved in more than four switch II regions and that are similar are shown against a black background and a shaded background , respectively. Only two amino acids ( arrowheads ) in the switch II region of Rab1A/B and Rab35 are different, and we replaced the Thr-76 and Thr-81 of Rab35 with Ser and Ala, respectively, by site-directed mutagenesis ( i.e. produced a T76S/T81A mutant, which mimics the switch II region of Rab1A). B ). C ) were streaked on SC-LW ( top panels ) and SC-AHLW (selection medium; bottom panels ) and incubated at 30 °C for 1 day and 1 week, respectively. D , two Thr residues of Rab35, Thr-76 and Thr-81, are critical for binding centaurin-β2 but not for binding other Rab35BPs. Yeast cells containing the pAct2 (or pGAD) plasmid expressing Rab35BP and pGBD plasmid expressing the constitutively active form (Rab35(QL)) of Rab35(WT) or Rab35(T76S/T81A) mutant were streaked on SC-LW ( left panels ) and SC-AHLW ( right panels ) and incubated at 30 °C for 1 day and 1 week, respectively. Note that the Rab35 containing the T76S/T81A mutations specifically abrogated binding activity toward centaurin-β2 ( bottom right panel ). E ). Co-immunoprecipitated T7-tagged Facsin1 ( middle panel ) and immunoprecipitated FLAG-tagged Rab35(WT) or Rab35(T76S/T81A) mutant ( bottom panel ) were detected with HRP-conjugated anti-T7 tag antibody and HRP-conjugated anti-FLAG tag antibody, respectively. Input , 1/50 of the volume of the reaction mixture used for immunoprecipitation ( IP ) ( top panel ). The positions of the molecular mass markers (in kilodaltons) are shown on the left. F , the T76S/T81A mutation dramatically decreased the centaurin-β2 binding activity of Rab35, a finding that was consistent with the results of the yeast two-hybrid assays shown in D . Co-immunoprecipitation assays were performed as described in E .

Techniques Used: Binding Assay, Mutagenesis, Sequencing, Produced, Selection, Incubation, Plasmid Preparation, Expressing, Activity Assay, Immunoprecipitation, FLAG-tag

16) Product Images from "The N-Terminal 24 Amino Acids of the p55 Gamma Regulatory Subunit of Phosphoinositide 3-Kinase Binds Rb and Induces Cell Cycle Arrest"

Article Title: The N-Terminal 24 Amino Acids of the p55 Gamma Regulatory Subunit of Phosphoinositide 3-Kinase Binds Rb and Induces Cell Cycle Arrest

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.5.1717-1725.2003

The N-terminal end of p55γ associates with Rb and blocks the binding of p55γ with Rb in MCF-7 cells (A) Two vectors coding for different GFP fusion proteins were constructed. One, designated N24-GFP, consisted of the 24-amino-acid N-terminal end of p55 fused to the N terminus of GFP. The other, designated GFP-N24, consisted of the N-terminal end of p55 fused to the C terminus of GFP. These two constructs and the GFP-N1 plasmid expressing GFP as a control were transiently transfected into MCF-7 cells. Cell lysates were immunoprecipitated with an anti-GFP antibody. The blots were probed with an anti-Rb antibody. (B) Lysates of these transfected cells were immunoprecipitated with an anti-p55γ antibody, and the immunocomplexes were separated on SDS-polyacrylamide gels. Proteins were analyzed by Western blotting using an anti-Rb antibody (G3-245; PharMingen) capable of recognizing hyper- and hypophosphorylated Rb. Lysates from GFP-N1-transfected cells were also used to determine the position of hypophosphorylated or hyperphosphorylated (ppRb) Rb by Western blot analysis.
Figure Legend Snippet: The N-terminal end of p55γ associates with Rb and blocks the binding of p55γ with Rb in MCF-7 cells (A) Two vectors coding for different GFP fusion proteins were constructed. One, designated N24-GFP, consisted of the 24-amino-acid N-terminal end of p55 fused to the N terminus of GFP. The other, designated GFP-N24, consisted of the N-terminal end of p55 fused to the C terminus of GFP. These two constructs and the GFP-N1 plasmid expressing GFP as a control were transiently transfected into MCF-7 cells. Cell lysates were immunoprecipitated with an anti-GFP antibody. The blots were probed with an anti-Rb antibody. (B) Lysates of these transfected cells were immunoprecipitated with an anti-p55γ antibody, and the immunocomplexes were separated on SDS-polyacrylamide gels. Proteins were analyzed by Western blotting using an anti-Rb antibody (G3-245; PharMingen) capable of recognizing hyper- and hypophosphorylated Rb. Lysates from GFP-N1-transfected cells were also used to determine the position of hypophosphorylated or hyperphosphorylated (ppRb) Rb by Western blot analysis.

Techniques Used: Binding Assay, Construct, Plasmid Preparation, Expressing, Transfection, Immunoprecipitation, Western Blot

The N terminal 24 amino acids of p55 γ bind to Rb in vitro. (A) A cDNA construct encoding a chimeric protein composed of the N-terminal 24 amino acid residues of p55γ and GFP (N24p55-GFP) was transfected into COS7 cells. Cell lysates were incubated with purified GST-Rb 1-928 or GST only. Blots were probed using an antibody to GFP. (B) (Left) COS7 cells were transfected with the cDNA construct expressing full-length p55γ. Cell lysates were analyzed by immunoblotting using an HRP-conjugated anti p55γ antibody. (Right) Cell lysates were also incubated with either purified GST-Rb 1-928, a truncated GST-Rb 379-928 fusion protein, or GST protein loaded onto glutathione-agarose beads. Associated proteins were analyzed by Western blotting using an anti-p55γ antibody.
Figure Legend Snippet: The N terminal 24 amino acids of p55 γ bind to Rb in vitro. (A) A cDNA construct encoding a chimeric protein composed of the N-terminal 24 amino acid residues of p55γ and GFP (N24p55-GFP) was transfected into COS7 cells. Cell lysates were incubated with purified GST-Rb 1-928 or GST only. Blots were probed using an antibody to GFP. (B) (Left) COS7 cells were transfected with the cDNA construct expressing full-length p55γ. Cell lysates were analyzed by immunoblotting using an HRP-conjugated anti p55γ antibody. (Right) Cell lysates were also incubated with either purified GST-Rb 1-928, a truncated GST-Rb 379-928 fusion protein, or GST protein loaded onto glutathione-agarose beads. Associated proteins were analyzed by Western blotting using an anti-p55γ antibody.

Techniques Used: In Vitro, Construct, Transfection, Incubation, Purification, Expressing, Western Blot

), both p55γ and p50γ are expressed. Cell lysates were analyzed by Western blotting using an HRP-conjugated anti-p55γ antibody. Arrows indicate the positions of p85, p55, and p50 subunits of PI 3-kinase. (Right) The cell lysates were also immunoprecipitated with an antibody to Rb, and immunoprecipitated proteins were analyzed by Western blotting using either HRP-conjugated anti-p55γ or anti-Rb antibodies as indicated. (C) (Left) A cDNA construct expressing a full-length C-terminal HA-tagged p55α was transfected into NIH 3T3 cells. Cell lysates were collected 48 h later and immunoprecipitated by an anti-Rb antibody or normal rabbit IgG. The immunocomplexes were analyzed using an anti-HA (upper panel) or anti-Rb antibody. (Right) COS7 cell lysates overexpressing p55α were incubated with purified GST or GST-Rb 1-928 . Bound proteins were analyzed by Western blotting using an anti-HA antibody.
Figure Legend Snippet: ), both p55γ and p50γ are expressed. Cell lysates were analyzed by Western blotting using an HRP-conjugated anti-p55γ antibody. Arrows indicate the positions of p85, p55, and p50 subunits of PI 3-kinase. (Right) The cell lysates were also immunoprecipitated with an antibody to Rb, and immunoprecipitated proteins were analyzed by Western blotting using either HRP-conjugated anti-p55γ or anti-Rb antibodies as indicated. (C) (Left) A cDNA construct expressing a full-length C-terminal HA-tagged p55α was transfected into NIH 3T3 cells. Cell lysates were collected 48 h later and immunoprecipitated by an anti-Rb antibody or normal rabbit IgG. The immunocomplexes were analyzed using an anti-HA (upper panel) or anti-Rb antibody. (Right) COS7 cell lysates overexpressing p55α were incubated with purified GST or GST-Rb 1-928 . Bound proteins were analyzed by Western blotting using an anti-HA antibody.

Techniques Used: Western Blot, Immunoprecipitation, Construct, Expressing, Transfection, Incubation, Purification

Rb and p55 colocalize in the nucleus. (A) A cDNA construct encoding FLAG-tagged p55γ (F-p55) was transfected into NIH 3T3 (upper panels) and MCF-7 (lower panels) cells. The cells on coverslips were stained with an anti-FLAG (M-2) antibody and phycoerythrin-labeled rabbit anti-mouse IgG (red) and with an anti-Rb (C-15) antibody and fluorescein-labeled sheep anti-goat IgG (green). The images were taken on a laser-scanning confocal microscope as described in the text. Areas of colocalization appear yellow. (B) AU565 cells were fractionated into cytoplasmic (C), organelle (O), or nuclear (N) fractions as described in the text. Equal amounts of proteins were resolved by SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting with antibodies to p55γ.
Figure Legend Snippet: Rb and p55 colocalize in the nucleus. (A) A cDNA construct encoding FLAG-tagged p55γ (F-p55) was transfected into NIH 3T3 (upper panels) and MCF-7 (lower panels) cells. The cells on coverslips were stained with an anti-FLAG (M-2) antibody and phycoerythrin-labeled rabbit anti-mouse IgG (red) and with an anti-Rb (C-15) antibody and fluorescein-labeled sheep anti-goat IgG (green). The images were taken on a laser-scanning confocal microscope as described in the text. Areas of colocalization appear yellow. (B) AU565 cells were fractionated into cytoplasmic (C), organelle (O), or nuclear (N) fractions as described in the text. Equal amounts of proteins were resolved by SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting with antibodies to p55γ.

Techniques Used: Construct, Transfection, Staining, Labeling, Microscopy, Polyacrylamide Gel Electrophoresis

Effect of growth factors on the interaction of Rb with p55γ. (A) AU565 cells were incubated in serum-free medium overnight and then treated with heregulin (HRG), EGF, or a combination for 20 h at the concentrations indicated. Cell extracts were immunoprecipitated with an anti-Rb antibody. Western blotting was performed using antibodies to p55γ or Rb as indicated. Aliquots of cell lysates were also analyzed using the HRP-conjugated anti-p55 antibody. (B) NIH 3T3 cells were cultured in serum-free DMEM-F-12 medium overnight (control). The medium was replaced by DMEM-F-12 medium containing 30% FBS, and cells were incubated for an additional 0.5, 3, or 5 h as indicated. Immunoprecipitation with an anti-Rb antibody and Western blotting with HRP-conjugated anti-p55 and anti-Rb were performed as described previously. Western blotting for p55γ of cell lysates was performed as described in the legend to panel A.
Figure Legend Snippet: Effect of growth factors on the interaction of Rb with p55γ. (A) AU565 cells were incubated in serum-free medium overnight and then treated with heregulin (HRG), EGF, or a combination for 20 h at the concentrations indicated. Cell extracts were immunoprecipitated with an anti-Rb antibody. Western blotting was performed using antibodies to p55γ or Rb as indicated. Aliquots of cell lysates were also analyzed using the HRP-conjugated anti-p55 antibody. (B) NIH 3T3 cells were cultured in serum-free DMEM-F-12 medium overnight (control). The medium was replaced by DMEM-F-12 medium containing 30% FBS, and cells were incubated for an additional 0.5, 3, or 5 h as indicated. Immunoprecipitation with an anti-Rb antibody and Western blotting with HRP-conjugated anti-p55 and anti-Rb were performed as described previously. Western blotting for p55γ of cell lysates was performed as described in the legend to panel A.

Techniques Used: Incubation, Immunoprecipitation, Western Blot, Cell Culture

17) Product Images from "Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids"

Article Title: Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids

Journal: PLoS ONE

doi: 10.1371/journal.pone.0043283

Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.
Figure Legend Snippet: Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.

Techniques Used: Transfection, Expressing, Luciferase, Plasmid Preparation, Cotransfection, Flow Cytometry, Cytometry, Fluorescence, Cell Counting

Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.
Figure Legend Snippet: Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.

Techniques Used: Expressing, Luciferase, Derivative Assay, Sequencing, Transfection, Activity Assay

18) Product Images from "Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids"

Article Title: Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids

Journal: PLoS ONE

doi: 10.1371/journal.pone.0043283

Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.
Figure Legend Snippet: Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.

Techniques Used: Transfection, Expressing, Luciferase, Plasmid Preparation, Cotransfection, Flow Cytometry, Cytometry, Fluorescence, Cell Counting

Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.
Figure Legend Snippet: Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.

Techniques Used: Expressing, Luciferase, Derivative Assay, Sequencing, Transfection, Activity Assay

19) Product Images from "Functional Inactivation of the Genome-Wide Association Study Obesity Gene Neuronal Growth Regulator 1 in Mice Causes a Body Mass Phenotype"

Article Title: Functional Inactivation of the Genome-Wide Association Study Obesity Gene Neuronal Growth Regulator 1 in Mice Causes a Body Mass Phenotype

Journal: PLoS ONE

doi: 10.1371/journal.pone.0041537

Ablation of NEGR1 in mouse. A. Generation of Negr1 knockout mice. Schematic diagram of the targeting vector, the wild-type and mutant alleles. Only relevant restriction sites are shown. The positions of the external southern blot probe, as well as the PCR primers, are indicated by asterisks and arrows, respectively. B. Genomic southern blot using the restriction enzymes EcoRI and SpeI. Bands resulting from introduction of a new EcoRI site and deletion of a SpeI site are indicated by asterisks. C. PCR genotyping of transgenic mice wild-type (+/+), heterozygous (+/−) and homozygous (−/−) mice. D. Western blot probed with antibodies specific to NEGR1 and β-actin showing complete loss of NEGR1 protein in knockout (−/−) mice. E. Sequencing of genomic DNA reveals a T-A mutation that converts an isoleucine residue to asparagine at position 87 (I87N). F. Western blots of brain lysate from Negr1 -I87N mice probed with an anti-NEGR1 antibody. G. NEGR1-immunoblotting of NSC-34 cell lysates overexpressing NEGR1-WT, NEGR1-I87N, and mock-transfected cells. H–I. The Negr1 -I87N mutation causes ER retention of NEGR1. Confocal images showing NSC-34 cells co-expressing NEGR1-WT (H) or NEGR1-I87N (I) together with DsRed-ER. NEGR1-WT is predominantly localized at the plasma membrane whereas distribution of NEGR1-I87N clearly overlaps with the DsRed-fluorophore-labeled ER. Nuclei are visualised by DAPI. Scale: 10 µm.
Figure Legend Snippet: Ablation of NEGR1 in mouse. A. Generation of Negr1 knockout mice. Schematic diagram of the targeting vector, the wild-type and mutant alleles. Only relevant restriction sites are shown. The positions of the external southern blot probe, as well as the PCR primers, are indicated by asterisks and arrows, respectively. B. Genomic southern blot using the restriction enzymes EcoRI and SpeI. Bands resulting from introduction of a new EcoRI site and deletion of a SpeI site are indicated by asterisks. C. PCR genotyping of transgenic mice wild-type (+/+), heterozygous (+/−) and homozygous (−/−) mice. D. Western blot probed with antibodies specific to NEGR1 and β-actin showing complete loss of NEGR1 protein in knockout (−/−) mice. E. Sequencing of genomic DNA reveals a T-A mutation that converts an isoleucine residue to asparagine at position 87 (I87N). F. Western blots of brain lysate from Negr1 -I87N mice probed with an anti-NEGR1 antibody. G. NEGR1-immunoblotting of NSC-34 cell lysates overexpressing NEGR1-WT, NEGR1-I87N, and mock-transfected cells. H–I. The Negr1 -I87N mutation causes ER retention of NEGR1. Confocal images showing NSC-34 cells co-expressing NEGR1-WT (H) or NEGR1-I87N (I) together with DsRed-ER. NEGR1-WT is predominantly localized at the plasma membrane whereas distribution of NEGR1-I87N clearly overlaps with the DsRed-fluorophore-labeled ER. Nuclei are visualised by DAPI. Scale: 10 µm.

Techniques Used: Knock-Out, Mouse Assay, Plasmid Preparation, Mutagenesis, Southern Blot, Polymerase Chain Reaction, Transgenic Assay, Western Blot, Sequencing, Transfection, Expressing, Labeling

Negr1 -I87N is a loss-of-function mutation. A–C. NSC-34 cells expressing EGFP alone (control) or together with Negr1 -WT or Negr1 -I87N mutants at 0 min (t1) and following 60 min (t2) of cell aggregation. D. Histogram showing cell aggregation expressed as the ratio of 0 min (t1) and 60 min (t2) time points. EGFP+empty vector (control): 1.06±0.05; NEGR1-WT+EGFP: 2.11±0.2; EGFP+NEGR1-I87N: 0.93±0.2. Data represent means ± sd calculated from three independent experiments. E–G. Confocal images showing hypothalamic neurons immunostained for the neuronal marker βIII-tubulin (red) cultured together with transfected NSC-34 cells (green). NSC34 cells were transfected with (E) pEGFP together with an empty pcDNA-vector (control), (F) pEGFP+pcDNA3-NEGR1-WT and (G) EGFP+NEGR1-I87N. H. Mean neurite lengths of hypothalamic neurons relative to control (set to 100%). Error bars represent SEM from three independent experiments (∼400 neurites per condition). Two-tailed Student's test; * P
Figure Legend Snippet: Negr1 -I87N is a loss-of-function mutation. A–C. NSC-34 cells expressing EGFP alone (control) or together with Negr1 -WT or Negr1 -I87N mutants at 0 min (t1) and following 60 min (t2) of cell aggregation. D. Histogram showing cell aggregation expressed as the ratio of 0 min (t1) and 60 min (t2) time points. EGFP+empty vector (control): 1.06±0.05; NEGR1-WT+EGFP: 2.11±0.2; EGFP+NEGR1-I87N: 0.93±0.2. Data represent means ± sd calculated from three independent experiments. E–G. Confocal images showing hypothalamic neurons immunostained for the neuronal marker βIII-tubulin (red) cultured together with transfected NSC-34 cells (green). NSC34 cells were transfected with (E) pEGFP together with an empty pcDNA-vector (control), (F) pEGFP+pcDNA3-NEGR1-WT and (G) EGFP+NEGR1-I87N. H. Mean neurite lengths of hypothalamic neurons relative to control (set to 100%). Error bars represent SEM from three independent experiments (∼400 neurites per condition). Two-tailed Student's test; * P

Techniques Used: Mutagenesis, Expressing, Plasmid Preparation, Marker, Cell Culture, Transfection, Two Tailed Test

20) Product Images from "Endophilin, Lamellipodin, and Mena cooperate to regulate F-actin-dependent EGF-receptor endocytosis"

Article Title: Endophilin, Lamellipodin, and Mena cooperate to regulate F-actin-dependent EGF-receptor endocytosis

Journal: The EMBO Journal

doi: 10.1038/emboj.2013.212

Lamellipodin, Mena, and F-actin regulate EGFR internalization. ( A ) Linear increase in the absorbance of biotinylated surface EGFR with increasing amounts of lysates of HeLa cells. Values are mean (±s.e.m.) of six independent experiments. ( B ) EGFR internalization in HeLa cells treated with Latrunculin B (Lat B) or DMSO control and 2 ng/ml EGF. ( C ) EGFR internalization in HeLa overexpressing Lpd-GFP or GFP as a control and treated with 2 ng/ml EGF. ( D – G ) EGFR internalization in HeLa cells expressing three Lpd-specific ( D , E ) or two Mena-specific ( F , G) or control shRNA and treated with 2 ng/ml EGF for indicated times. ( B – G ) Results are mean±s.e.m. of at least three independent experiments. ( B – D ) t -test: * P
Figure Legend Snippet: Lamellipodin, Mena, and F-actin regulate EGFR internalization. ( A ) Linear increase in the absorbance of biotinylated surface EGFR with increasing amounts of lysates of HeLa cells. Values are mean (±s.e.m.) of six independent experiments. ( B ) EGFR internalization in HeLa cells treated with Latrunculin B (Lat B) or DMSO control and 2 ng/ml EGF. ( C ) EGFR internalization in HeLa overexpressing Lpd-GFP or GFP as a control and treated with 2 ng/ml EGF. ( D – G ) EGFR internalization in HeLa cells expressing three Lpd-specific ( D , E ) or two Mena-specific ( F , G) or control shRNA and treated with 2 ng/ml EGF for indicated times. ( B – G ) Results are mean±s.e.m. of at least three independent experiments. ( B – D ) t -test: * P

Techniques Used: Expressing, shRNA

Lamellipodin is recruited to CCPs and interacts with the EGFR. ( A ) HeLa cells expressing mCherry-Lpd and EGFR-GFP were imaged using TIRFM. Single colour (magnified square) and merged images of one representative cell are shown. Scale bar: 30 μm (left image) and 5 μm (right image). ( B ) IP of EGFR from HEK-293 cells overexpressing EGFR-GFP using Lpd-specific antibodies or IgG control. EGFR was detected using anti-GFP antibodies (left panels). Reprobe of the same blot with Lpd-specific antibodies (right panels). ( C , D ) Co-IP of endogenous EGFR and Lpd from A431 cell lysate using Lpd ( C ) or EGFR-specific antibodies ( D ) or IgG control. EGFR and Lpd were detected using specific antibodies. ( E , F ) Co-IP of endogenous EGFR and Lpd from HeLa cell lysate using Lpd ( E ) and EGFR-specific antibodies ( F ) or IgG control. Cells were stimulated with 2 ng/ml EGF (+) for 5 min or not stimulated (−). ( B – F ) A representative blot each from at least three independent experiments is shown. ( G , H ) HeLa cells expressing mCherry-Lpd and ( G ) GFP-Lpd or ( H ) GFP-Mena and mRFP-Clc were imaged using TIRFM. Single colour (magnified square) and merged images of one representative cell are shown. ( G , H ) Scale bar: 30 μm ( G ) and 10 μm ( H ) (left image) and 5 μm ( G ) and 2 μm ( H ) (right image). ( G , H ) Quantification of the percentage of colocalization of mRFP-Clc with Lpd-GFP (Clathrin versus Lpd) ( G ) or GFP-Mena (Clathrin versus Mena) ( H ) and Lpd-GFP with mRFP-Clc (Lpd versus Clathrin) ( G ) or GFP-Mena (Mena versus Clathrin) ( H) . Each time point of TIRF movies from four cells were analysed containing on average 850 Clc-positive and 450 Lpd-positive spots each. ( I , J ) Dynamics of Lpd-GFP and mRFP-Clc in HeLa cells was assessed every 5 s using TIRFM. Single colour and merged images of an area of a representative cell are shown. Arrows show recruitment of Lpd-GFP to mRFP-Clc shortly before scission. Scale bar: 1 μm (see also Supplementary Movie S3 ). ( J ) Quantification of the percentage of scission events of CCPs containing mRFP-Clc and Lpd-GFP. In total, 700 scission events of 3 different cells were analysed for each experiment.
Figure Legend Snippet: Lamellipodin is recruited to CCPs and interacts with the EGFR. ( A ) HeLa cells expressing mCherry-Lpd and EGFR-GFP were imaged using TIRFM. Single colour (magnified square) and merged images of one representative cell are shown. Scale bar: 30 μm (left image) and 5 μm (right image). ( B ) IP of EGFR from HEK-293 cells overexpressing EGFR-GFP using Lpd-specific antibodies or IgG control. EGFR was detected using anti-GFP antibodies (left panels). Reprobe of the same blot with Lpd-specific antibodies (right panels). ( C , D ) Co-IP of endogenous EGFR and Lpd from A431 cell lysate using Lpd ( C ) or EGFR-specific antibodies ( D ) or IgG control. EGFR and Lpd were detected using specific antibodies. ( E , F ) Co-IP of endogenous EGFR and Lpd from HeLa cell lysate using Lpd ( E ) and EGFR-specific antibodies ( F ) or IgG control. Cells were stimulated with 2 ng/ml EGF (+) for 5 min or not stimulated (−). ( B – F ) A representative blot each from at least three independent experiments is shown. ( G , H ) HeLa cells expressing mCherry-Lpd and ( G ) GFP-Lpd or ( H ) GFP-Mena and mRFP-Clc were imaged using TIRFM. Single colour (magnified square) and merged images of one representative cell are shown. ( G , H ) Scale bar: 30 μm ( G ) and 10 μm ( H ) (left image) and 5 μm ( G ) and 2 μm ( H ) (right image). ( G , H ) Quantification of the percentage of colocalization of mRFP-Clc with Lpd-GFP (Clathrin versus Lpd) ( G ) or GFP-Mena (Clathrin versus Mena) ( H ) and Lpd-GFP with mRFP-Clc (Lpd versus Clathrin) ( G ) or GFP-Mena (Mena versus Clathrin) ( H) . Each time point of TIRF movies from four cells were analysed containing on average 850 Clc-positive and 450 Lpd-positive spots each. ( I , J ) Dynamics of Lpd-GFP and mRFP-Clc in HeLa cells was assessed every 5 s using TIRFM. Single colour and merged images of an area of a representative cell are shown. Arrows show recruitment of Lpd-GFP to mRFP-Clc shortly before scission. Scale bar: 1 μm (see also Supplementary Movie S3 ). ( J ) Quantification of the percentage of scission events of CCPs containing mRFP-Clc and Lpd-GFP. In total, 700 scission events of 3 different cells were analysed for each experiment.

Techniques Used: Expressing, Co-Immunoprecipitation Assay

21) Product Images from "MicroRNA-195 Inhibits the Proliferation of Human Glioma Cells by Directly Targeting Cyclin D1 and Cyclin E1"

Article Title: MicroRNA-195 Inhibits the Proliferation of Human Glioma Cells by Directly Targeting Cyclin D1 and Cyclin E1

Journal: PLoS ONE

doi: 10.1371/journal.pone.0054932

miR-195 downregulates cyclin D1 and cyclin E1 by directly targeting their 3 ′ -UTRs. A , Predicted miR-195 target sequence in the 3′-UTR of cyclin D1 and cyclin E1 (cyclin D1 3′-UTR and cyclin E1 3′-UTR) and illustration of the three altered nucleotides in miR-195-mut. B , Western blotting analysis of expression of phosphorylated Rb (p-Rb), total Rb, PCNA, cyclin D1, cyclin E1, CDK2 and p21 in indicated cells. α-Tubulin served as the loading control. C , Western blotting analysis of GFP expression in indicated cells. D , Luciferase assay of indicated cells transfected with the pGL3-cyclin D1-3′UTR reporter (left) or the pGL3-cyclin E1-3′UTR reporter (right) with increasing amounts (10, 50 nM) of miR-195 mimic, or miR-195 inhibitor, or miR-195 mutant. Each bar represents the mean ± SD of three independent experiments. * P
Figure Legend Snippet: miR-195 downregulates cyclin D1 and cyclin E1 by directly targeting their 3 ′ -UTRs. A , Predicted miR-195 target sequence in the 3′-UTR of cyclin D1 and cyclin E1 (cyclin D1 3′-UTR and cyclin E1 3′-UTR) and illustration of the three altered nucleotides in miR-195-mut. B , Western blotting analysis of expression of phosphorylated Rb (p-Rb), total Rb, PCNA, cyclin D1, cyclin E1, CDK2 and p21 in indicated cells. α-Tubulin served as the loading control. C , Western blotting analysis of GFP expression in indicated cells. D , Luciferase assay of indicated cells transfected with the pGL3-cyclin D1-3′UTR reporter (left) or the pGL3-cyclin E1-3′UTR reporter (right) with increasing amounts (10, 50 nM) of miR-195 mimic, or miR-195 inhibitor, or miR-195 mutant. Each bar represents the mean ± SD of three independent experiments. * P

Techniques Used: Sequencing, Western Blot, Expressing, Luciferase, Transfection, Mutagenesis

22) Product Images from "Internal Ribosome Entry Segment Activity of ATXN8 Opposite Strand RNA"

Article Title: Internal Ribosome Entry Segment Activity of ATXN8 Opposite Strand RNA

Journal: PLoS ONE

doi: 10.1371/journal.pone.0073885

IRES activity of the ATXN8OS transcript. (A) ATXN8OS organization with promoter (open arrow), exons (open boxes) and functional splice donor sequences (GT) of D exons (D5, D4, D, D″ and D′) indicated. The CTG repeat tract is located in exon A. Transcription start site of exon D5 and exon D are represented by +1 and +801, respectively. (B) ATXN8OS RNA (NR_002717) generated from the splicing events represented by the wavy lines. The putative ORF initiated from AUG +1247 is indicated by the open boxes inside the RNA. The restriction enzymes and the cutting sites used to generate +801∼+1195 cDNA fragment of ATXN8OS are shown on the bottom of the cDNA. (C) The dual luciferase reporter plasmid had Renilla luciferase and firefly luciferase genes between the TK promoter and polyadenylation signal. The locations of Xba I, Xho I and Bam HI sites used for construction are shown on the top. (D) Relative luciferase activities generated by dual luciferase constructs with ECMV IRES and ATXN8OS +801∼+1195 cDNA fragment in HEK-293 and IMR-32 cells. Forty-eight hours following transfection, cells were harvested and luciferases activities were measured. IRES activity is expressed as percentages of the activity of the ECMV IRES, which was set at 100%. In addition, relative luciferase activities with ATXN8OS +801∼+953 and +953∼+1195 cDNA fragments were measured in HEK-293 cells, with IRES activity of +801∼+1195 set at 100%. Each value is the mean ± SD of three independent experiments each performed in duplicate.
Figure Legend Snippet: IRES activity of the ATXN8OS transcript. (A) ATXN8OS organization with promoter (open arrow), exons (open boxes) and functional splice donor sequences (GT) of D exons (D5, D4, D, D″ and D′) indicated. The CTG repeat tract is located in exon A. Transcription start site of exon D5 and exon D are represented by +1 and +801, respectively. (B) ATXN8OS RNA (NR_002717) generated from the splicing events represented by the wavy lines. The putative ORF initiated from AUG +1247 is indicated by the open boxes inside the RNA. The restriction enzymes and the cutting sites used to generate +801∼+1195 cDNA fragment of ATXN8OS are shown on the bottom of the cDNA. (C) The dual luciferase reporter plasmid had Renilla luciferase and firefly luciferase genes between the TK promoter and polyadenylation signal. The locations of Xba I, Xho I and Bam HI sites used for construction are shown on the top. (D) Relative luciferase activities generated by dual luciferase constructs with ECMV IRES and ATXN8OS +801∼+1195 cDNA fragment in HEK-293 and IMR-32 cells. Forty-eight hours following transfection, cells were harvested and luciferases activities were measured. IRES activity is expressed as percentages of the activity of the ECMV IRES, which was set at 100%. In addition, relative luciferase activities with ATXN8OS +801∼+953 and +953∼+1195 cDNA fragments were measured in HEK-293 cells, with IRES activity of +801∼+1195 set at 100%. Each value is the mean ± SD of three independent experiments each performed in duplicate.

Techniques Used: Activity Assay, Functional Assay, CTG Assay, Generated, Luciferase, Plasmid Preparation, Construct, Transfection

Transient expression of ATXN8OS ORF-EGFP constructs in HEK-293 cells. (A) ORF-EGFP constructs. A 752-bp cDNA fragment containing exon D, C2 and portion of C1 was inserted into pEGFP-N1 MCS so that ATXN8OS ORF was fused in-frame with the EGFP gene to generate pCMV/+801. A +1∼+800 ATXN8OS fragment was inserted between CMV promoter and exon D of pCMV/+801 to generate pCMV/+1. In pATXN8OS/−114 and/−481, 114 and 481-bp ATXN8OS promoter fragments was used to replace the CMV promoter in pCMV/+1. (B) Real-time PCR quantification of ORF-EGFP RNA level relative to endogenous HPRT1 RNA. To normalize, expression level in pATXN8OS/−481 transfected cells is set as 1.0. (C) FACS analysis of EGFP fluorescence. Levels of EGFP were expressed as percentages of pIRES2-EGFP, which was set at 100%. Each value is the mean ± SD of three independent experiments each performed in duplicate.
Figure Legend Snippet: Transient expression of ATXN8OS ORF-EGFP constructs in HEK-293 cells. (A) ORF-EGFP constructs. A 752-bp cDNA fragment containing exon D, C2 and portion of C1 was inserted into pEGFP-N1 MCS so that ATXN8OS ORF was fused in-frame with the EGFP gene to generate pCMV/+801. A +1∼+800 ATXN8OS fragment was inserted between CMV promoter and exon D of pCMV/+801 to generate pCMV/+1. In pATXN8OS/−114 and/−481, 114 and 481-bp ATXN8OS promoter fragments was used to replace the CMV promoter in pCMV/+1. (B) Real-time PCR quantification of ORF-EGFP RNA level relative to endogenous HPRT1 RNA. To normalize, expression level in pATXN8OS/−481 transfected cells is set as 1.0. (C) FACS analysis of EGFP fluorescence. Levels of EGFP were expressed as percentages of pIRES2-EGFP, which was set at 100%. Each value is the mean ± SD of three independent experiments each performed in duplicate.

Techniques Used: Expressing, Construct, Real-time Polymerase Chain Reaction, Transfection, FACS, Fluorescence

23) Product Images from "Phosphatase of regenerating liver-3 (PRL-3) is associated with metastasis and poor prognosis in gastric carcinoma"

Article Title: Phosphatase of regenerating liver-3 (PRL-3) is associated with metastasis and poor prognosis in gastric carcinoma

Journal: Journal of Translational Medicine

doi: 10.1186/1479-5876-11-309

Kaplan–Meier estimates of overall survival (OS) with respect to PRL-3 expression. A . OS curves stratified by PRL-3 expression in gastric cancer tissues. B . OS curves stratified by PRL-3 expression in the subgroup of well and moderately differentiated carcinoma. C . OS curves for the subgroups of patients without distant metastasis stratified by PRL-3 expression.
Figure Legend Snippet: Kaplan–Meier estimates of overall survival (OS) with respect to PRL-3 expression. A . OS curves stratified by PRL-3 expression in gastric cancer tissues. B . OS curves stratified by PRL-3 expression in the subgroup of well and moderately differentiated carcinoma. C . OS curves for the subgroups of patients without distant metastasis stratified by PRL-3 expression.

Techniques Used: Expressing

Effects of PRL-3-WT/mutations on migration and invasion. A . Migration assay in BGC823 cells expressing Myc-PRL-3-WT, mutant Myc-PRL-3(C104S) and Myc-PRL-3(ΔCAAX) using transwell chamber; B . Invasion assay in BGC823 cells expressing Myc-PRL-3-WT, mutant Myc-PRL-3(C104S) and Myc-PRL-3(ΔCAAX). Quantitative analysis of the number of the cells migrated to the lower side of the membrane in migration assay (C) and invasion assay (D) is shown. All Data are mean ± SE of three independent experiments. *, P
Figure Legend Snippet: Effects of PRL-3-WT/mutations on migration and invasion. A . Migration assay in BGC823 cells expressing Myc-PRL-3-WT, mutant Myc-PRL-3(C104S) and Myc-PRL-3(ΔCAAX) using transwell chamber; B . Invasion assay in BGC823 cells expressing Myc-PRL-3-WT, mutant Myc-PRL-3(C104S) and Myc-PRL-3(ΔCAAX). Quantitative analysis of the number of the cells migrated to the lower side of the membrane in migration assay (C) and invasion assay (D) is shown. All Data are mean ± SE of three independent experiments. *, P

Techniques Used: Migration, Expressing, Mutagenesis, Invasion Assay

Immunohistochemical staining of PRL-3 expression in gastric cancer. A . Negative control; B . PRL-3 mild expression in gastric carcinoma (x200); C . PRL-3 moderate expression in gastric carcinoma (x200); D . Cellular location of PRL-3 expression, mainly localized in the cytomembrane with some granulated loci in cytoplasm (x400, magnification of D .); E . Intense expression of PRL-3 in the primary gastric carcinoma (x200); F . PRL-3 expression in matched liver metastasis developed 2 years after surgery (x200, E and F are from the same patient).
Figure Legend Snippet: Immunohistochemical staining of PRL-3 expression in gastric cancer. A . Negative control; B . PRL-3 mild expression in gastric carcinoma (x200); C . PRL-3 moderate expression in gastric carcinoma (x200); D . Cellular location of PRL-3 expression, mainly localized in the cytomembrane with some granulated loci in cytoplasm (x400, magnification of D .); E . Intense expression of PRL-3 in the primary gastric carcinoma (x200); F . PRL-3 expression in matched liver metastasis developed 2 years after surgery (x200, E and F are from the same patient).

Techniques Used: Immunohistochemistry, Staining, Expressing, Negative Control

Level of Myc-PRL-3-WT, Myc-PRL-3(C104S), Myc-PRL-3( Δ CAAX) expression in stable BGC823 cell pools. They are detected by RT-PCR (A) and Western blot (B) . Results shown are representative of three independent experiments.
Figure Legend Snippet: Level of Myc-PRL-3-WT, Myc-PRL-3(C104S), Myc-PRL-3( Δ CAAX) expression in stable BGC823 cell pools. They are detected by RT-PCR (A) and Western blot (B) . Results shown are representative of three independent experiments.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Immunofluorescence of wild type PRL-3 and its mutants. Intracellular localization of PRL-3. BGC823 cells were transfected with pEGFP-C1-PRL-3-WT, pEGFP-C1-PRL-3(C104S) and pEGFP-C1-PRL-3(ΔCAAX) expression vectors. Nuclei were stained with 4, 6-diamidino-2-phemylindole (blue). PRL-3 expression was presented as green fluorescence.
Figure Legend Snippet: Immunofluorescence of wild type PRL-3 and its mutants. Intracellular localization of PRL-3. BGC823 cells were transfected with pEGFP-C1-PRL-3-WT, pEGFP-C1-PRL-3(C104S) and pEGFP-C1-PRL-3(ΔCAAX) expression vectors. Nuclei were stained with 4, 6-diamidino-2-phemylindole (blue). PRL-3 expression was presented as green fluorescence.

Techniques Used: Immunofluorescence, Transfection, Expressing, Staining, Fluorescence

24) Product Images from "Involvement of decreased hypoxia-inducible factor 1 activity and resultant G1-S cell cycle transition in radioresistance of perinecrotic tumor cells"

Article Title: Involvement of decreased hypoxia-inducible factor 1 activity and resultant G1-S cell cycle transition in radioresistance of perinecrotic tumor cells

Journal: Oncogene

doi: 10.1038/onc.2012.223

Radioresistance of cells under low-glucose and hypoxic conditions. ( a – e) With ( d ) or without ( a – c , e ) transient transfection using an empty vector or pcDNA4/p27 Kip1 , HEK293/EGFP-53BP1-M ( a , b , d , e ) and A549 ( c ) cells were cultured in medium containing a low (L: 0.45 g/l) or high (H: 4.5 g/l) concentration of glucose under normoxic (20% oxygen) or hypoxic (0.02% oxygen) conditions for 20 h, and treated with the indicated dose of X-radiation. ( a , d) The radiation-induced DNA double-stranded breaks (DNA DSBs) were then observed as EGFP-53BP1-M foci under a fluorescence microscope. The numbers of DNA DSB per nucleus in each group were quantified as the mean of 50 nuclei in 10 independent fields. Results are the mean±s.d. * P
Figure Legend Snippet: Radioresistance of cells under low-glucose and hypoxic conditions. ( a – e) With ( d ) or without ( a – c , e ) transient transfection using an empty vector or pcDNA4/p27 Kip1 , HEK293/EGFP-53BP1-M ( a , b , d , e ) and A549 ( c ) cells were cultured in medium containing a low (L: 0.45 g/l) or high (H: 4.5 g/l) concentration of glucose under normoxic (20% oxygen) or hypoxic (0.02% oxygen) conditions for 20 h, and treated with the indicated dose of X-radiation. ( a , d) The radiation-induced DNA double-stranded breaks (DNA DSBs) were then observed as EGFP-53BP1-M foci under a fluorescence microscope. The numbers of DNA DSB per nucleus in each group were quantified as the mean of 50 nuclei in 10 independent fields. Results are the mean±s.d. * P

Techniques Used: Transfection, Plasmid Preparation, Cell Culture, Concentration Assay, Fluorescence, Microscopy

25) Product Images from "p62/SQSTM1 Enhances NOD2-Mediated Signaling and Cytokine Production through Stabilizing NOD2 Oligomerization"

Article Title: p62/SQSTM1 Enhances NOD2-Mediated Signaling and Cytokine Production through Stabilizing NOD2 Oligomerization

Journal: PLoS ONE

doi: 10.1371/journal.pone.0057138

The NBD of NOD2 interacts with both TRAF6 and UBA domains of p62. A . NOD2 (left top panel) and p62 (right top panel) structures, and their mutant constructs are schematically presented. B . HEK293T cells were transfected with GFP-p62 and Myc-NBD (left middle panel), GFP-p62 and Myc-CARD (center middle panel) or GFP-p62 and Myc-ΔLRR (LRR region-deleted NOD2) (right middle panel). C . Similarly, HEK293T cells were transiently transfected with GFP-TRAF6 domain of p62 and Myc-ΔLRR (left bottom panel), GFP-UBA domain of p62 and Myc-ΔLRR (middle bottom panel), and GFP-PB1 domain of p62 and Myc-ΔLRR (right bottom panel); co-immunoprecipitation assays were performed as described in legend to Fig. 2. Data shown are representative images of 3 independent experiments.
Figure Legend Snippet: The NBD of NOD2 interacts with both TRAF6 and UBA domains of p62. A . NOD2 (left top panel) and p62 (right top panel) structures, and their mutant constructs are schematically presented. B . HEK293T cells were transfected with GFP-p62 and Myc-NBD (left middle panel), GFP-p62 and Myc-CARD (center middle panel) or GFP-p62 and Myc-ΔLRR (LRR region-deleted NOD2) (right middle panel). C . Similarly, HEK293T cells were transiently transfected with GFP-TRAF6 domain of p62 and Myc-ΔLRR (left bottom panel), GFP-UBA domain of p62 and Myc-ΔLRR (middle bottom panel), and GFP-PB1 domain of p62 and Myc-ΔLRR (right bottom panel); co-immunoprecipitation assays were performed as described in legend to Fig. 2. Data shown are representative images of 3 independent experiments.

Techniques Used: Mutagenesis, Construct, Transfection, Immunoprecipitation

p62 stabilizes gMDP-induced NOD2 oligomers. A . HEK293T cells were stably transfected with pLNCX-NOD2 as described in “Methods”. These cells were treated with scramble (si-Scramble) or p62 targeting (si-p62) small interference RNAs for 24 h. Cells were then treated with the translation inhibitor cyclohexamide (CHX, 100 µg/ml) and gMDP (5 µg/ml) for the time indicated, and immunoblots against NOD2 were performed. Intensities of NOD2 bands in comparison with p38 bands (loading control) were expressed as 100% for control samples (right panel). The ImageJ (NIH) program was used for densitometry analysis and data were expressed as mean ± S.D. (n≥4). *p
Figure Legend Snippet: p62 stabilizes gMDP-induced NOD2 oligomers. A . HEK293T cells were stably transfected with pLNCX-NOD2 as described in “Methods”. These cells were treated with scramble (si-Scramble) or p62 targeting (si-p62) small interference RNAs for 24 h. Cells were then treated with the translation inhibitor cyclohexamide (CHX, 100 µg/ml) and gMDP (5 µg/ml) for the time indicated, and immunoblots against NOD2 were performed. Intensities of NOD2 bands in comparison with p38 bands (loading control) were expressed as 100% for control samples (right panel). The ImageJ (NIH) program was used for densitometry analysis and data were expressed as mean ± S.D. (n≥4). *p

Techniques Used: Stable Transfection, Transfection, Western Blot

p62 co-localizes with NOD2 through the NBD domain of NOD2. A . HEK293T cells were transfected for 24 h with scramble siRNA (left top panel), si-p62 (right top panel), and GFP-NOD2. GFP-NOD2 was visualized using confocal microscopy as described in “Methods”. B . Similarly, DsRed-NBD domain, LRR region or full-length NOD2 and GFP-p62 expression vectors were transfected in HEK293T cells and co-localization of these proteins was examined using confocal microscopy. C . Immunogold staining of co-localized pCMV-HA-p62 (18 nm colloidal gold) and GFP-NOD2 (10 nm colloidal gold) in HEK293T cells. Cells on grids were viewed using a transmission electron microscope. Scale bars: 500 nm (left bottom), 100 nm (middle, right bottom). D . HEK293T cells were transfected with DsRed-NOD2 and GFP-LC3 plasmids. Cells were observed by confocal microscopy and images were acquired using ZEN software.
Figure Legend Snippet: p62 co-localizes with NOD2 through the NBD domain of NOD2. A . HEK293T cells were transfected for 24 h with scramble siRNA (left top panel), si-p62 (right top panel), and GFP-NOD2. GFP-NOD2 was visualized using confocal microscopy as described in “Methods”. B . Similarly, DsRed-NBD domain, LRR region or full-length NOD2 and GFP-p62 expression vectors were transfected in HEK293T cells and co-localization of these proteins was examined using confocal microscopy. C . Immunogold staining of co-localized pCMV-HA-p62 (18 nm colloidal gold) and GFP-NOD2 (10 nm colloidal gold) in HEK293T cells. Cells on grids were viewed using a transmission electron microscope. Scale bars: 500 nm (left bottom), 100 nm (middle, right bottom). D . HEK293T cells were transfected with DsRed-NOD2 and GFP-LC3 plasmids. Cells were observed by confocal microscopy and images were acquired using ZEN software.

Techniques Used: Transfection, Confocal Microscopy, Expressing, Staining, Transmission Assay, Microscopy, Software

NOD2 physically interacts with p62. A–C . HEK293T cells were transiently transfected with expression vectors encoding GFP-tagged p62 (GFP-p62) and/or Myc-tagged NOD2 (Myc-NOD2). After 24 h, total cell lysates were subjected to immunoprecipitation using anti-GFP (A) or anti-Myc (B) antibodies and the immune complexes were resolved by SDS-PAGE followed by immunoblotting against GFP and HA. C . Similar experiments as A-B were performed but with or without N-glycorylated muramyldipeptide (gMDP: 5 µg/mL) treatments for 4 h. Data shown are representative images of 3 independent experiments.
Figure Legend Snippet: NOD2 physically interacts with p62. A–C . HEK293T cells were transiently transfected with expression vectors encoding GFP-tagged p62 (GFP-p62) and/or Myc-tagged NOD2 (Myc-NOD2). After 24 h, total cell lysates were subjected to immunoprecipitation using anti-GFP (A) or anti-Myc (B) antibodies and the immune complexes were resolved by SDS-PAGE followed by immunoblotting against GFP and HA. C . Similar experiments as A-B were performed but with or without N-glycorylated muramyldipeptide (gMDP: 5 µg/mL) treatments for 4 h. Data shown are representative images of 3 independent experiments.

Techniques Used: Transfection, Expressing, Immunoprecipitation, SDS Page

p62 is required for the activation of NF-κB and p38 MAPK, and ubiquitination of RIP2 and TRAF6. A . HEK293T cells were transfected with pCMV-Myc-NOD2 and NF-κB luciferase reporter constructs in the presence of scrambled or p62-targeting small interference RNAs (si-p62). Cells were then treated with gMDP (5 µg/mL) for 4 h and NF-κB activity was measured. Data are expressed as the fold of luciferase activity ± SD (n = 3). B-C . HEK293T cells stably expressing NOD2 were first treated with scramble siRNA or si-p62 for 24 h, and then transfected with expression vectors for HA-ubiquitin (HA-Ub) and pcDNA3-Myc-RIP2 or pCMV-Myc-TRAF6 for another 24 h. After treating the cells with gMDP (5 µg/mL) for 4 h, RIP2 (B) or TRAF6 (C) was immunoprecipitated with Myc antibodies from total cell lysates and the immune complexes were resolved by SDS-PAGE followed by immunoblotting against HA. Myc-RIP2 or Myc-TRAF6, NOD2 and HA-ubiquitin were analyzed by immunoblot as the inputs (bottom panels). p62 protein levels were also measured by immunoblot. Data shown are representative images of 3 independent experiments. D , HEK293T cells stably expressing NOD2 were first treated with scramble siRNA or si-p62 for 24 h, and then treated with gMDP (5 µg/ml) for the times indicated. Activation of p38 was analysed through immunoblotting against tyrosine phosphoryled p38. The ImageJ (NIH) program was used for densitometry analysis of phosphor-p83 bands and data were expressed as mean ± S.D. (n = 3).
Figure Legend Snippet: p62 is required for the activation of NF-κB and p38 MAPK, and ubiquitination of RIP2 and TRAF6. A . HEK293T cells were transfected with pCMV-Myc-NOD2 and NF-κB luciferase reporter constructs in the presence of scrambled or p62-targeting small interference RNAs (si-p62). Cells were then treated with gMDP (5 µg/mL) for 4 h and NF-κB activity was measured. Data are expressed as the fold of luciferase activity ± SD (n = 3). B-C . HEK293T cells stably expressing NOD2 were first treated with scramble siRNA or si-p62 for 24 h, and then transfected with expression vectors for HA-ubiquitin (HA-Ub) and pcDNA3-Myc-RIP2 or pCMV-Myc-TRAF6 for another 24 h. After treating the cells with gMDP (5 µg/mL) for 4 h, RIP2 (B) or TRAF6 (C) was immunoprecipitated with Myc antibodies from total cell lysates and the immune complexes were resolved by SDS-PAGE followed by immunoblotting against HA. Myc-RIP2 or Myc-TRAF6, NOD2 and HA-ubiquitin were analyzed by immunoblot as the inputs (bottom panels). p62 protein levels were also measured by immunoblot. Data shown are representative images of 3 independent experiments. D , HEK293T cells stably expressing NOD2 were first treated with scramble siRNA or si-p62 for 24 h, and then treated with gMDP (5 µg/ml) for the times indicated. Activation of p38 was analysed through immunoblotting against tyrosine phosphoryled p38. The ImageJ (NIH) program was used for densitometry analysis of phosphor-p83 bands and data were expressed as mean ± S.D. (n = 3).

Techniques Used: Activation Assay, Transfection, Luciferase, Construct, Activity Assay, Stable Transfection, Expressing, Immunoprecipitation, SDS Page

26) Product Images from "An engineered three-dimensional gastric tumor culture model for evaluating the antitumor activity of immune cells in vitro"

Article Title: An engineered three-dimensional gastric tumor culture model for evaluating the antitumor activity of immune cells in vitro

Journal: Oncology Letters

doi: 10.3892/ol.2012.1021

FACS analysis of the transfected tumor cells and the PKH26-labeled CIK cells. BGC823 cells were transfected with EGFP-expressing plasmid and the CIK cells were labeled with PKH26. The efficacy of transfection and PKH-labeling was characterized by FACS analysis. Data are representative histograms of each type of cells from three separate experiments. (A) FACS analysis of EGFP + BGC823 cells; (B) FACS analysis of PKH26-labeled CIK cells. CIK, cytokine-induced killer.
Figure Legend Snippet: FACS analysis of the transfected tumor cells and the PKH26-labeled CIK cells. BGC823 cells were transfected with EGFP-expressing plasmid and the CIK cells were labeled with PKH26. The efficacy of transfection and PKH-labeling was characterized by FACS analysis. Data are representative histograms of each type of cells from three separate experiments. (A) FACS analysis of EGFP + BGC823 cells; (B) FACS analysis of PKH26-labeled CIK cells. CIK, cytokine-induced killer.

Techniques Used: FACS, Transfection, Labeling, Expressing, Plasmid Preparation

27) Product Images from "TIAF1 self-aggregation in peritumor capsule formation, spontaneous activation of SMAD-responsive promoter in p53-deficient environment, and cell death"

Article Title: TIAF1 self-aggregation in peritumor capsule formation, spontaneous activation of SMAD-responsive promoter in p53-deficient environment, and cell death

Journal: Cell Death & Disease

doi: 10.1038/cddis.2012.36

TIAF1 is essential for apoptosis mediated by WOX1, p53 and dominant-negative JNK1. ( a ) Mv1Lu cells were transfected with expression plasmid constructs of WOX1 and/or TIAF1si by electroporation, followed by culturing for 48 h. The extent of cell growth was measured by MTS proliferation assay. TIAF1 knockdown cells resisted WOX1-induced growth inhibition ( n =8; mean±S.D.; Student's t -test: experiments versus scramble controls). Scram=‘scrambled RNA' control plasmid. ( b ) L929 cells were transfected with WOX1 and/or TIAF1si plasmids, and then grown in soft agarose for 3 weeks to allow colony formation (measured by MTS proliferation assay) ( n =3; Student's t -test; experiments versus scramble controls). ( c ) When L929 cells were transfected with WOX1 and TIAF1 plasmids (1.25 μ g per 10 6 cells), both expressed proteins synergistically caused cell death (∼40%, n=8). Dominant-negative WOX1 (dnWOX1) 17 and phospho-WOX1 mutants (Y33R and Y61R) 17 failed to induce cell death, in the absence or presence of TIAF1 ( n =8; Student's t -test: experiments versus scramble controls). ( d ) L929 stable transfectants, expressing a scramble, TIAF1si or WOX1si construct, were established. Transient overexpression of these cells with an empty vector, p53 or dnJNK1 construct was carried out, and the extent of cell death was measured in 48 h ( n =8; Student's t -test; experiments versus scramble controls). When TIAF1 and WOX1 were knocked down, ectopic p53 and dnJNK1-induced cell death was blocked. ( e ) In contrast, when TIAF1 was knocked down, Smad4-induced apoptosis of Mv1Lu cells was enhanced (see subG1 phase; a representative data from two experiments). a , SubG1 phase; b , G0/G1 phase; c , S phase; d , G2/M phase
Figure Legend Snippet: TIAF1 is essential for apoptosis mediated by WOX1, p53 and dominant-negative JNK1. ( a ) Mv1Lu cells were transfected with expression plasmid constructs of WOX1 and/or TIAF1si by electroporation, followed by culturing for 48 h. The extent of cell growth was measured by MTS proliferation assay. TIAF1 knockdown cells resisted WOX1-induced growth inhibition ( n =8; mean±S.D.; Student's t -test: experiments versus scramble controls). Scram=‘scrambled RNA' control plasmid. ( b ) L929 cells were transfected with WOX1 and/or TIAF1si plasmids, and then grown in soft agarose for 3 weeks to allow colony formation (measured by MTS proliferation assay) ( n =3; Student's t -test; experiments versus scramble controls). ( c ) When L929 cells were transfected with WOX1 and TIAF1 plasmids (1.25 μ g per 10 6 cells), both expressed proteins synergistically caused cell death (∼40%, n=8). Dominant-negative WOX1 (dnWOX1) 17 and phospho-WOX1 mutants (Y33R and Y61R) 17 failed to induce cell death, in the absence or presence of TIAF1 ( n =8; Student's t -test: experiments versus scramble controls). ( d ) L929 stable transfectants, expressing a scramble, TIAF1si or WOX1si construct, were established. Transient overexpression of these cells with an empty vector, p53 or dnJNK1 construct was carried out, and the extent of cell death was measured in 48 h ( n =8; Student's t -test; experiments versus scramble controls). When TIAF1 and WOX1 were knocked down, ectopic p53 and dnJNK1-induced cell death was blocked. ( e ) In contrast, when TIAF1 was knocked down, Smad4-induced apoptosis of Mv1Lu cells was enhanced (see subG1 phase; a representative data from two experiments). a , SubG1 phase; b , G0/G1 phase; c , S phase; d , G2/M phase

Techniques Used: Dominant Negative Mutation, Transfection, Expressing, Plasmid Preparation, Construct, Electroporation, Proliferation Assay, Inhibition, Over Expression

28) Product Images from "TIAF1 self-aggregation in peritumor capsule formation, spontaneous activation of SMAD-responsive promoter in p53-deficient environment, and cell death"

Article Title: TIAF1 self-aggregation in peritumor capsule formation, spontaneous activation of SMAD-responsive promoter in p53-deficient environment, and cell death

Journal: Cell Death & Disease

doi: 10.1038/cddis.2012.36

TIAF1 is essential for apoptosis mediated by WOX1, p53 and dominant-negative JNK1. ( a ) Mv1Lu cells were transfected with expression plasmid constructs of WOX1 and/or TIAF1si by electroporation, followed by culturing for 48 h. The extent of cell growth was measured by MTS proliferation assay. TIAF1 knockdown cells resisted WOX1-induced growth inhibition ( n =8; mean±S.D.; Student's t -test: experiments versus scramble controls). Scram=‘scrambled RNA' control plasmid. ( b ) L929 cells were transfected with WOX1 and/or TIAF1si plasmids, and then grown in soft agarose for 3 weeks to allow colony formation (measured by MTS proliferation assay) ( n =3; Student's t -test; experiments versus scramble controls). ( c ) When L929 cells were transfected with WOX1 and TIAF1 plasmids (1.25 μ g per 10 6 cells), both expressed proteins synergistically caused cell death (∼40%, n=8). Dominant-negative WOX1 (dnWOX1) 17 and phospho-WOX1 mutants (Y33R and Y61R) 17 failed to induce cell death, in the absence or presence of TIAF1 ( n =8; Student's t -test: experiments versus scramble controls). ( d ) L929 stable transfectants, expressing a scramble, TIAF1si or WOX1si construct, were established. Transient overexpression of these cells with an empty vector, p53 or dnJNK1 construct was carried out, and the extent of cell death was measured in 48 h ( n =8; Student's t -test; experiments versus scramble controls). When TIAF1 and WOX1 were knocked down, ectopic p53 and dnJNK1-induced cell death was blocked. ( e ) In contrast, when TIAF1 was knocked down, Smad4-induced apoptosis of Mv1Lu cells was enhanced (see subG1 phase; a representative data from two experiments). a , SubG1 phase; b , G0/G1 phase; c , S phase; d , G2/M phase
Figure Legend Snippet: TIAF1 is essential for apoptosis mediated by WOX1, p53 and dominant-negative JNK1. ( a ) Mv1Lu cells were transfected with expression plasmid constructs of WOX1 and/or TIAF1si by electroporation, followed by culturing for 48 h. The extent of cell growth was measured by MTS proliferation assay. TIAF1 knockdown cells resisted WOX1-induced growth inhibition ( n =8; mean±S.D.; Student's t -test: experiments versus scramble controls). Scram=‘scrambled RNA' control plasmid. ( b ) L929 cells were transfected with WOX1 and/or TIAF1si plasmids, and then grown in soft agarose for 3 weeks to allow colony formation (measured by MTS proliferation assay) ( n =3; Student's t -test; experiments versus scramble controls). ( c ) When L929 cells were transfected with WOX1 and TIAF1 plasmids (1.25 μ g per 10 6 cells), both expressed proteins synergistically caused cell death (∼40%, n=8). Dominant-negative WOX1 (dnWOX1) 17 and phospho-WOX1 mutants (Y33R and Y61R) 17 failed to induce cell death, in the absence or presence of TIAF1 ( n =8; Student's t -test: experiments versus scramble controls). ( d ) L929 stable transfectants, expressing a scramble, TIAF1si or WOX1si construct, were established. Transient overexpression of these cells with an empty vector, p53 or dnJNK1 construct was carried out, and the extent of cell death was measured in 48 h ( n =8; Student's t -test; experiments versus scramble controls). When TIAF1 and WOX1 were knocked down, ectopic p53 and dnJNK1-induced cell death was blocked. ( e ) In contrast, when TIAF1 was knocked down, Smad4-induced apoptosis of Mv1Lu cells was enhanced (see subG1 phase; a representative data from two experiments). a , SubG1 phase; b , G0/G1 phase; c , S phase; d , G2/M phase

Techniques Used: Dominant Negative Mutation, Transfection, Expressing, Plasmid Preparation, Construct, Electroporation, Proliferation Assay, Inhibition, Over Expression

29) Product Images from "Casein kinase I delta controls centrosome positioning during T cell activation"

Article Title: Casein kinase I delta controls centrosome positioning during T cell activation

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201106025

CKIδ is required for centrosome translocation to the IS. (A) Still images from time-lapse videos showing conjugate formation between Jurkat cells transfected with GFP-centrin1 (green) and SEE-pulsed Raji cells (blue). Control (EV; top; Videos 1 and 2 ) and CKIδ-depleted Jurkat cells (shdelta; bottom; Videos 3 and 4 ) are shown. Bar, 10 µm. The percentages next to panels indicate the frequency of the particular phenotype. The plots below depict the distance between the centrosome and the IS as a function of time; time 0 is when the conjugate forms. Tracks are symbol- and color-matched to individual cells. The shdelta cell (orange) polarizes its centrosome with a delay. (B) Summary of the time-lapse data collected. (C) Distribution of the time required for centrosome polarization to the IS in control (EV) and CKIδ-depleted (shdelta) cells. Median values are marked by horizontal lines.
Figure Legend Snippet: CKIδ is required for centrosome translocation to the IS. (A) Still images from time-lapse videos showing conjugate formation between Jurkat cells transfected with GFP-centrin1 (green) and SEE-pulsed Raji cells (blue). Control (EV; top; Videos 1 and 2 ) and CKIδ-depleted Jurkat cells (shdelta; bottom; Videos 3 and 4 ) are shown. Bar, 10 µm. The percentages next to panels indicate the frequency of the particular phenotype. The plots below depict the distance between the centrosome and the IS as a function of time; time 0 is when the conjugate forms. Tracks are symbol- and color-matched to individual cells. The shdelta cell (orange) polarizes its centrosome with a delay. (B) Summary of the time-lapse data collected. (C) Distribution of the time required for centrosome polarization to the IS in control (EV) and CKIδ-depleted (shdelta) cells. Median values are marked by horizontal lines.

Techniques Used: Translocation Assay, Transfection

CKIδ promotes microtubule growth in Jurkat cells. (A) Analysis of EB3-GFP comets in control (EV), CKIδ-depleted (shdelta) Jurkat cell clones, and colcemid-treated EV cells. The box plot shows the distribution of mean growth speeds of tracked EB3-GFP comets per cell. On the right, an overlay of EB3 growth tracks collected over 1 min is shown in individual cells (dots mark centrosome position). Bar, 5 μm. (B) Maximum intensity projection of EB3-GFP signal in individual cells over 28 sequential frames (0.5 s per frame). Corresponding kymographs on the right show the growth of individual microtubules in control (EV) and CKIδ-depleted (shdelta) Jurkat cells. Bars, 5 µm. (C) Analysis of EB3-GFP comets in nonconjugated (Non-conj) or Raji-conjugated (Conj) Jurkat cells. The box plot shows the distribution of mean growth speeds of tracked EB3-GFP comets per cell. In box plots, whiskers are set at 5–95 percentiles; horizontal lines mark the median, and boxes indicate interquartile range (25–75%).
Figure Legend Snippet: CKIδ promotes microtubule growth in Jurkat cells. (A) Analysis of EB3-GFP comets in control (EV), CKIδ-depleted (shdelta) Jurkat cell clones, and colcemid-treated EV cells. The box plot shows the distribution of mean growth speeds of tracked EB3-GFP comets per cell. On the right, an overlay of EB3 growth tracks collected over 1 min is shown in individual cells (dots mark centrosome position). Bar, 5 μm. (B) Maximum intensity projection of EB3-GFP signal in individual cells over 28 sequential frames (0.5 s per frame). Corresponding kymographs on the right show the growth of individual microtubules in control (EV) and CKIδ-depleted (shdelta) Jurkat cells. Bars, 5 µm. (C) Analysis of EB3-GFP comets in nonconjugated (Non-conj) or Raji-conjugated (Conj) Jurkat cells. The box plot shows the distribution of mean growth speeds of tracked EB3-GFP comets per cell. In box plots, whiskers are set at 5–95 percentiles; horizontal lines mark the median, and boxes indicate interquartile range (25–75%).

Techniques Used: Clone Assay

Identification of a CKIδ phosphorylation site on EB1. (A) A table summarizing the experimental conditions and results for assay 1. Fragmentation spectra were obtained and analyzed for all conditions. The spectrum on the right corresponds to 10 units of kinase. Ponceau S staining shows purified recombinant GST-EB1 used in the assay. Molecular mass (MM) is indicated in kilodaltons. (B) A table summarizing the experimental conditions and results for assay 2. Fragmentation spectra were obtained and analyzed for all conditions. The spectrum on the right corresponds to 10 units of kinase. Ponceau S staining shows the purified recombinant EB1 used in the assay. (A and B) Fragmentation spectra of the triply charged [M + 3H] 3+ peptide ion at m/z 700.3676 (A) and m/z 700.367 (B) correspond to the phosphorylated peptide of aa 131–150 with the sequence phospho-QGQETAVAPSLVAPALNKPK of EB1. The predominant y-ion series for both spectra, extending from y11 to y17, confirm the sequence ETAVAP-phosphoS (dots). The fragment ions observed at m/z 667.93 (A) and 668.14 (B) correspond to the neutral loss of H 3 PO 4 from the precursor ions, whereas those observed at m/z 662.14 (A) and 662.14 (B) correspond to the neutral loss of H 3 PO 4 and H 2 O. GST-CKIδ and CKIδ[1–318] were commercially sourced. (C) A schematic diagram shows the position of the phosphorylation site in EB1. The domains shown are calponin homology (CH) and end binding homology (EBH).
Figure Legend Snippet: Identification of a CKIδ phosphorylation site on EB1. (A) A table summarizing the experimental conditions and results for assay 1. Fragmentation spectra were obtained and analyzed for all conditions. The spectrum on the right corresponds to 10 units of kinase. Ponceau S staining shows purified recombinant GST-EB1 used in the assay. Molecular mass (MM) is indicated in kilodaltons. (B) A table summarizing the experimental conditions and results for assay 2. Fragmentation spectra were obtained and analyzed for all conditions. The spectrum on the right corresponds to 10 units of kinase. Ponceau S staining shows the purified recombinant EB1 used in the assay. (A and B) Fragmentation spectra of the triply charged [M + 3H] 3+ peptide ion at m/z 700.3676 (A) and m/z 700.367 (B) correspond to the phosphorylated peptide of aa 131–150 with the sequence phospho-QGQETAVAPSLVAPALNKPK of EB1. The predominant y-ion series for both spectra, extending from y11 to y17, confirm the sequence ETAVAP-phosphoS (dots). The fragment ions observed at m/z 667.93 (A) and 668.14 (B) correspond to the neutral loss of H 3 PO 4 from the precursor ions, whereas those observed at m/z 662.14 (A) and 662.14 (B) correspond to the neutral loss of H 3 PO 4 and H 2 O. GST-CKIδ and CKIδ[1–318] were commercially sourced. (C) A schematic diagram shows the position of the phosphorylation site in EB1. The domains shown are calponin homology (CH) and end binding homology (EBH).

Techniques Used: Staining, Purification, Recombinant, Sequencing, Binding Assay

CKIδ is dispensable for IS formation and early TCR signaling. (A) Conjugates of control (EV) or CKIδ-depleted (shdelta) Jurkat and SEE-pulsed Raji cells are stained with anti–LFA-1 antibody (top), phalloidin (F-actin; middle), or anti-CD3 antibody (bottom). Centrosomes are stained with anti-CDK5RAP2 antibody. Raji cells are in blue in merge. (B) Cytoplasmic cell extracts of control (EV) or CKIδ-depleted (shdelta) Jurkat cells were lysed at different time points after conjugate formation with SEE-pulsed Raji cells and were immunoblotted with the indicated antibodies. WB, Western blotting. (C) Conjugates between control (EV; left) or CKIδ-depleted (shdelta; right) Jurkat and SEE-pulsed Raji cells are stained with anti-DIC (green in merge) and anti-CDK5RAP2 (red in merge) antibodies. Arrowheads highlight DIC accumulation at the IS. (D and E) Cytoplasmic cell extracts (CCE) were prepared from control (EV) and CKIδ-depleted (shdelta; D) or DMSO- and D4476-treated (E) Jurkat cells that were unconjugated (−) or conjugated (+) to anti-CD3 antibody beads. Immunoprecipitations (ip) were performed with random IgG (ip con) or anti-p150 glued (ip p150 glued ) antibodies. Bars: 10 µm; (en face) 5 µm.
Figure Legend Snippet: CKIδ is dispensable for IS formation and early TCR signaling. (A) Conjugates of control (EV) or CKIδ-depleted (shdelta) Jurkat and SEE-pulsed Raji cells are stained with anti–LFA-1 antibody (top), phalloidin (F-actin; middle), or anti-CD3 antibody (bottom). Centrosomes are stained with anti-CDK5RAP2 antibody. Raji cells are in blue in merge. (B) Cytoplasmic cell extracts of control (EV) or CKIδ-depleted (shdelta) Jurkat cells were lysed at different time points after conjugate formation with SEE-pulsed Raji cells and were immunoblotted with the indicated antibodies. WB, Western blotting. (C) Conjugates between control (EV; left) or CKIδ-depleted (shdelta; right) Jurkat and SEE-pulsed Raji cells are stained with anti-DIC (green in merge) and anti-CDK5RAP2 (red in merge) antibodies. Arrowheads highlight DIC accumulation at the IS. (D and E) Cytoplasmic cell extracts (CCE) were prepared from control (EV) and CKIδ-depleted (shdelta; D) or DMSO- and D4476-treated (E) Jurkat cells that were unconjugated (−) or conjugated (+) to anti-CD3 antibody beads. Immunoprecipitations (ip) were performed with random IgG (ip con) or anti-p150 glued (ip p150 glued ) antibodies. Bars: 10 µm; (en face) 5 µm.

Techniques Used: Staining, Western Blot

CKIδ interacts with the microtubule plus-end–binding proteins EB1 and p150 glued . (A) Still images from time-lapse imaging ( Video 5 ) showing conjugate formation between a Jurkat cell expressing GFP-CKIδ (green) and an SEE-pulsed Raji cell (blue). Insets correspond to higher magnifications of the centrosomal region. Asterisks mark fiberlike extrusions near the centrosome. Bars: 10 µm; (inset) 1 µm. (B) In vitro microtubule-pelleting assay. Pure tubulin was incubated in the presence of GTP without (−) or with (+) taxol and then added to cytoplasmic extracts of Jurkat cells transfected with GFP-CKIδ. High-speed supernatants (S) and pellets (P) were collected and immunoblotted with antibodies as indicated. WB, Western blotting. (C) A schematic view of key functional domains of CKIδ: kinase, autoinhibitory ( Longenecker et al., 1998 ), and centrosome-targeting ( Greer and Rubin, 2011 ) domains. Sequence alignments of the extreme C termini of two CKIδ isoforms and CKIε are shown below. Acidic and basic amino acids are in red and blue, respectively. Hs, Homo sapiens ; Mm, Mus musculus ; Gg, Gallus gallus ; Xl, Xenopus laevis ; Dr, Danio rerio . (D) Cytoplasmic cell extracts (CCE) of Jurkat cells were subjected to pull-down assays with GST, GST-EB1 (EB1), or GST-EB3 (EB3) and were immunoblotted with antibodies as indicated. Recombinant GST products are shown in Ponceau S staining below. (E) Jurkat cells were unconjugated (−) or conjugated (+) with anti-CD3–coated beads. Cytoplasmic cell extracts were processed for immunoprecipitation (ip) with random IgG (ip con) or anti-p150 glued (ip p150 glued ) antibodies and immunoblotted with the indicated antibodies. (F) Cytoplasmic cell extracts of Jurkat cells were mock depleted with random IgG (−) or immunodepleted of p150 glued (+). Depleted extracts were then processed for pull-down assays with GST or GST-EB1 (EB1) and immunoblotted with the indicated antibodies. Recombinant GST products are visible in Ponceau S staining below.
Figure Legend Snippet: CKIδ interacts with the microtubule plus-end–binding proteins EB1 and p150 glued . (A) Still images from time-lapse imaging ( Video 5 ) showing conjugate formation between a Jurkat cell expressing GFP-CKIδ (green) and an SEE-pulsed Raji cell (blue). Insets correspond to higher magnifications of the centrosomal region. Asterisks mark fiberlike extrusions near the centrosome. Bars: 10 µm; (inset) 1 µm. (B) In vitro microtubule-pelleting assay. Pure tubulin was incubated in the presence of GTP without (−) or with (+) taxol and then added to cytoplasmic extracts of Jurkat cells transfected with GFP-CKIδ. High-speed supernatants (S) and pellets (P) were collected and immunoblotted with antibodies as indicated. WB, Western blotting. (C) A schematic view of key functional domains of CKIδ: kinase, autoinhibitory ( Longenecker et al., 1998 ), and centrosome-targeting ( Greer and Rubin, 2011 ) domains. Sequence alignments of the extreme C termini of two CKIδ isoforms and CKIε are shown below. Acidic and basic amino acids are in red and blue, respectively. Hs, Homo sapiens ; Mm, Mus musculus ; Gg, Gallus gallus ; Xl, Xenopus laevis ; Dr, Danio rerio . (D) Cytoplasmic cell extracts (CCE) of Jurkat cells were subjected to pull-down assays with GST, GST-EB1 (EB1), or GST-EB3 (EB3) and were immunoblotted with antibodies as indicated. Recombinant GST products are shown in Ponceau S staining below. (E) Jurkat cells were unconjugated (−) or conjugated (+) with anti-CD3–coated beads. Cytoplasmic cell extracts were processed for immunoprecipitation (ip) with random IgG (ip con) or anti-p150 glued (ip p150 glued ) antibodies and immunoblotted with the indicated antibodies. (F) Cytoplasmic cell extracts of Jurkat cells were mock depleted with random IgG (−) or immunodepleted of p150 glued (+). Depleted extracts were then processed for pull-down assays with GST or GST-EB1 (EB1) and immunoblotted with the indicated antibodies. Recombinant GST products are visible in Ponceau S staining below.

Techniques Used: Binding Assay, Imaging, Expressing, In Vitro, Incubation, Transfection, Western Blot, Functional Assay, Sequencing, Recombinant, Staining, Immunoprecipitation

CKIδ regulates TCR-mediated centrosome polarization to the IS. (A) Conjugates of Jurkat and SEE-pulsed Raji cells in the presence of the indicated drugs. Centrosomes are stained with anti–γ-tubulin antibody. Raji cells are blue in merge. The box plot shows quantification of centrosome polarization to the IS based on scoring criteria in Fig. S1 A ( n = 4 experiments; 200 conjugates/experiment). (B) Conjugates of Jurkat and SEE-pulsed Raji cells in the presence of the indicated drugs. Cells are stained with anti–PY174-Vav and anti–γ-tubulin antibodies. Raji cells are blue in merge. (C) Cytoplasmic cell extracts of Jurkat cells containing stably integrated EV (control), vector encoding CSNK1D shRNA (clones shdelta1 and shdelta2), or CSNK1E shRNA (clone sheps1) are immunoblotted with antibodies against CKIδ or CKIε. Actin serves as a loading control. WB, Western blotting. (D) Conjugates of control (EV), CKIε- or CKIδ-depleted Jurkat cell clones, and SEE-pulsed Raji cells. Centrosomes are stained with anti–γ-tubulin antibody. Raji cells are blue in merge. The box plot shows quantification of centrosome polarization to the IS ( n = 4 experiments; 200 conjugates/experiment). In the box plots, whiskers are set at minimum and maximum, and horizontal lines mark the median, whereas boxes indicate the interquartile range (25–75%). Bars:10 µm; (en face) 5 µm.
Figure Legend Snippet: CKIδ regulates TCR-mediated centrosome polarization to the IS. (A) Conjugates of Jurkat and SEE-pulsed Raji cells in the presence of the indicated drugs. Centrosomes are stained with anti–γ-tubulin antibody. Raji cells are blue in merge. The box plot shows quantification of centrosome polarization to the IS based on scoring criteria in Fig. S1 A ( n = 4 experiments; 200 conjugates/experiment). (B) Conjugates of Jurkat and SEE-pulsed Raji cells in the presence of the indicated drugs. Cells are stained with anti–PY174-Vav and anti–γ-tubulin antibodies. Raji cells are blue in merge. (C) Cytoplasmic cell extracts of Jurkat cells containing stably integrated EV (control), vector encoding CSNK1D shRNA (clones shdelta1 and shdelta2), or CSNK1E shRNA (clone sheps1) are immunoblotted with antibodies against CKIδ or CKIε. Actin serves as a loading control. WB, Western blotting. (D) Conjugates of control (EV), CKIε- or CKIδ-depleted Jurkat cell clones, and SEE-pulsed Raji cells. Centrosomes are stained with anti–γ-tubulin antibody. Raji cells are blue in merge. The box plot shows quantification of centrosome polarization to the IS ( n = 4 experiments; 200 conjugates/experiment). In the box plots, whiskers are set at minimum and maximum, and horizontal lines mark the median, whereas boxes indicate the interquartile range (25–75%). Bars:10 µm; (en face) 5 µm.

Techniques Used: Staining, Stable Transfection, Plasmid Preparation, shRNA, Western Blot, Clone Assay

The SQIP motif of CKIδ is required for centrosome polarization. (A) A schematic view of various GFP-fused CKIδ constructs is shown on top. Examples for conjugates formed between SEE-pulsed Raji cells and CKIδ-depleted Jurkat cells (shdelta) expressing the indicated GFP fusion product (green in merge). Asterisks mark cells with GFP signal. Centrosomes are stained with anti-CDK5RAP2 antibodies (red in merge). Raji cells are blue in merge. Bars, 10 µm. (B) A graph showing quantification of centrosome polarization to the IS in cells expressing the different GFP constructs ( n = 4 experiments; at least 50 GFP-positive cells were scored per experiment). Error bars represent SD.
Figure Legend Snippet: The SQIP motif of CKIδ is required for centrosome polarization. (A) A schematic view of various GFP-fused CKIδ constructs is shown on top. Examples for conjugates formed between SEE-pulsed Raji cells and CKIδ-depleted Jurkat cells (shdelta) expressing the indicated GFP fusion product (green in merge). Asterisks mark cells with GFP signal. Centrosomes are stained with anti-CDK5RAP2 antibodies (red in merge). Raji cells are blue in merge. Bars, 10 µm. (B) A graph showing quantification of centrosome polarization to the IS in cells expressing the different GFP constructs ( n = 4 experiments; at least 50 GFP-positive cells were scored per experiment). Error bars represent SD.

Techniques Used: Construct, Expressing, Staining

30) Product Images from "Visualizing the proteome of Escherichia coli: an efficient and versatile method for labeling chromosomal coding DNA sequences (CDSs) with fluorescent protein genes"

Article Title: Visualizing the proteome of Escherichia coli: an efficient and versatile method for labeling chromosomal coding DNA sequences (CDSs) with fluorescent protein genes

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkl1158

Representative fluorescence microscopy images of live, immobilized E. coli cells containing C-terminal fluorescent fusions to one or two CDSs on the chromosome. All panels are at the same magnification, and show a phase contrast image of cells (gray); DAPI fluorescent staining of (nucleoid) DNA (blue); mRFP1 fluorescence (red), EGFP or EYFP fluorescence (green). Panel 1: DY380 adk -mRFP1; AdK distributed diffusely throughout cytoplasm. Panel 2: DY380 yeaA -EYFP; YeaA located evenly throughout cytoplasm. Panel 3a and 3b: DY380 metK -mRFP1/ pyrH -EGFP; cells containing MetK and PyrH in varying proportions of diffuse cytoplasmic and punctate forms (foci). Panel 4: DY380 yfjP -EYFP/ pyrH -mRFP1; PyrH located in one membrane-associated foci per cell, NadK distributed irregularly throughout the cytoplasm, with some clusters formed. Panel 5: DY380 metK -mRFP1; cells containing almost exclusively diffuse cytoplasmic MetK, or foci. Panel 6: DY380 nusA -mRFP1; NusA located predominantly in 2 clusters associated with each nucleoid. Panel 7: DY380 pyrH -RFP; PyrH distributed diffusely throughout cytoplasm, and in 1–4 foci per cell. Panel 8: DY380 yaeL -EYFP; YaeL generally associated with the membrane. Panel 9: DY380 yqiK -EGFP; YqiK distributed diffusely throughout cytoplasm. Panel 10: DY380 yihA -EGFP; YihA distributed diffusely throughout cytoplasm. Panel 11: DY380 ygeH -EGFP; YgeH located predominantly beside the membrane.
Figure Legend Snippet: Representative fluorescence microscopy images of live, immobilized E. coli cells containing C-terminal fluorescent fusions to one or two CDSs on the chromosome. All panels are at the same magnification, and show a phase contrast image of cells (gray); DAPI fluorescent staining of (nucleoid) DNA (blue); mRFP1 fluorescence (red), EGFP or EYFP fluorescence (green). Panel 1: DY380 adk -mRFP1; AdK distributed diffusely throughout cytoplasm. Panel 2: DY380 yeaA -EYFP; YeaA located evenly throughout cytoplasm. Panel 3a and 3b: DY380 metK -mRFP1/ pyrH -EGFP; cells containing MetK and PyrH in varying proportions of diffuse cytoplasmic and punctate forms (foci). Panel 4: DY380 yfjP -EYFP/ pyrH -mRFP1; PyrH located in one membrane-associated foci per cell, NadK distributed irregularly throughout the cytoplasm, with some clusters formed. Panel 5: DY380 metK -mRFP1; cells containing almost exclusively diffuse cytoplasmic MetK, or foci. Panel 6: DY380 nusA -mRFP1; NusA located predominantly in 2 clusters associated with each nucleoid. Panel 7: DY380 pyrH -RFP; PyrH distributed diffusely throughout cytoplasm, and in 1–4 foci per cell. Panel 8: DY380 yaeL -EYFP; YaeL generally associated with the membrane. Panel 9: DY380 yqiK -EGFP; YqiK distributed diffusely throughout cytoplasm. Panel 10: DY380 yihA -EGFP; YihA distributed diffusely throughout cytoplasm. Panel 11: DY380 ygeH -EGFP; YgeH located predominantly beside the membrane.

Techniques Used: Fluorescence, Microscopy, Staining

Fluorescence microscopy images of PyrH fluorescence within live E. coli cells. Panel 1: Preferential localization of PyrH-RFP foci (red) at the old cell pole in DY380 pyrH -RFP. Left image: merge of phase contrast and red fluorescence; right: same image including DAPI staining (blue) of DNA. Panels 2 and 3: Typical images of PyrH-mRFP1 fluorescence (red) and nucleoids (DAPI stained blue) in filamentous DY380 pyrH -mRFP1/ ftsZ -C ext cells (both at the same magnification), showing PyrH protein existing predominantly in foci beside the membranes, located at the poles and at positions of potential cell division.
Figure Legend Snippet: Fluorescence microscopy images of PyrH fluorescence within live E. coli cells. Panel 1: Preferential localization of PyrH-RFP foci (red) at the old cell pole in DY380 pyrH -RFP. Left image: merge of phase contrast and red fluorescence; right: same image including DAPI staining (blue) of DNA. Panels 2 and 3: Typical images of PyrH-mRFP1 fluorescence (red) and nucleoids (DAPI stained blue) in filamentous DY380 pyrH -mRFP1/ ftsZ -C ext cells (both at the same magnification), showing PyrH protein existing predominantly in foci beside the membranes, located at the poles and at positions of potential cell division.

Techniques Used: Fluorescence, Microscopy, Staining

31) Product Images from "Lamin B2 prevents chromosome instability by ensuring proper mitotic chromosome segregation"

Article Title: Lamin B2 prevents chromosome instability by ensuring proper mitotic chromosome segregation

Journal: Oncogenesis

doi: 10.1038/oncsis.2014.6

Ectopic expression of lamin B2 in CIN cancer cells prevents mitotic defects. ( a – c ) WiDr cells stably expressing histone H2B-GFP were transfected with lamin B2-mCherry or mCherry (control) expression plasmid, and expression of lamin B2-mCherry ( M r , 96 kDa) was examined by western blotting with anti-lamin B2 and anti-DsRed antibodies ( a ). Anti-DsRed antibody can detect mCherry. Living cells expressing lamin B2-mCherry or mCherry were monitored by confocal microscopy. Representative images of living cells are shown ( b ). Elapsed times from nuclear envelope breakdown (NEB) are indicated at the bottom of mitotic images ( b ). Dotted arrow indicates chromosome mis-segregation in anaphase. Scale bars, 10 μm. Percentages of cells exhibiting chromosome mis-segregation during anaphase (mCherry, n =42; lamin B2-mCherry, n =41) are indicated below mitotic images ( b ). ( c ) Plot represents the value of the time from NEB to anaphase onset in each cell, and the means±s.d. are shown (mCherry, n =28; lamin B2-mCherry, n =13). Asterisk indicates a significant difference from the control, calculated by Student's t -test (* P
Figure Legend Snippet: Ectopic expression of lamin B2 in CIN cancer cells prevents mitotic defects. ( a – c ) WiDr cells stably expressing histone H2B-GFP were transfected with lamin B2-mCherry or mCherry (control) expression plasmid, and expression of lamin B2-mCherry ( M r , 96 kDa) was examined by western blotting with anti-lamin B2 and anti-DsRed antibodies ( a ). Anti-DsRed antibody can detect mCherry. Living cells expressing lamin B2-mCherry or mCherry were monitored by confocal microscopy. Representative images of living cells are shown ( b ). Elapsed times from nuclear envelope breakdown (NEB) are indicated at the bottom of mitotic images ( b ). Dotted arrow indicates chromosome mis-segregation in anaphase. Scale bars, 10 μm. Percentages of cells exhibiting chromosome mis-segregation during anaphase (mCherry, n =42; lamin B2-mCherry, n =41) are indicated below mitotic images ( b ). ( c ) Plot represents the value of the time from NEB to anaphase onset in each cell, and the means±s.d. are shown (mCherry, n =28; lamin B2-mCherry, n =13). Asterisk indicates a significant difference from the control, calculated by Student's t -test (* P

Techniques Used: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Confocal Microscopy

Ectopic expression of lamin B2 in CIN cancer cells prevents aberrant mitotic spindle formation. WiDr cells stably expressing mCherry-α-tubulin were transfected with lamin B2-GFP or GFP (control) expression plasmid. Living cells expressing lamin B2-GFP or GFP were monitored by confocal microscopy. Images shown are representative cells (arrows). Scale bars, 10 μm. Elapsed times during chromosome segregation are indicated at the top of mitotic images. Percentages of cells exhibiting aberrant spindle formation during mitosis (GFP, n =29; lamin B2-GFP, n =29) are indicated below mitotic images.
Figure Legend Snippet: Ectopic expression of lamin B2 in CIN cancer cells prevents aberrant mitotic spindle formation. WiDr cells stably expressing mCherry-α-tubulin were transfected with lamin B2-GFP or GFP (control) expression plasmid. Living cells expressing lamin B2-GFP or GFP were monitored by confocal microscopy. Images shown are representative cells (arrows). Scale bars, 10 μm. Elapsed times during chromosome segregation are indicated at the top of mitotic images. Percentages of cells exhibiting aberrant spindle formation during mitosis (GFP, n =29; lamin B2-GFP, n =29) are indicated below mitotic images.

Techniques Used: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Confocal Microscopy

Repression of lamin B2 induces mitotic defects and aneuploidy. ( a ) Lamin B2-siRNA alone or together with lamin B2-GFP expression plasmid was transfected into MIN-type HCT116 cells and, 48 h later, the expressions of lamin B2 and lamin B2-GFP were checked by western blotting. Lane 1, control-siRNA; lane 2, lamin B2-siRNA alone; lane 3, lamin B2-siRNA+lamin B2-GFP expression plasmid. ( b ) FISH analysis using centromere probes (CEP7, 8 and 12) was performed 48 h after transfection with lamin B2-siRNA in HCT116 cells. White arrows indicate aneuploid cells. ( c ) Frequency of aneuploid cells in HCT116 cells transfected with either lamin B2-siRNA alone or together with lamin B2-GFP expression plasmid. Centromere singles (CEP7, 8, 12 and 15) were counted in at least 200 cells. ( d ) HCT116 cells stably expressing histone H2B-GFP were treated with control or lamin B2-siRNA for 24 h. Time-lapse images of the cells were taken at 3- to 5-min intervals. Images shown are representative of control or lamin B2-siRNA-treated cells from prophase to anaphase. Control cells took 12.5±5.6 min ( n =81) from congression to separation of sister chromatids, whereas lamin B2-siRNA-treated cells took 28.9±18.2 min ( n =84). Arrowheads indicate a chromosomal bridge. Scale bars, 10 μm.
Figure Legend Snippet: Repression of lamin B2 induces mitotic defects and aneuploidy. ( a ) Lamin B2-siRNA alone or together with lamin B2-GFP expression plasmid was transfected into MIN-type HCT116 cells and, 48 h later, the expressions of lamin B2 and lamin B2-GFP were checked by western blotting. Lane 1, control-siRNA; lane 2, lamin B2-siRNA alone; lane 3, lamin B2-siRNA+lamin B2-GFP expression plasmid. ( b ) FISH analysis using centromere probes (CEP7, 8 and 12) was performed 48 h after transfection with lamin B2-siRNA in HCT116 cells. White arrows indicate aneuploid cells. ( c ) Frequency of aneuploid cells in HCT116 cells transfected with either lamin B2-siRNA alone or together with lamin B2-GFP expression plasmid. Centromere singles (CEP7, 8, 12 and 15) were counted in at least 200 cells. ( d ) HCT116 cells stably expressing histone H2B-GFP were treated with control or lamin B2-siRNA for 24 h. Time-lapse images of the cells were taken at 3- to 5-min intervals. Images shown are representative of control or lamin B2-siRNA-treated cells from prophase to anaphase. Control cells took 12.5±5.6 min ( n =81) from congression to separation of sister chromatids, whereas lamin B2-siRNA-treated cells took 28.9±18.2 min ( n =84). Arrowheads indicate a chromosomal bridge. Scale bars, 10 μm.

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Fluorescence In Situ Hybridization, Stable Transfection

Immunohistochemical analysis of lamin B2 in sporadic colorectal cancer and HNPCC. Paraffin-embedded tissue sections of sporadic colorectal cancer (CIN) and HNPCC (MIN) were stained with rabbit polyclonal anti-lamin B2 antibody. Using the TissueFAXS system and HistoQuest software, images of cancer cells and adjacent normal epithelial cells in each tissue section were obtained, and the intensity of lamin B2 staining in these cells was quantitated. ( a ) Representative images at low and high magnification of the dotted square regions are shown. Bars, 100 μm. ( b ) Plot represents the value of the relative intensity of lamin B2 staining in cancer cells in each tissue section compared with the intensity in adjacent normal epithelial cells in the same tissue section, and the means±s.d. are shown (sporadic colorectal cancer, n =8; HNPCC, n =8). The P -value was calculated by Student's t -test.
Figure Legend Snippet: Immunohistochemical analysis of lamin B2 in sporadic colorectal cancer and HNPCC. Paraffin-embedded tissue sections of sporadic colorectal cancer (CIN) and HNPCC (MIN) were stained with rabbit polyclonal anti-lamin B2 antibody. Using the TissueFAXS system and HistoQuest software, images of cancer cells and adjacent normal epithelial cells in each tissue section were obtained, and the intensity of lamin B2 staining in these cells was quantitated. ( a ) Representative images at low and high magnification of the dotted square regions are shown. Bars, 100 μm. ( b ) Plot represents the value of the relative intensity of lamin B2 staining in cancer cells in each tissue section compared with the intensity in adjacent normal epithelial cells in the same tissue section, and the means±s.d. are shown (sporadic colorectal cancer, n =8; HNPCC, n =8). The P -value was calculated by Student's t -test.

Techniques Used: Immunohistochemistry, Staining, Software

Repression of lamin B2 abrogates mitotic spindle formation. MIN cell lines, HCT116 and RKO, were transfected with control- or lamin B2-siRNA. Forty-eight hours after transfection, the cells were synchronized with thymidine for 16 h and then released into fresh medium. Cells that entered mitosis were analyzed by immunostaining using anti-β-tubulin ( a ) and α-tubulin ( b ) antibodies in HCT116 ( a ) and RKO ( b ) cells, respectively. DNA was marked by DAPI. Scale bars, 10 μm.
Figure Legend Snippet: Repression of lamin B2 abrogates mitotic spindle formation. MIN cell lines, HCT116 and RKO, were transfected with control- or lamin B2-siRNA. Forty-eight hours after transfection, the cells were synchronized with thymidine for 16 h and then released into fresh medium. Cells that entered mitosis were analyzed by immunostaining using anti-β-tubulin ( a ) and α-tubulin ( b ) antibodies in HCT116 ( a ) and RKO ( b ) cells, respectively. DNA was marked by DAPI. Scale bars, 10 μm.

Techniques Used: Transfection, Immunostaining

Proteomic analysis of colorectal cancer cell lines revealed that lamin B2 is downregulated in CIN cell lines. ( a ) Proteomic analysis of CIN and MIN cell lines using agarose 2D-DIGE. Nuclear extracts prepared from the cell lines were labeled with Cy3 (MIN) and Cy5 (CIN), and separated with two-dimensional electrophoresis. Increased and decreased protein spots in CIN nuclei are displayed as red (Cy5) and green (Cy3), respectively. Yellow arrow shows the protein spot corresponding to lamin B2. ( b ) Western blot analysis of nuclear extracts (10 μg) from CIN and MIN cell lines using antibodies to lamin B2, B1 and lamin A/C. ( c ) Immunostaining of CIN and MIN cell lines using anti-lamin B2 antibody and DAPI for DNA. Images show interphase cells. Scale bars, 10 μm.
Figure Legend Snippet: Proteomic analysis of colorectal cancer cell lines revealed that lamin B2 is downregulated in CIN cell lines. ( a ) Proteomic analysis of CIN and MIN cell lines using agarose 2D-DIGE. Nuclear extracts prepared from the cell lines were labeled with Cy3 (MIN) and Cy5 (CIN), and separated with two-dimensional electrophoresis. Increased and decreased protein spots in CIN nuclei are displayed as red (Cy5) and green (Cy3), respectively. Yellow arrow shows the protein spot corresponding to lamin B2. ( b ) Western blot analysis of nuclear extracts (10 μg) from CIN and MIN cell lines using antibodies to lamin B2, B1 and lamin A/C. ( c ) Immunostaining of CIN and MIN cell lines using anti-lamin B2 antibody and DAPI for DNA. Images show interphase cells. Scale bars, 10 μm.

Techniques Used: Labeling, Electrophoresis, Western Blot, Immunostaining

Disappearance of immunostaining for mitotic lamin B2 in CIN cell lines. ( a ) CIN (SW837 and HT29) and MIN (HCT116 and DLD1) cell lines were stained with anti-lamin B2 (red) and anti-α-tubulin (green) antibodies. Images show mitotic cells. ( b – d , f ) HCT116 cells were treated with siRNA targeting lamin B2 ( b ), B1( c ) or A/C ( d ) or SUN1 ( f ) and stained with the indicated antibodies. ( e ) HCT116 cells were stained with anti-lamin B2 and anti-Eg5 antibodies. DNA was stained with DAPI (blue). Scale bars, 10 μm. The areas indicated by arrows are shown at high magnification.
Figure Legend Snippet: Disappearance of immunostaining for mitotic lamin B2 in CIN cell lines. ( a ) CIN (SW837 and HT29) and MIN (HCT116 and DLD1) cell lines were stained with anti-lamin B2 (red) and anti-α-tubulin (green) antibodies. Images show mitotic cells. ( b – d , f ) HCT116 cells were treated with siRNA targeting lamin B2 ( b ), B1( c ) or A/C ( d ) or SUN1 ( f ) and stained with the indicated antibodies. ( e ) HCT116 cells were stained with anti-lamin B2 and anti-Eg5 antibodies. DNA was stained with DAPI (blue). Scale bars, 10 μm. The areas indicated by arrows are shown at high magnification.

Techniques Used: Immunostaining, Staining

32) Product Images from "The RNA-binding protein Fus directs translation of localized mRNAs in APC-RNP granules"

Article Title: The RNA-binding protein Fus directs translation of localized mRNAs in APC-RNP granules

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201306058

Preferential recruitment of APC-RNPs in cytoplasmic granules formed by an ALS-associated mutant of Fus. (a) NIH/3T3 cells were transfected with GFP-Fus(R521C) (top panels). Middle panels show distribution of coexpressed fluorescently tagged proteins or immunostaining of endogenous proteins. (b) Primary hippocampal neurons were transfected with GFP or GFP-Fus(R521C) and were immunostained at DIV5 to detect APC. (c) Hippocampal sections from a patient with FTLD-Fus (sporadic NIFID) and a control donor were stained for Fus and APC. Nuclei were stained with Draq5. Arrows point to Fus granules. Note that likely both glial cells and neurons are present in these sections. (d) NIH/3T3 cells transfected with GFP- or RFP-Fus(R521C) were analyzed by FISH to detect the Ddr2 or RhoA mRNAs or were cotransfected with DsRed-MS2 and an MS2 reporter RNA carrying the Pkp4 3′UTR (β24bs/Pkp4). Graph shows relative fluorescence intensities of Ddr2 and RhoA mRNAs within the cytoplasm or Fus granules. P-value is by Student’s t test. Bars, 8 µm.
Figure Legend Snippet: Preferential recruitment of APC-RNPs in cytoplasmic granules formed by an ALS-associated mutant of Fus. (a) NIH/3T3 cells were transfected with GFP-Fus(R521C) (top panels). Middle panels show distribution of coexpressed fluorescently tagged proteins or immunostaining of endogenous proteins. (b) Primary hippocampal neurons were transfected with GFP or GFP-Fus(R521C) and were immunostained at DIV5 to detect APC. (c) Hippocampal sections from a patient with FTLD-Fus (sporadic NIFID) and a control donor were stained for Fus and APC. Nuclei were stained with Draq5. Arrows point to Fus granules. Note that likely both glial cells and neurons are present in these sections. (d) NIH/3T3 cells transfected with GFP- or RFP-Fus(R521C) were analyzed by FISH to detect the Ddr2 or RhoA mRNAs or were cotransfected with DsRed-MS2 and an MS2 reporter RNA carrying the Pkp4 3′UTR (β24bs/Pkp4). Graph shows relative fluorescence intensities of Ddr2 and RhoA mRNAs within the cytoplasm or Fus granules. P-value is by Student’s t test. Bars, 8 µm.

Techniques Used: Mutagenesis, Transfection, Immunostaining, Staining, Fluorescence In Situ Hybridization, Fluorescence

The RNA-binding protein Fus is a component of APC-RNPs at cell protrusions. NIH/3T3 cells untransfected (a and c) or transfected with GFP or GFP-Fus (b) were immunoprecipitated (IP) with the indicated antibodies and analyzed by Western blot (a–c, top panel) or by RT-PCR (c, bottom panels). (d) NIH/3T3 cells were plated on microporous filters, induced to migrate by addition of LPA, and protrusions and cell bodies were isolated and analyzed by Western blot. (e) NIH/3T3 cells were immunostained to detect endogenous Fus and APC. Insets: magnification of the boxed protrusive area. Yellow line in overlay panel: cell outline (f) GFP-Fus–expressing cells immunostained to detect APC. Bars, 10 µm (insets, 3 µm).
Figure Legend Snippet: The RNA-binding protein Fus is a component of APC-RNPs at cell protrusions. NIH/3T3 cells untransfected (a and c) or transfected with GFP or GFP-Fus (b) were immunoprecipitated (IP) with the indicated antibodies and analyzed by Western blot (a–c, top panel) or by RT-PCR (c, bottom panels). (d) NIH/3T3 cells were plated on microporous filters, induced to migrate by addition of LPA, and protrusions and cell bodies were isolated and analyzed by Western blot. (e) NIH/3T3 cells were immunostained to detect endogenous Fus and APC. Insets: magnification of the boxed protrusive area. Yellow line in overlay panel: cell outline (f) GFP-Fus–expressing cells immunostained to detect APC. Bars, 10 µm (insets, 3 µm).

Techniques Used: RNA Binding Assay, Transfection, Immunoprecipitation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Isolation, Expressing

33) Product Images from "Ago2 facilitates Rad51 recruitment and DNA double-strand break repair by homologous recombination"

Article Title: Ago2 facilitates Rad51 recruitment and DNA double-strand break repair by homologous recombination

Journal: Cell Research

doi: 10.1038/cr.2014.36

Ago2 accumulates at DSBs and interacts with Rad51. (A) U2OS/DR-GFP cells were transfected with empty vector or HA-Ago2 constructs, followed by transfection with I- Sce I or empty vector control. ChIP assays were performed using HA antibody. Bound DNA was analyzed by PCR. (B) Quantification of ChIP data shown in A . The quantification is based on three independent experiments. Data are represented as mean ± SEM. (C) Myc-Ago2 or empty vector were overexpressed in 293T cells by transfection as indicated and the lysates were subjected to immunoprecipitation using Myc-coupled beads. Immunoprecipitates (IP) and whole-cell extracts (WCE) were immunoblotted with the indicated antibodies. (D) GFP-Rad51 was overexpressed in 293T cells by transfection as indicated and the lysates were subjected to immunoprecipitation using GFP beads. Immunoprecipitates and WCE were immunoblotted with the indicated antibodies. (E) 293T cells were transfected with control or Rad51 siRNAs, lysed and the lysates were subjected to Rad51 immunoprecipitation. Immunoprecipitates and WCE were immunoblotted with the indicated antibodies. (F) Two days post transfection with the indicated DNA constructs, 293T cells were treated with IR (5 Gy) and left to recover for 1 h. Cells were then lysed and the lysates were subjected to immunoprecipitation using Myc-beads. Immunoprecipitates and WCE were immunoblotted with the indicated antibodies ( * P
Figure Legend Snippet: Ago2 accumulates at DSBs and interacts with Rad51. (A) U2OS/DR-GFP cells were transfected with empty vector or HA-Ago2 constructs, followed by transfection with I- Sce I or empty vector control. ChIP assays were performed using HA antibody. Bound DNA was analyzed by PCR. (B) Quantification of ChIP data shown in A . The quantification is based on three independent experiments. Data are represented as mean ± SEM. (C) Myc-Ago2 or empty vector were overexpressed in 293T cells by transfection as indicated and the lysates were subjected to immunoprecipitation using Myc-coupled beads. Immunoprecipitates (IP) and whole-cell extracts (WCE) were immunoblotted with the indicated antibodies. (D) GFP-Rad51 was overexpressed in 293T cells by transfection as indicated and the lysates were subjected to immunoprecipitation using GFP beads. Immunoprecipitates and WCE were immunoblotted with the indicated antibodies. (E) 293T cells were transfected with control or Rad51 siRNAs, lysed and the lysates were subjected to Rad51 immunoprecipitation. Immunoprecipitates and WCE were immunoblotted with the indicated antibodies. (F) Two days post transfection with the indicated DNA constructs, 293T cells were treated with IR (5 Gy) and left to recover for 1 h. Cells were then lysed and the lysates were subjected to immunoprecipitation using Myc-beads. Immunoprecipitates and WCE were immunoblotted with the indicated antibodies ( * P

Techniques Used: Transfection, Plasmid Preparation, Construct, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Immunoprecipitation

34) Product Images from "The Downregulation of GFI1 by the EZH2-NDY1/KDM2B-JARID2 Axis and by Human Cytomegalovirus (HCMV) Associated Factors Allows the Activation of the HCMV Major IE Promoter and the Transition to Productive Infection"

Article Title: The Downregulation of GFI1 by the EZH2-NDY1/KDM2B-JARID2 Axis and by Human Cytomegalovirus (HCMV) Associated Factors Allows the Activation of the HCMV Major IE Promoter and the Transition to Productive Infection

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004136

The abundance of H3K4me3 and H3K27me3 at the HCMV MIEP in shControl and shEZH2 HFFs, at the pre-immediate early stage of the infection. A. Graphical illustration of the MIEP of HCMV. Open boxes represent regions probed by ChIP for association with histone H3K27me3 and histone H3K4me3. Enh stands for Enhancer and crs stands for cis-repressive sequence. B: HFFs were transduced with either the pLKO.1 empty vector or the shEZH2 lentiviral construct. A western blot of cell lysates was probed with the EZH2 antibody. C-E. HFFs transduced with a lentivirus construct of shEZH2 or with the empty vector, were infected with HCMV (MOI 0.5 PFUs/cell). ChIPs using antibodies against H3K27me3 and H3K4me3 were analysed by qPCR using primers specific for the indicated loci (see materials and methods ), at 0.5 h, 1.5 h and 3 h.p.i. The results are given as mean + SD for triplicate Real Time PCRs. The asterisks indicate statistical significance. * indicates p
Figure Legend Snippet: The abundance of H3K4me3 and H3K27me3 at the HCMV MIEP in shControl and shEZH2 HFFs, at the pre-immediate early stage of the infection. A. Graphical illustration of the MIEP of HCMV. Open boxes represent regions probed by ChIP for association with histone H3K27me3 and histone H3K4me3. Enh stands for Enhancer and crs stands for cis-repressive sequence. B: HFFs were transduced with either the pLKO.1 empty vector or the shEZH2 lentiviral construct. A western blot of cell lysates was probed with the EZH2 antibody. C-E. HFFs transduced with a lentivirus construct of shEZH2 or with the empty vector, were infected with HCMV (MOI 0.5 PFUs/cell). ChIPs using antibodies against H3K27me3 and H3K4me3 were analysed by qPCR using primers specific for the indicated loci (see materials and methods ), at 0.5 h, 1.5 h and 3 h.p.i. The results are given as mean + SD for triplicate Real Time PCRs. The asterisks indicate statistical significance. * indicates p

Techniques Used: Infection, Chromatin Immunoprecipitation, Sequencing, Transduction, Plasmid Preparation, Construct, Western Blot, Real-time Polymerase Chain Reaction

Infection by HCMV depends on the downregulation of GFI1, a repressor of immediate-early gene transcription. Model summarizing the data on the interaction between the virus and the host. (Panel A) This panel describes the infection of wild type HFFs. The incoming virus rapidly degrades GFI1 to allow the activation of the MIEP of HCMV, and viral infection. In addition, virus infection alters the expression of NDY1/KDM2B, EZH2, JARID2 and JMJD3. The solid lines from these molecules to GFI1 indicate that they actively repress GFI1 both before and after infection, although due to HCMV-induced changes in their expression, the repression is enhanced after infection. (Panel B, Left) The repression of GFI1 in uninfected cells was blocked by the knockdown of NDY1/KDM2B, EZH2 or JARID2 and by the overexpression of JMJD3, resulting in significant up-regulation of GFI1 (dotted lines). (Panel B, Right) describes the infection of HFFs in the left side of panel B. The virus continues to degrade GFI1. However, the degradation of GFI1 by the virus is insufficient to downregulate it to levels that allow the activation of the MIEP and viral infection.
Figure Legend Snippet: Infection by HCMV depends on the downregulation of GFI1, a repressor of immediate-early gene transcription. Model summarizing the data on the interaction between the virus and the host. (Panel A) This panel describes the infection of wild type HFFs. The incoming virus rapidly degrades GFI1 to allow the activation of the MIEP of HCMV, and viral infection. In addition, virus infection alters the expression of NDY1/KDM2B, EZH2, JARID2 and JMJD3. The solid lines from these molecules to GFI1 indicate that they actively repress GFI1 both before and after infection, although due to HCMV-induced changes in their expression, the repression is enhanced after infection. (Panel B, Left) The repression of GFI1 in uninfected cells was blocked by the knockdown of NDY1/KDM2B, EZH2 or JARID2 and by the overexpression of JMJD3, resulting in significant up-regulation of GFI1 (dotted lines). (Panel B, Right) describes the infection of HFFs in the left side of panel B. The virus continues to degrade GFI1. However, the degradation of GFI1 by the virus is insufficient to downregulate it to levels that allow the activation of the MIEP and viral infection.

Techniques Used: Infection, Activation Assay, Expressing, Over Expression

NDY1/KDM2B, EZH2 JARID2 and JMJD3 control the expression of GFI1, a direct repressor of the HCMV MIEP, by regulating histone H3K27 tri-methylation in the GFI1 promoter. A . (Upper panel). Schematic diagram of the major immediate-early promoter of HCMV, showing the relative location of the binding sites of the indicated transcriptional regulators (activators and repressors). (Lower panel). The expression of the indicated transcriptional regulators in HFFs in which EZH2, NDY1/KDM2B, or JARID2 were knocked down, or JMJD3 was overexpressed via transduction with the indicated constructs, was measured by real time RT-PCR. The bars show the relative expression of GFI1 (mean ± SD) in the cells transduced with these constructs. The western blot in the inset shows that the GFI1 protein, the only transcriptional regulator whose expression at the RNA level was induced by these constructs, is also upregulated. B . The knock down of NDY1/KDM2B, EZH2 or JARID2 enhances the binding of GFI1 to the HCMV promoter. HFFs were transduced with shEZH2, shNDY1/KDM2, shJARID2 or the empty lentiviral vector and they were subsequently infected with HCMV. ChIP assays addressing the binding of GFI1 on the two known GFI1 binding sites in the HCMV promoter or in exon 1 of the immediate-early region were carried out using lysates harvested from these cells 1 hour post-infection The bars show the fold increase in GFI1 binding (mean ± SD) in the shEZH2, shNDY1/KDM2B and shJARID2-transduced cells relative to the cells transduced with the empty vector. C . GFI1 is a direct repressor of the HCMV MIE promoter. HEK 293T cells transduced with the indicated lentiviral or retroviral constructs were transfected with an HCMV MIEP-EGFP reporter in which the HCMV MIEP was either wild type or mutated in the two known GFI1 binding sites. The activity of the HCMV MIEP was monitored by both fluorescence microscopy (upper panel) and fluorescence densitometric analyses (lower panel). Bars in the lower panel show the relative EGFP fluorescence in the indicated cells (mean ± SD). D . The knockdown of NDY1/KDM2B, EZH2 and JARID2 decrease the abundance of histone H3K27me3 in a negative regulatory domain of the GFI1 promoter (site # 1). ChIP analyses addressing the abundance of H3K27me3 at five different sites within the GFI1 promoter in HFFs transduced with the indicated constructs. The p16 Ink4a locus was used as the positive control. The upper panel shows the position of the five selected sites, relative to the transcription start site in the GFI1 promoter (arrow). The bars in the lower panel show the fold change in the abundance of H3K27me3 (mean ± SD) at these sites, and in the p16 Ink4a locus. NRE: Negative Regulatory Element.
Figure Legend Snippet: NDY1/KDM2B, EZH2 JARID2 and JMJD3 control the expression of GFI1, a direct repressor of the HCMV MIEP, by regulating histone H3K27 tri-methylation in the GFI1 promoter. A . (Upper panel). Schematic diagram of the major immediate-early promoter of HCMV, showing the relative location of the binding sites of the indicated transcriptional regulators (activators and repressors). (Lower panel). The expression of the indicated transcriptional regulators in HFFs in which EZH2, NDY1/KDM2B, or JARID2 were knocked down, or JMJD3 was overexpressed via transduction with the indicated constructs, was measured by real time RT-PCR. The bars show the relative expression of GFI1 (mean ± SD) in the cells transduced with these constructs. The western blot in the inset shows that the GFI1 protein, the only transcriptional regulator whose expression at the RNA level was induced by these constructs, is also upregulated. B . The knock down of NDY1/KDM2B, EZH2 or JARID2 enhances the binding of GFI1 to the HCMV promoter. HFFs were transduced with shEZH2, shNDY1/KDM2, shJARID2 or the empty lentiviral vector and they were subsequently infected with HCMV. ChIP assays addressing the binding of GFI1 on the two known GFI1 binding sites in the HCMV promoter or in exon 1 of the immediate-early region were carried out using lysates harvested from these cells 1 hour post-infection The bars show the fold increase in GFI1 binding (mean ± SD) in the shEZH2, shNDY1/KDM2B and shJARID2-transduced cells relative to the cells transduced with the empty vector. C . GFI1 is a direct repressor of the HCMV MIE promoter. HEK 293T cells transduced with the indicated lentiviral or retroviral constructs were transfected with an HCMV MIEP-EGFP reporter in which the HCMV MIEP was either wild type or mutated in the two known GFI1 binding sites. The activity of the HCMV MIEP was monitored by both fluorescence microscopy (upper panel) and fluorescence densitometric analyses (lower panel). Bars in the lower panel show the relative EGFP fluorescence in the indicated cells (mean ± SD). D . The knockdown of NDY1/KDM2B, EZH2 and JARID2 decrease the abundance of histone H3K27me3 in a negative regulatory domain of the GFI1 promoter (site # 1). ChIP analyses addressing the abundance of H3K27me3 at five different sites within the GFI1 promoter in HFFs transduced with the indicated constructs. The p16 Ink4a locus was used as the positive control. The upper panel shows the position of the five selected sites, relative to the transcription start site in the GFI1 promoter (arrow). The bars in the lower panel show the fold change in the abundance of H3K27me3 (mean ± SD) at these sites, and in the p16 Ink4a locus. NRE: Negative Regulatory Element.

Techniques Used: Expressing, Methylation, Binding Assay, Transduction, Construct, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Infection, Chromatin Immunoprecipitation, Transfection, Activity Assay, Fluorescence, Microscopy, Positive Control

35) Product Images from "Generation of single-chain LAGLIDADG homing endonucleases from native homodimeric precursor proteins"

Article Title: Generation of single-chain LAGLIDADG homing endonucleases from native homodimeric precursor proteins

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkp004

mCreI and mMsoI induce catalytic activity-dependent recombination in human cells. ( a ) In vivo recombination activity was assayed by co-transfecting coding plasmids for I-CreI or I-MsoI proteins together with a recombination reporter plasmid that contains a direct repeat of two genetically inactive copies of GFP. In vivo cleavage of the I-CreI or I-MsoI target site initiates gene conversion and repair of the cleaved copy to generate GFP+ cells that can be detected and quantified by flow cytometry (Materials and methods section). ( b ) Flow histograms of cells mock-transfected, transfected with reporter plasmid alone, or co-transfected with reporter: endonuclease plasmid pairs. The gate for GFP+ cells is shown by the boxed area, and the GFP+ frequency is given in the lower right of each histogram. ( c ) Frequency and fold increase in GFP+ cells for different reporter/coding plasmid combination. The frequency of GFP+ cells generated by in vitro Xho I-linearized DR-GFPCre reporter DNA (*) indicates that a substantial fraction of reporter molecules are likely cleaved in vivo by I-CreI and mCreI. D20N I-CreI and D22N I-MsoI are catalytically inactive mutants of I-CreI and I-MsoI which fail to induce GFP+ cells when cotransfected with a Cre or Mso-specific reporter plasmid. Error bars are means ± SDs.
Figure Legend Snippet: mCreI and mMsoI induce catalytic activity-dependent recombination in human cells. ( a ) In vivo recombination activity was assayed by co-transfecting coding plasmids for I-CreI or I-MsoI proteins together with a recombination reporter plasmid that contains a direct repeat of two genetically inactive copies of GFP. In vivo cleavage of the I-CreI or I-MsoI target site initiates gene conversion and repair of the cleaved copy to generate GFP+ cells that can be detected and quantified by flow cytometry (Materials and methods section). ( b ) Flow histograms of cells mock-transfected, transfected with reporter plasmid alone, or co-transfected with reporter: endonuclease plasmid pairs. The gate for GFP+ cells is shown by the boxed area, and the GFP+ frequency is given in the lower right of each histogram. ( c ) Frequency and fold increase in GFP+ cells for different reporter/coding plasmid combination. The frequency of GFP+ cells generated by in vitro Xho I-linearized DR-GFPCre reporter DNA (*) indicates that a substantial fraction of reporter molecules are likely cleaved in vivo by I-CreI and mCreI. D20N I-CreI and D22N I-MsoI are catalytically inactive mutants of I-CreI and I-MsoI which fail to induce GFP+ cells when cotransfected with a Cre or Mso-specific reporter plasmid. Error bars are means ± SDs.

Techniques Used: Activity Assay, In Vivo, Plasmid Preparation, Flow Cytometry, Cytometry, Transfection, Generated, In Vitro

36) Product Images from "Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7-RILP-p150Glued and late endosome positioning"

Article Title: Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7-RILP-p150Glued and late endosome positioning

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200811005

ORP1L controls recruitment of p150 Glued to the Rab7–RILP receptor. (A) ORP1L domain structure and constructs. Five domains are predicted in ORP1L. Numbers indicate amino acid residue positions. Constructs were N-terminally tagged with mRFP. The ΔORDPHDPHD chimera had ORD exchanged for a tandem PH domain derived from ORP1L. (B) Effect of ORP1L deletion or chimeric constructs on RILP-mediated p150 Glued recruitment. (left) Mel JuSo cells transfected with GFP-RILP and mRFP-ORP1L constructs and stained with anti-p150 Glued antibodies before CLSM. For pixel analyses of the images, see Fig. S2 E . n > 200 for each condition. (right) Mel JuSo cells transfected with the indicated mRFP-ORP1L constructs and whole cell lysates analyzed by immunoblotting with anti-mRFP antibodies (WB: α-mRFP). Molecular standards are indicated. WB, Western blot. (C) Effect of ORP1L constructs on RILP recruitment of the p150 Glued -associated dynein motor adapter DIC. Mel JuSo cells were transfected with GFP-RILP and the mRFP-ORP1L constructs indicated and stained with anti-DIC antibodies. n > 100 for each condition. Bars, 10 µm.
Figure Legend Snippet: ORP1L controls recruitment of p150 Glued to the Rab7–RILP receptor. (A) ORP1L domain structure and constructs. Five domains are predicted in ORP1L. Numbers indicate amino acid residue positions. Constructs were N-terminally tagged with mRFP. The ΔORDPHDPHD chimera had ORD exchanged for a tandem PH domain derived from ORP1L. (B) Effect of ORP1L deletion or chimeric constructs on RILP-mediated p150 Glued recruitment. (left) Mel JuSo cells transfected with GFP-RILP and mRFP-ORP1L constructs and stained with anti-p150 Glued antibodies before CLSM. For pixel analyses of the images, see Fig. S2 E . n > 200 for each condition. (right) Mel JuSo cells transfected with the indicated mRFP-ORP1L constructs and whole cell lysates analyzed by immunoblotting with anti-mRFP antibodies (WB: α-mRFP). Molecular standards are indicated. WB, Western blot. (C) Effect of ORP1L constructs on RILP recruitment of the p150 Glued -associated dynein motor adapter DIC. Mel JuSo cells were transfected with GFP-RILP and the mRFP-ORP1L constructs indicated and stained with anti-DIC antibodies. n > 100 for each condition. Bars, 10 µm.

Techniques Used: Construct, Derivative Assay, Transfection, Staining, Confocal Laser Scanning Microscopy, Western Blot

Cholesterol-dependent ORP1L-mediated contacts between LEs and the ER. (A) ΔORD recruits endogenous VAP-A. Mel JuSo cells expressing mRFP-ΔORD, -ORP1L, or -ΔORDPHDPHD were stained for VAP-A. The merge panels show an overlay of the two channels, and the boxes indicate the zoomed-in areas. Pixel analyses of the zoomed-in areas for fluorescence distribution of the mRFP-ORP1L variants and VAP-A are shown. n > 100. (B) Mel JuSo cells expressing TAP1-GFP (red) were exposed to different cholesterol manipulating conditions, as indicated. Cells were stained with antibodies for CD63 (green). Colocalization of the markers was determined by computational pixel analysis and visualized in blue. (right) Quantification of the colocalizing surface of the CD63-positive area. The mean ± SEM for > 20 cells analyzed per condition is shown (*, P = 0.068; **, P = 1.55 × 10 −6 ; ***, P = 0.0003). Bars: (A) 10 µm; (B) 5 µm.
Figure Legend Snippet: Cholesterol-dependent ORP1L-mediated contacts between LEs and the ER. (A) ΔORD recruits endogenous VAP-A. Mel JuSo cells expressing mRFP-ΔORD, -ORP1L, or -ΔORDPHDPHD were stained for VAP-A. The merge panels show an overlay of the two channels, and the boxes indicate the zoomed-in areas. Pixel analyses of the zoomed-in areas for fluorescence distribution of the mRFP-ORP1L variants and VAP-A are shown. n > 100. (B) Mel JuSo cells expressing TAP1-GFP (red) were exposed to different cholesterol manipulating conditions, as indicated. Cells were stained with antibodies for CD63 (green). Colocalization of the markers was determined by computational pixel analysis and visualized in blue. (right) Quantification of the colocalizing surface of the CD63-positive area. The mean ± SEM for > 20 cells analyzed per condition is shown (*, P = 0.068; **, P = 1.55 × 10 −6 ; ***, P = 0.0003). Bars: (A) 10 µm; (B) 5 µm.

Techniques Used: Expressing, Staining, Fluorescence

ORP1L conformation and LE positioning. (A and B, top) Mel JuSo cells expressing mRFP-ORP1L-GFP (A) or mRFP-ΔORD-GFP (B) were imaged for FRET by CLSM. The GFP image, mRFP image, calculated FRET, and donor FRET efficiency E D images are indicated. E D values are shown in false colors (the LUT shows the corresponding values). The position of the nucleus (N) and cell boundaries are drawn in the GFP channel. The cytosol is divided by five concentric rings, as indicated in the E D panel. (bottom) E D values for the constructs were collected and binned for the different rings. Values are shown for the different rings under the corresponding image. Mean ± SEM of E D values collected for > 10 cells analyzed per construct are shown. The asterisks indicate a statistically significant difference in E D of that bin compared to bin 1 (the most internal ring; *, P = 0.024; **, P = 0.026; ***, P = 0.0063). Bars, 10 µm.
Figure Legend Snippet: ORP1L conformation and LE positioning. (A and B, top) Mel JuSo cells expressing mRFP-ORP1L-GFP (A) or mRFP-ΔORD-GFP (B) were imaged for FRET by CLSM. The GFP image, mRFP image, calculated FRET, and donor FRET efficiency E D images are indicated. E D values are shown in false colors (the LUT shows the corresponding values). The position of the nucleus (N) and cell boundaries are drawn in the GFP channel. The cytosol is divided by five concentric rings, as indicated in the E D panel. (bottom) E D values for the constructs were collected and binned for the different rings. Values are shown for the different rings under the corresponding image. Mean ± SEM of E D values collected for > 10 cells analyzed per construct are shown. The asterisks indicate a statistically significant difference in E D of that bin compared to bin 1 (the most internal ring; *, P = 0.024; **, P = 0.026; ***, P = 0.0063). Bars, 10 µm.

Techniques Used: Expressing, Confocal Laser Scanning Microscopy, Construct

ORP1L controls cholesterol-dependent LE positioning. (A) Cholesterol-dependent ORP1L vesicle positioning. Mel JuSo cells expressing mRFP-ORP1L were cultured under control conditions (FCS) or conditions causing decreased (statin) or increased (U-18666A) cholesterol levels. Actin was stained with falloidin (green) to mark the cell boundaries before analyses by CLSM. n > 100. (B) ORP1L is dominant in vesicle repositioning as the result of cholesterol manipulations. Mel JuSo cells expressing mRFP-ΔORDPHDPHD or mRFP-ΔORD were treated and imaged as described in A. n > 100. Bars, 10 µm.
Figure Legend Snippet: ORP1L controls cholesterol-dependent LE positioning. (A) Cholesterol-dependent ORP1L vesicle positioning. Mel JuSo cells expressing mRFP-ORP1L were cultured under control conditions (FCS) or conditions causing decreased (statin) or increased (U-18666A) cholesterol levels. Actin was stained with falloidin (green) to mark the cell boundaries before analyses by CLSM. n > 100. (B) ORP1L is dominant in vesicle repositioning as the result of cholesterol manipulations. Mel JuSo cells expressing mRFP-ΔORDPHDPHD or mRFP-ΔORD were treated and imaged as described in A. n > 100. Bars, 10 µm.

Techniques Used: Expressing, Cell Culture, Staining, Confocal Laser Scanning Microscopy

LE cholesterol alters the conformation of ORP1L. (A) Intramolecular FRET for mRFP-ORP1L-GFP. GFP and mRFP are attached to the same molecule, allowing FRET from donor GFP to acceptor mRFP. FRET depends on distance and orientation and thus indicates conformational changes. FRET can be detected by sensitized emission. GFP is excited by 488-nm light, and then, after energy transfer, 582–675-nm light emission by mRFP is detected. (B) Cholesterol effects on ORP1L conformation detected by sensitized emission. Mel JuSo cells expressing mRFP-ORP1L-GFP were cultured under control (FCS) or cholesterol-depleting (statin) or -enhancing (U-18666A) conditions before imaging by CLSM for FRET determination. Panels show the GFP signal, the mRFP signal, calculated FRET, and FRET related to donor fluorophore input: the donor FRET efficiency (E D ). The color LUT visualizes the differences in the E D panels. (right) quantification of the donor FRET efficiency detected for mRFP-ORP1L-GFP under the different conditions of cholesterol manipulation. The mean and SD from two experiments ( > 10 cells analyzed) are shown (*, P = 0.05; **, P = 0.03). (C) ORP1L controls LE positioning in NPC1-silenced cells. Mel JuSo cells were transfected with mRFP-ORP1L, -ΔORD, or -ΔORDPHDPHD and siRNA for NPC1 and analyzed by CLSM. n > 100. (D) Sensitized emission and ORP1L conformation in NPC1-deficient cells. mRFP-ORP1L-GFP–expressing MelJuSo cells were transfected with control (siCTRL) or NPC1 (siNPC1) siRNAs before imaging by confocal FRET. (right) Donor FRET efficiencies determined in > 10 control siRNA– or NPC1 siRNA–transfected cells. The mean ± SD is shown (**, P = 5.1 × 10 −6 ). (E) NPC1, cholesterol, and LE clustering. Mel JuSo cells were transfected with control or NPC1 siRNA and then mRFP-ΔORD before staining with filipin for cholesterol. Pixel analyses are shown in Fig. S2 E . n > 50 for each condition. Bars, 10 µm.
Figure Legend Snippet: LE cholesterol alters the conformation of ORP1L. (A) Intramolecular FRET for mRFP-ORP1L-GFP. GFP and mRFP are attached to the same molecule, allowing FRET from donor GFP to acceptor mRFP. FRET depends on distance and orientation and thus indicates conformational changes. FRET can be detected by sensitized emission. GFP is excited by 488-nm light, and then, after energy transfer, 582–675-nm light emission by mRFP is detected. (B) Cholesterol effects on ORP1L conformation detected by sensitized emission. Mel JuSo cells expressing mRFP-ORP1L-GFP were cultured under control (FCS) or cholesterol-depleting (statin) or -enhancing (U-18666A) conditions before imaging by CLSM for FRET determination. Panels show the GFP signal, the mRFP signal, calculated FRET, and FRET related to donor fluorophore input: the donor FRET efficiency (E D ). The color LUT visualizes the differences in the E D panels. (right) quantification of the donor FRET efficiency detected for mRFP-ORP1L-GFP under the different conditions of cholesterol manipulation. The mean and SD from two experiments ( > 10 cells analyzed) are shown (*, P = 0.05; **, P = 0.03). (C) ORP1L controls LE positioning in NPC1-silenced cells. Mel JuSo cells were transfected with mRFP-ORP1L, -ΔORD, or -ΔORDPHDPHD and siRNA for NPC1 and analyzed by CLSM. n > 100. (D) Sensitized emission and ORP1L conformation in NPC1-deficient cells. mRFP-ORP1L-GFP–expressing MelJuSo cells were transfected with control (siCTRL) or NPC1 (siNPC1) siRNAs before imaging by confocal FRET. (right) Donor FRET efficiencies determined in > 10 control siRNA– or NPC1 siRNA–transfected cells. The mean ± SD is shown (**, P = 5.1 × 10 −6 ). (E) NPC1, cholesterol, and LE clustering. Mel JuSo cells were transfected with control or NPC1 siRNA and then mRFP-ΔORD before staining with filipin for cholesterol. Pixel analyses are shown in Fig. S2 E . n > 50 for each condition. Bars, 10 µm.

Techniques Used: Expressing, Cell Culture, Imaging, Confocal Laser Scanning Microscopy, Transfection, Staining

ORP1L requires VAP-A to remove p150 Glued from Rab7–RILP. (A) Effect of the FFAT motif in ΔORD on RILP-mediated p150 Glued recruitment. (left) Mel JuSo cells were transfected with GFP-RILP and mRFP-ΔORD containing a point mutation in its FFAT motif, ΔORDFFAT(D478A). Fixed cells were stained with anti-p150 Glued antibodies. n > 200. (right) Mel JuSo cells were transfected with the mRFP-ΔORD or mRFP-ΔORDFFAT(D478A) constructs, and whole cell lysates were analyzed by immunoblotting with anti-mRFP antibodies (WB: α-mRFP). (B) p150 Glued exclusion by ΔORD and VAP-A. VAP-A was silenced by siRNA (siVAP-A) in cells expressing GFP-RILP and mRFP-ΔORD. (left) Cells stained for p150 Glued . Pixel analyses are shown in Fig. S2 E . n > 100 for each condition. (right) Western blot analysis of cells transfected with transfection reagent (mock), control (siCTRL), or VAP-A siRNA (siVAP-A) and probed for α-tubulin (as loading control) and anti–VAP-A antibodies. (C) VAP-A removes p150 Glued from Rab7–RILP. GTP-locked His-Rab7(Q67L) was GTP loaded and coupled to Talon beads before adding purified RILP, ORP1L, and p150 Glued (C25) fragments in equimolar amounts to form a preassembled ORP1L–Rab7–RILP–p150 Glued (C25) complex. Subsequently, the complex was incubated in the presence or absence of purified VAP-A. After washing, the bead-bound proteins were analyzed by SDS-PAGE and Western blotting. (D) ORP1L requirements for p150 Glued removal by VAP-A. GTP-loaded His-Rab7(Q67L) was coupled to Talon Co 2+ beads before adding the isolated proteins indicated in equimolar amounts to form a preassembled complex. After washing, these complexes were exposed to isolated VAP-A or the p150 Glued (C25) fragment, as indicated, and the effects on the preformed complex were assessed by SDS-PAGE and Western blot analyses with antibodies, as indicated. (E) VAP-A interacts with p150 Glued . GST or GST–VAP-A was coupled to beads before exposure to the p150 Glued (C25) fragment. After washing, the complexes were analyzed by SDS-PAGE and Western blotting. (A–E) Molecular masses are indicated. WB, Western blot. Bars, 10 µm.
Figure Legend Snippet: ORP1L requires VAP-A to remove p150 Glued from Rab7–RILP. (A) Effect of the FFAT motif in ΔORD on RILP-mediated p150 Glued recruitment. (left) Mel JuSo cells were transfected with GFP-RILP and mRFP-ΔORD containing a point mutation in its FFAT motif, ΔORDFFAT(D478A). Fixed cells were stained with anti-p150 Glued antibodies. n > 200. (right) Mel JuSo cells were transfected with the mRFP-ΔORD or mRFP-ΔORDFFAT(D478A) constructs, and whole cell lysates were analyzed by immunoblotting with anti-mRFP antibodies (WB: α-mRFP). (B) p150 Glued exclusion by ΔORD and VAP-A. VAP-A was silenced by siRNA (siVAP-A) in cells expressing GFP-RILP and mRFP-ΔORD. (left) Cells stained for p150 Glued . Pixel analyses are shown in Fig. S2 E . n > 100 for each condition. (right) Western blot analysis of cells transfected with transfection reagent (mock), control (siCTRL), or VAP-A siRNA (siVAP-A) and probed for α-tubulin (as loading control) and anti–VAP-A antibodies. (C) VAP-A removes p150 Glued from Rab7–RILP. GTP-locked His-Rab7(Q67L) was GTP loaded and coupled to Talon beads before adding purified RILP, ORP1L, and p150 Glued (C25) fragments in equimolar amounts to form a preassembled ORP1L–Rab7–RILP–p150 Glued (C25) complex. Subsequently, the complex was incubated in the presence or absence of purified VAP-A. After washing, the bead-bound proteins were analyzed by SDS-PAGE and Western blotting. (D) ORP1L requirements for p150 Glued removal by VAP-A. GTP-loaded His-Rab7(Q67L) was coupled to Talon Co 2+ beads before adding the isolated proteins indicated in equimolar amounts to form a preassembled complex. After washing, these complexes were exposed to isolated VAP-A or the p150 Glued (C25) fragment, as indicated, and the effects on the preformed complex were assessed by SDS-PAGE and Western blot analyses with antibodies, as indicated. (E) VAP-A interacts with p150 Glued . GST or GST–VAP-A was coupled to beads before exposure to the p150 Glued (C25) fragment. After washing, the complexes were analyzed by SDS-PAGE and Western blotting. (A–E) Molecular masses are indicated. WB, Western blot. Bars, 10 µm.

Techniques Used: Transfection, Mutagenesis, Staining, Construct, Western Blot, Expressing, Purification, Incubation, SDS Page, Isolation

37) Product Images from "Promotion of embryonic cortico-cerebral neuronogenesis by miR-124"

Article Title: Promotion of embryonic cortico-cerebral neuronogenesis by miR-124

Journal: Neural Development

doi: 10.1186/1749-8104-4-40

Overexpression of miR-124 in vitro and in vivo . (A) Backbone of the expression plasmids pPri-miR-124(2) and pPri-miR-155/neg_control; miR-124-responsive sensor plasmid pCMV-DsRed2/ Lhx2 _3'UTR. The asterisk indicates the position of Pri-miR cDNA fragments. (B, C) Specific attenuation of DsRed2 expression in HeLa cells cotransfected with pPri-miR-124(2) and pCMV-DsRed2/ Lhx2 _3'UTR. PT, post-transfection. (D) Backbone of lentivectors LV_Pri-miR-124(2) and LV_Pri-miR-155/neg_control. The asterisk indicates the position of Pri-miR cDNA fragments. (E) DsRed2 expression in E12.5 primary cortico-cerebral progenitors infected by lentiviruses LV_Pri-miR-124(2) and LV_Pri-miR-155/neg_control at a multiplicity of infection of 40 and kept for 30 h in DMEM:F12:N2 medium supplemented with 2.5% fetal calf serum. PI, post-infection. BF, bright field. (F, G) Differential β-tubulin immunoprofiling of acutely infected, E12.5 dissociated cortical progenitor cells, at 72 h after infection. (H) Example of neurite outgrowth evaluation by immunostaining and subsequent NeuriteTracer ® analysis. (I) Differential neurite outgrowth in low density cortical progenitor cells 72 h after infection at E12.5, calculated for three different experiments by NeuriteTracer ® . (J, K) In vivo overexpression of miR-124 in lateral neocortex. Distribution of miR-124 and pCMV-driven EmGFP on E14.5 midfrontal telencephalic sections from brains electroporated at E12.5 with the plasmids pPri-miR-124(2) and pPri-miR-155/neg_control, respectively. Magnifications of boxed insets of electronic merges are shown to the right. Abbreviations: bg, basal ganglia. In (C) error bars represent the standard error of the mean calculated among the means of each experiment; ** P
Figure Legend Snippet: Overexpression of miR-124 in vitro and in vivo . (A) Backbone of the expression plasmids pPri-miR-124(2) and pPri-miR-155/neg_control; miR-124-responsive sensor plasmid pCMV-DsRed2/ Lhx2 _3'UTR. The asterisk indicates the position of Pri-miR cDNA fragments. (B, C) Specific attenuation of DsRed2 expression in HeLa cells cotransfected with pPri-miR-124(2) and pCMV-DsRed2/ Lhx2 _3'UTR. PT, post-transfection. (D) Backbone of lentivectors LV_Pri-miR-124(2) and LV_Pri-miR-155/neg_control. The asterisk indicates the position of Pri-miR cDNA fragments. (E) DsRed2 expression in E12.5 primary cortico-cerebral progenitors infected by lentiviruses LV_Pri-miR-124(2) and LV_Pri-miR-155/neg_control at a multiplicity of infection of 40 and kept for 30 h in DMEM:F12:N2 medium supplemented with 2.5% fetal calf serum. PI, post-infection. BF, bright field. (F, G) Differential β-tubulin immunoprofiling of acutely infected, E12.5 dissociated cortical progenitor cells, at 72 h after infection. (H) Example of neurite outgrowth evaluation by immunostaining and subsequent NeuriteTracer ® analysis. (I) Differential neurite outgrowth in low density cortical progenitor cells 72 h after infection at E12.5, calculated for three different experiments by NeuriteTracer ® . (J, K) In vivo overexpression of miR-124 in lateral neocortex. Distribution of miR-124 and pCMV-driven EmGFP on E14.5 midfrontal telencephalic sections from brains electroporated at E12.5 with the plasmids pPri-miR-124(2) and pPri-miR-155/neg_control, respectively. Magnifications of boxed insets of electronic merges are shown to the right. Abbreviations: bg, basal ganglia. In (C) error bars represent the standard error of the mean calculated among the means of each experiment; ** P

Techniques Used: Over Expression, In Vitro, In Vivo, Expressing, Plasmid Preparation, Transfection, Infection, Immunostaining

38) Product Images from "G protein ? interacts with the glucocorticoid receptor and suppresses its transcriptional activity in the nucleus"

Article Title: G protein ? interacts with the glucocorticoid receptor and suppresses its transcriptional activity in the nucleus

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200409150

Gβ1, Gβ2, and Rack1 suppress the transcriptional activity of GR on the MMTV promoter, and endogenous Gβ and Gγ are associated with GR in vivo. (A) Gβ1, Gβ2, and Rack1 dose dependently suppress the transcriptional activity of GR on the MMTV promoter in HCT116 cells. HCT116 cells were transfected with indicated amounts of Gβ1, Gβ2, or Rack1-expressing plasmids together with pRShGRα, pMMTV-Luc, and pSV40-β-Gal. Bars represent mean ± SEM values of the luciferase activity normalized for β-galactosidase activity in the absence or presence of 10 −6 M of dexamethasone. (B) GR(Δ262-404) has stronger transcriptional activity than the wild-type GR and Gβ2 loses its suppressive effect on GR(Δ262-404)-induced transactivation in HCT116 cells. HCT116 cells were transfected with Gβ2-expressing plasmids and pRShGRα or pRShGRα(Δ262-404), together with pMMTV-Luc and pSV40-β-Gal. Bars represent mean ± SEM values of the luciferase activity normalized for β-galactosidase activity in the absence or presence of 10 −6 M of dexamethasone. (*), P
Figure Legend Snippet: Gβ1, Gβ2, and Rack1 suppress the transcriptional activity of GR on the MMTV promoter, and endogenous Gβ and Gγ are associated with GR in vivo. (A) Gβ1, Gβ2, and Rack1 dose dependently suppress the transcriptional activity of GR on the MMTV promoter in HCT116 cells. HCT116 cells were transfected with indicated amounts of Gβ1, Gβ2, or Rack1-expressing plasmids together with pRShGRα, pMMTV-Luc, and pSV40-β-Gal. Bars represent mean ± SEM values of the luciferase activity normalized for β-galactosidase activity in the absence or presence of 10 −6 M of dexamethasone. (B) GR(Δ262-404) has stronger transcriptional activity than the wild-type GR and Gβ2 loses its suppressive effect on GR(Δ262-404)-induced transactivation in HCT116 cells. HCT116 cells were transfected with Gβ2-expressing plasmids and pRShGRα or pRShGRα(Δ262-404), together with pMMTV-Luc and pSV40-β-Gal. Bars represent mean ± SEM values of the luciferase activity normalized for β-galactosidase activity in the absence or presence of 10 −6 M of dexamethasone. (*), P

Techniques Used: Activity Assay, In Vivo, Transfection, Expressing, Luciferase

Forced cytoplasmic localization of Gβ2 attenuates the suppressive effect of the wild-type Gβ on GR-induced transactivation, whereas forced nuclear localization enhances it. (A and B) EGFP-fused NES-Gβ2 and NLS-Gβ2 are exclusively localized in the cytoplasm and the nucleus, respectively, in HCT116 cells. HCT116 cells were transfected with pEGFP-C1-NES-Gβ2- or pEGFP-C1-NLS-Gβ2-expressing plasmid. The cells were fixed and their confocal images were obtained. Representative images are shown in A, whereas mean ± SEM values of signal intensities in the nucleus (black bars) and the cytoplasm (white bars) obtained from over 20 cells are shown in B. (C) NES-Gβ2 loses the suppressive effect on GR transactivation, whereas NLS-Gβ2 has a stronger inhibitory effect than the wild-type Gβ2 on GR transactivation in HCT116 cells. HCT116 cells were transfected with pCDNA4His/MaxB-NES-Gβ2- or -NLS-Gβ2 together with pRShGRα, pMMTV-Luc, and pSV40-β-Gal. Bars represent mean ± SEM values of the luciferase activity normalized for β-galactosidase activity in the absence or presence of 10 −6 M of dexamethasone. (*), P
Figure Legend Snippet: Forced cytoplasmic localization of Gβ2 attenuates the suppressive effect of the wild-type Gβ on GR-induced transactivation, whereas forced nuclear localization enhances it. (A and B) EGFP-fused NES-Gβ2 and NLS-Gβ2 are exclusively localized in the cytoplasm and the nucleus, respectively, in HCT116 cells. HCT116 cells were transfected with pEGFP-C1-NES-Gβ2- or pEGFP-C1-NLS-Gβ2-expressing plasmid. The cells were fixed and their confocal images were obtained. Representative images are shown in A, whereas mean ± SEM values of signal intensities in the nucleus (black bars) and the cytoplasm (white bars) obtained from over 20 cells are shown in B. (C) NES-Gβ2 loses the suppressive effect on GR transactivation, whereas NLS-Gβ2 has a stronger inhibitory effect than the wild-type Gβ2 on GR transactivation in HCT116 cells. HCT116 cells were transfected with pCDNA4His/MaxB-NES-Gβ2- or -NLS-Gβ2 together with pRShGRα, pMMTV-Luc, and pSV40-β-Gal. Bars represent mean ± SEM values of the luciferase activity normalized for β-galactosidase activity in the absence or presence of 10 −6 M of dexamethasone. (*), P

Techniques Used: Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay

Somatostatin suppressed dexamethasone-stimulated transcriptional activity of the Kv1.5 potassium channel gene, whereas abrogation of Gβ1 and Gβ2 attenuated the somatostatin effect in GH3 cells. GH3 cells were transfected with control or Gβ1 and Gβ2 siRNAs, and were treated with 10 −6 M of dexamethasone and/or the indicated amounts of somatostatin for 24 h. Total RNA was then purified from the cells and the amounts of Kv1.5 potassium channel (A), Gβ1 (B), Gβ2 (C), or RPLP0 mRNAs were determined by RT-PCR. Bars show mean ± SEM of their fold induction over baseline.
Figure Legend Snippet: Somatostatin suppressed dexamethasone-stimulated transcriptional activity of the Kv1.5 potassium channel gene, whereas abrogation of Gβ1 and Gβ2 attenuated the somatostatin effect in GH3 cells. GH3 cells were transfected with control or Gβ1 and Gβ2 siRNAs, and were treated with 10 −6 M of dexamethasone and/or the indicated amounts of somatostatin for 24 h. Total RNA was then purified from the cells and the amounts of Kv1.5 potassium channel (A), Gβ1 (B), Gβ2 (C), or RPLP0 mRNAs were determined by RT-PCR. Bars show mean ± SEM of their fold induction over baseline.

Techniques Used: Activity Assay, Transfection, Purification, Reverse Transcription Polymerase Chain Reaction

Subcellular localization of Gβ2 and Gγ2 in HCT116 cells. (A) Endogenous Gβ and Gγ are visualized in the nucleus as well as in the cytoplasm/plasma membrane in HCT116 cells. Endogenous Gβ (left, top two panels) and Gγ (right, top two panels) were visualized by treatment with anti-Gβ or -Gγ2 antibodies, and FITC-labeled secondary antibody, and their confocal images were obtained. Nuclei were also stained with DAPI. Co-treatment of the samples with blocking peptides for anti-Gβ (left, bottom) or anti-Gγ2 (right, bottom) antibodies abolished their specific staining. Cells, expressing Gβ or Gγ exclusively in the cytoplasm, are indicated as “ℵ” and “a”, respectively, whereas cells retaining these molecules weakly or strongly in the nucleus are indicated as “ℑ” and “b”, and “ℜ” and “c”, respectively. (B) Endogenous Gβ and Gγ are detected in the nuclear fraction as well as in the cytoplasm and membrane fractions in HCT116 cells. HCT116 cells were lysed and their subcellular fractions were separated by centrifugation. 0.1 μg of protein of indicated subcellular fractions was run on SDS-PAGE gels, blotted to the nitrocellulose membranes, and Gβ and Gγ were visualized with their specific antibodies by reprobing the same membrane. Intracellular adhesion molecule 1 (ICAM1), α-tubulin, and Oct1, detected also by reprobing the same membrane with their specific antibodies, were respectively shown as positive controls for the membrane, cytoplasmic and nuclear fractions to indicate that the subcellular fractionation did not produce cross-contamination. (C and D) EGFP-Gβ2 was localized in the nucleus in addition to the cytoplasm, whereas EGFP-Gγ2 was detected in the nucleus and the cytoplasm, and at the plasma membrane in HCT116 cells. HCT116 cells were transfected with pEGFP-C-1-Gβ2 or -Gγ2, and the cells were fixed and their confocal images were obtained. Nuclei were also stained with DAPI. Representative images of EGFP-Gβ2 and -Gγ2 are respectively shown in C, whereas mean ± SEM values of their signal intensities in the nucleus and the cytoplasm obtained from over 20 cells are shown in D. (E and F) EGFP-Gβ2 translocated into the nucleus with DsRed2-GR in response to 10 −6 M of dexamethasone in HCT116 cells. HCT116 cells were transfected with pEGFP-C1-Gβ2 and pDsRed2-GRα. Confocal images of EGFP-Gβ2 and DsRed2-GR were obtained before and 30 min after the treatment with 10 −6 M of dexamethasone. Representative images are shown in D, whereas mean ± SEM values of signal intensities in the nucleus (black bars) and the cytoplasm (white bars) obtained from over 20 cells is shown in E. (G) EGFP-Gβ2 and DsRed2-GR are colocalized at the plasma membrane in response to somatostatin in HCT116 cells. HCT116 cells were transfected with pEGFP-C1-Gβ2, pDSRed2-GRα, and Gγ2- and SSTR2-expressing plasmids. Confocal images of EGFP-Gβ2 and DsRed2-GR were obtained before and 30 min after the treatment with 100 nM of somatostatin. Blue and orange arrows indicate signals of EGFP-Gβ2, DsRed2-GR, which are localized at the plasma membrane, whereas yellow arrows indicate their colocalization.
Figure Legend Snippet: Subcellular localization of Gβ2 and Gγ2 in HCT116 cells. (A) Endogenous Gβ and Gγ are visualized in the nucleus as well as in the cytoplasm/plasma membrane in HCT116 cells. Endogenous Gβ (left, top two panels) and Gγ (right, top two panels) were visualized by treatment with anti-Gβ or -Gγ2 antibodies, and FITC-labeled secondary antibody, and their confocal images were obtained. Nuclei were also stained with DAPI. Co-treatment of the samples with blocking peptides for anti-Gβ (left, bottom) or anti-Gγ2 (right, bottom) antibodies abolished their specific staining. Cells, expressing Gβ or Gγ exclusively in the cytoplasm, are indicated as “ℵ” and “a”, respectively, whereas cells retaining these molecules weakly or strongly in the nucleus are indicated as “ℑ” and “b”, and “ℜ” and “c”, respectively. (B) Endogenous Gβ and Gγ are detected in the nuclear fraction as well as in the cytoplasm and membrane fractions in HCT116 cells. HCT116 cells were lysed and their subcellular fractions were separated by centrifugation. 0.1 μg of protein of indicated subcellular fractions was run on SDS-PAGE gels, blotted to the nitrocellulose membranes, and Gβ and Gγ were visualized with their specific antibodies by reprobing the same membrane. Intracellular adhesion molecule 1 (ICAM1), α-tubulin, and Oct1, detected also by reprobing the same membrane with their specific antibodies, were respectively shown as positive controls for the membrane, cytoplasmic and nuclear fractions to indicate that the subcellular fractionation did not produce cross-contamination. (C and D) EGFP-Gβ2 was localized in the nucleus in addition to the cytoplasm, whereas EGFP-Gγ2 was detected in the nucleus and the cytoplasm, and at the plasma membrane in HCT116 cells. HCT116 cells were transfected with pEGFP-C-1-Gβ2 or -Gγ2, and the cells were fixed and their confocal images were obtained. Nuclei were also stained with DAPI. Representative images of EGFP-Gβ2 and -Gγ2 are respectively shown in C, whereas mean ± SEM values of their signal intensities in the nucleus and the cytoplasm obtained from over 20 cells are shown in D. (E and F) EGFP-Gβ2 translocated into the nucleus with DsRed2-GR in response to 10 −6 M of dexamethasone in HCT116 cells. HCT116 cells were transfected with pEGFP-C1-Gβ2 and pDsRed2-GRα. Confocal images of EGFP-Gβ2 and DsRed2-GR were obtained before and 30 min after the treatment with 10 −6 M of dexamethasone. Representative images are shown in D, whereas mean ± SEM values of signal intensities in the nucleus (black bars) and the cytoplasm (white bars) obtained from over 20 cells is shown in E. (G) EGFP-Gβ2 and DsRed2-GR are colocalized at the plasma membrane in response to somatostatin in HCT116 cells. HCT116 cells were transfected with pEGFP-C1-Gβ2, pDSRed2-GRα, and Gγ2- and SSTR2-expressing plasmids. Confocal images of EGFP-Gβ2 and DsRed2-GR were obtained before and 30 min after the treatment with 100 nM of somatostatin. Blue and orange arrows indicate signals of EGFP-Gβ2, DsRed2-GR, which are localized at the plasma membrane, whereas yellow arrows indicate their colocalization.

Techniques Used: Labeling, Staining, Blocking Assay, Expressing, Centrifugation, SDS Page, Fractionation, Transfection

Gβ1, Gβ2 and Rack1 interact with GR(263-419) in yeast two-hybrid assays. (A) Full-length Gβ1, Gβ2, and Rack1, as well as Rack1(139-317) interact with GR(263-420) in a yeast two-hybrid assay. EGY48 yeast cells were transformed with p8OP-LacZ, pLexA-GRα(263-419) and the indicated full-length Gβ1-, Gβ2-, Rack1-, or Rack1(139-317)-expressing pB42AD-derived plasmids. Bars represent mean ± SEM values of fold activation compared with the baseline. (B) Gβ2(143-270) (blades 3–5) interacts with GR(263-420) in a yeast two-hybrid assay. EGY48 yeast cells were transformed with p8OP-LacZ, pLexA-GRα(263-419) and the indicated Gβ2 fragment-expressing pB42AD plasmids. Bars represent mean ± SEM values of fold activation compared with the baseline. (C) Summary of yeast two-hybrid assays, which demonstrates domains of Gβ2 and Rack1 that are necessary for the interaction with GR(263-419).
Figure Legend Snippet: Gβ1, Gβ2 and Rack1 interact with GR(263-419) in yeast two-hybrid assays. (A) Full-length Gβ1, Gβ2, and Rack1, as well as Rack1(139-317) interact with GR(263-420) in a yeast two-hybrid assay. EGY48 yeast cells were transformed with p8OP-LacZ, pLexA-GRα(263-419) and the indicated full-length Gβ1-, Gβ2-, Rack1-, or Rack1(139-317)-expressing pB42AD-derived plasmids. Bars represent mean ± SEM values of fold activation compared with the baseline. (B) Gβ2(143-270) (blades 3–5) interacts with GR(263-420) in a yeast two-hybrid assay. EGY48 yeast cells were transformed with p8OP-LacZ, pLexA-GRα(263-419) and the indicated Gβ2 fragment-expressing pB42AD plasmids. Bars represent mean ± SEM values of fold activation compared with the baseline. (C) Summary of yeast two-hybrid assays, which demonstrates domains of Gβ2 and Rack1 that are necessary for the interaction with GR(263-419).

Techniques Used: Y2H Assay, Transformation Assay, Expressing, Derivative Assay, Activation Assay

Gβ2 is attracted to GREs and directly suppresses GR-induced transactivation by inhibiting its AF-2 function. (A) Gβ2 was attracted to the chromatin-integrated MMTV GREs via interaction with GR in COS7 cells. COS7 cells, which have genomically integrated MMTV-Luc, were transfected with Gβ2-expressing plasmid and pRShGRα or pRShGRα(Δ262-404). 24 h after addition of 10 −6 M of dexamethasone, the cells were fixed and the ChIP reaction was performed with anti-Gβ or control antibodies. The portion of the MMTV promoter that contains two GREs was amplified by PCR with a specific primer pair. Two images obtained from separate gels were combined to produce the Input gel image. (B) Gβ2 suppresses the transcriptional activity of GR on the chromatin-integrated MMTV promoter in COS7 cells. COS7 cells with genomically integrated MMTV-Luc, were transfected with the indicated amounts of the Gβ2-expressing plasmid, together with pRShGRα and pSV40-β-Gal. Bars represent mean ± SEM values of the luciferase activity normalized for β-galactosidase activity in the absence or presence of 10 −6 M of dexamethasone. (*), P
Figure Legend Snippet: Gβ2 is attracted to GREs and directly suppresses GR-induced transactivation by inhibiting its AF-2 function. (A) Gβ2 was attracted to the chromatin-integrated MMTV GREs via interaction with GR in COS7 cells. COS7 cells, which have genomically integrated MMTV-Luc, were transfected with Gβ2-expressing plasmid and pRShGRα or pRShGRα(Δ262-404). 24 h after addition of 10 −6 M of dexamethasone, the cells were fixed and the ChIP reaction was performed with anti-Gβ or control antibodies. The portion of the MMTV promoter that contains two GREs was amplified by PCR with a specific primer pair. Two images obtained from separate gels were combined to produce the Input gel image. (B) Gβ2 suppresses the transcriptional activity of GR on the chromatin-integrated MMTV promoter in COS7 cells. COS7 cells with genomically integrated MMTV-Luc, were transfected with the indicated amounts of the Gβ2-expressing plasmid, together with pRShGRα and pSV40-β-Gal. Bars represent mean ± SEM values of the luciferase activity normalized for β-galactosidase activity in the absence or presence of 10 −6 M of dexamethasone. (*), P

Techniques Used: Transfection, Expressing, Plasmid Preparation, Chromatin Immunoprecipitation, Amplification, Polymerase Chain Reaction, Activity Assay, Luciferase

39) Product Images from "GEF-H1 Mediated Control of NOD1 Dependent NF-?B Activation by Shigella Effectors"

Article Title: GEF-H1 Mediated Control of NOD1 Dependent NF-?B Activation by Shigella Effectors

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000228

GEF-H1 and NOD1 mediate NF-κB activation induced by S. flexneri effectors. (A) Confocal microscopic image analysis of MDCK cells transfected with indicated GFP-tagged S. flexneri effectors. Bars indicate 10 µm. (B) NF-κB activation in response to GFP-tagged S. flexneri effectors in HEK293 cells. (C) Three-dimensional reconstructions of confocal microscopic image series of MDCK cells transfected with indicated GFP-tagged S. flexneri effectors and immunostained for endogenously expressed GEF-H1. Bars indicate 10 µm, arrows indicate co-localization of GEF-H1 and S. flexneri effectors. (D) NF-κB activation in response to IpgB1, IpgB2 and OspB expression in the absence or presence of control or GEF-H1 specific siRNAs (*p
Figure Legend Snippet: GEF-H1 and NOD1 mediate NF-κB activation induced by S. flexneri effectors. (A) Confocal microscopic image analysis of MDCK cells transfected with indicated GFP-tagged S. flexneri effectors. Bars indicate 10 µm. (B) NF-κB activation in response to GFP-tagged S. flexneri effectors in HEK293 cells. (C) Three-dimensional reconstructions of confocal microscopic image series of MDCK cells transfected with indicated GFP-tagged S. flexneri effectors and immunostained for endogenously expressed GEF-H1. Bars indicate 10 µm, arrows indicate co-localization of GEF-H1 and S. flexneri effectors. (D) NF-κB activation in response to IpgB1, IpgB2 and OspB expression in the absence or presence of control or GEF-H1 specific siRNAs (*p

Techniques Used: Activation Assay, Transfection, Expressing

40) Product Images from "Synapse formation is regulated by the signaling adaptor GIT1"

Article Title: Synapse formation is regulated by the signaling adaptor GIT1

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200211002

Synaptic targeting of GIT1. (A) Schematic diagram of the full-length and deletion constructs of GIT1 (all GFP-tagged at the NH 2 terminus except N-SLD and C-SLD, which are FLAG-tagged at the NH 2 terminus). The indicated domains are as follows: ARF-GAP domain (ARF-GAP), ankyrin repeats (Ank), Spa2 homology domain 1(SHD1), and paxillin binding site (paxillin). The fusion proteins with synaptic localization are indicated with “+”. N-SLD shows weak localization to synapses which is indicated with “+/−”. (B) GIT1 fusion proteins that show specific localization to synapses. Hippocampal neurons were transfected with the various GIT1 fusion proteins (left column) and stained for synaptic markers (middle column). GIT1Δc and CD-GIT1 were coimmunostained for SV2. CDΔAnk was coimmunostained for PSD-95. CDΔAS/SLD was coimmunostained for synapsin1. Overlays are shown in the right column. The GIT1 constructs were pseudocolored green, and the synaptic markers were pseudocolored red. Bar, 2 μm. (C) FLAG–N-SLD– expressing neurons were coimmunostained for FLAG and synapsin1. N-SLD shows partial localization to synapses (arrows). The overlaid picture is shown in the right column. N-SLD was pseudocolored green, and synapsin1 was pseudocolored red. Bar, 2 μm. (D) GIT1 fusion proteins that fail to localize to synapses. Hippocampal neurons expressing the GFP-tagged GIT1 deletion constructs (left column) were stained for SV2 (middle column). The GIT1 constructs lacking SLD (cGIT1, GIT1ΔCD, nGIT1, and GIT1ΔSLD) do not show synaptic localization. Deletion of the NH 2 -terminal 32 aa of SLD (SLDΔ32) dramatically reduces localization to the synapse. For localization of C-SLD, neurons expressing FLAG–C-SLD were coimmunostained for FLAG and synapsin1. C-SLD shows a diffuse labeling pattern. The overlays are shown in the right column. The GIT1 constructs were pseudocolored green, and the synaptic markers were pseudocolored red. Bar, 2 μm.
Figure Legend Snippet: Synaptic targeting of GIT1. (A) Schematic diagram of the full-length and deletion constructs of GIT1 (all GFP-tagged at the NH 2 terminus except N-SLD and C-SLD, which are FLAG-tagged at the NH 2 terminus). The indicated domains are as follows: ARF-GAP domain (ARF-GAP), ankyrin repeats (Ank), Spa2 homology domain 1(SHD1), and paxillin binding site (paxillin). The fusion proteins with synaptic localization are indicated with “+”. N-SLD shows weak localization to synapses which is indicated with “+/−”. (B) GIT1 fusion proteins that show specific localization to synapses. Hippocampal neurons were transfected with the various GIT1 fusion proteins (left column) and stained for synaptic markers (middle column). GIT1Δc and CD-GIT1 were coimmunostained for SV2. CDΔAnk was coimmunostained for PSD-95. CDΔAS/SLD was coimmunostained for synapsin1. Overlays are shown in the right column. The GIT1 constructs were pseudocolored green, and the synaptic markers were pseudocolored red. Bar, 2 μm. (C) FLAG–N-SLD– expressing neurons were coimmunostained for FLAG and synapsin1. N-SLD shows partial localization to synapses (arrows). The overlaid picture is shown in the right column. N-SLD was pseudocolored green, and synapsin1 was pseudocolored red. Bar, 2 μm. (D) GIT1 fusion proteins that fail to localize to synapses. Hippocampal neurons expressing the GFP-tagged GIT1 deletion constructs (left column) were stained for SV2 (middle column). The GIT1 constructs lacking SLD (cGIT1, GIT1ΔCD, nGIT1, and GIT1ΔSLD) do not show synaptic localization. Deletion of the NH 2 -terminal 32 aa of SLD (SLDΔ32) dramatically reduces localization to the synapse. For localization of C-SLD, neurons expressing FLAG–C-SLD were coimmunostained for FLAG and synapsin1. C-SLD shows a diffuse labeling pattern. The overlays are shown in the right column. The GIT1 constructs were pseudocolored green, and the synaptic markers were pseudocolored red. Bar, 2 μm.

Techniques Used: Construct, Binding Assay, Transfection, Staining, Expressing, Labeling

PIX is targeted to the synapses by GIT1. (A) HA-tagged βPIX localizes to the synapses. Hippocampal neurons were transfected with βPIX-HA and stained for HA and synapsin1. Arrows indicate PIX puncta that colocalize with synapsin1 puncta. Bar, 20 μm. (B) The localization of PIX to synapses is inhibited by coexpression of GFP-SLD. Hippocampal neurons were cotransfected with either GFP-GIT1 and PIX-HA or GFP-SLD and PIX-HA. They were fixed and stained for HA and synapsin1. Note the localization of PIX in the synapses when coexpressed with GIT1 (arrows, top) and the decreased localization of PIX to synapses when coexpressed with SLD (arrows, bottom). Bar, 20 μm. (C) A GIT1 binding-deficient PIX mutant (PIXΔGBD) does not localized to synapses. Hippocampal neurons were transfected with HA-PIXΔGBD and coimmunostained for HA and synapsin1. Note the lack of localization to synapses with PIXΔGBD (arrows). Bar, 2 μm.
Figure Legend Snippet: PIX is targeted to the synapses by GIT1. (A) HA-tagged βPIX localizes to the synapses. Hippocampal neurons were transfected with βPIX-HA and stained for HA and synapsin1. Arrows indicate PIX puncta that colocalize with synapsin1 puncta. Bar, 20 μm. (B) The localization of PIX to synapses is inhibited by coexpression of GFP-SLD. Hippocampal neurons were cotransfected with either GFP-GIT1 and PIX-HA or GFP-SLD and PIX-HA. They were fixed and stained for HA and synapsin1. Note the localization of PIX in the synapses when coexpressed with GIT1 (arrows, top) and the decreased localization of PIX to synapses when coexpressed with SLD (arrows, bottom). Bar, 20 μm. (C) A GIT1 binding-deficient PIX mutant (PIXΔGBD) does not localized to synapses. Hippocampal neurons were transfected with HA-PIXΔGBD and coimmunostained for HA and synapsin1. Note the lack of localization to synapses with PIXΔGBD (arrows). Bar, 2 μm.

Techniques Used: Transfection, Staining, Binding Assay, Mutagenesis

Overexpression of the SLD from GIT1 regulates spine morphology and synaptic density. (A) Hippocampal neurons were cotransfected with GFP-SLD and GIT1-FLAG and stained for synapsin1. Note the localization of GFP-SLD to the synapses in relatively low expressing cells causes a decreased localization of GIT1 in the synapses (arrows). Bar, 20 μm. (B) Hippocampal neurons were transfected with various GIT1 constructs at 1 wk in culture and stained for SV2 at 2 wk in culture. Note the increase in dendritic protrusions (left column) and the decrease in synaptic density (right column) in neurons expressing high levels of GFP-SLD. Bar, 20 μm. (C) Quantification of the number of spines and dendritic protrusions in hippocampal neurons transfected with either GFP-GIT1 or GFP-SLD. 80–100 dendrites from independent transfections were quantified for each construct. The definitions of spines and dendritic protrusions are provided in Materials and methods. (D) Quantification of synaptic density in hippocampal neurons transfected with GIT1, nGIT1, CD-GIT1, or SLD. 85–110 dendrites from independent transfections were quantified for each construct (as described in Materials and methods). The difference between SLD and other GIT1 constructs was statistically significant as determined by Student's t test (*P
Figure Legend Snippet: Overexpression of the SLD from GIT1 regulates spine morphology and synaptic density. (A) Hippocampal neurons were cotransfected with GFP-SLD and GIT1-FLAG and stained for synapsin1. Note the localization of GFP-SLD to the synapses in relatively low expressing cells causes a decreased localization of GIT1 in the synapses (arrows). Bar, 20 μm. (B) Hippocampal neurons were transfected with various GIT1 constructs at 1 wk in culture and stained for SV2 at 2 wk in culture. Note the increase in dendritic protrusions (left column) and the decrease in synaptic density (right column) in neurons expressing high levels of GFP-SLD. Bar, 20 μm. (C) Quantification of the number of spines and dendritic protrusions in hippocampal neurons transfected with either GFP-GIT1 or GFP-SLD. 80–100 dendrites from independent transfections were quantified for each construct. The definitions of spines and dendritic protrusions are provided in Materials and methods. (D) Quantification of synaptic density in hippocampal neurons transfected with GIT1, nGIT1, CD-GIT1, or SLD. 85–110 dendrites from independent transfections were quantified for each construct (as described in Materials and methods). The difference between SLD and other GIT1 constructs was statistically significant as determined by Student's t test (*P

Techniques Used: Over Expression, Staining, Expressing, Transfection, Construct

GIT1 Δ SHD-expressing neurons show a phenotype similar to SLD-expressing neurons. (A) Hippocampal neurons were transfected with either GFP-GIT1 or GFP-GIT1ΔSHD at day 7 in culture and imaged at day 14 in culture. Note the increase in dendritic protrusions in GIT1ΔSHD-expressing neurons. Bar, 2 μm. (B) Quantification of the number of spines and dendritic protrusions in GIT1- and GIT1ΔSHD-expressing neurons. (C) Quantification of the synaptic density in GIT1- and GIT1ΔSHD-expressing neurons. The difference between GIT1 and GIT1ΔSHD was statistically significant as determined by Student's t test (*P
Figure Legend Snippet: GIT1 Δ SHD-expressing neurons show a phenotype similar to SLD-expressing neurons. (A) Hippocampal neurons were transfected with either GFP-GIT1 or GFP-GIT1ΔSHD at day 7 in culture and imaged at day 14 in culture. Note the increase in dendritic protrusions in GIT1ΔSHD-expressing neurons. Bar, 2 μm. (B) Quantification of the number of spines and dendritic protrusions in GIT1- and GIT1ΔSHD-expressing neurons. (C) Quantification of the synaptic density in GIT1- and GIT1ΔSHD-expressing neurons. The difference between GIT1 and GIT1ΔSHD was statistically significant as determined by Student's t test (*P

Techniques Used: Expressing, Transfection

GIT1 is expressed in cultured hippocampal neurons and enriched in synapses. (A) Western blot of a lysate from day 10 cultured hippocampal neurons. The blot was probed with a GIT1 antibody. A specific band at ∼95 kD confirms the presence of the GIT1 protein in these neurons. (B) Hippocampal neurons at 2–3 wk in culture were double immunostained for endogenous GIT1 (left column) and various synaptic proteins (right column). GIT1 colocalizes with the presynaptic marker SV2 (top). GIT1 shows colocalization with PSD-95 in some puncta (arrowheads), but some GIT1 puncta, especially those on the cell body, do not overlap with PSD-95 (middle, arrows). These puncta show colocalization with the inhibitory synapse marker GAD-6 (bottom). Enlargements of the boxed regions are shown in insets at the bottom right of each panel. Bar, 20 μm. (C) Hippocampal neurons were transfected with either GFP-GIT1 (top) or GIT1-FLAG (bottom) and immunostained for the presynaptic marker synapsin1 at 3 wk in culture. Both GFP-GIT1 and GIT1-FLAG colocalize with synapsin1 in dendritic spines and shafts (arrows). Bar, 20 μm. (D) Hippocampal neurons were transfected with GFP-GIT1 and immunostained for the appropriate synaptic markers at 2–3 wk in culture. The GIT1 clusters on the dendrites (Dendritic) almost completely merge with the postsynaptic marker PSD-95 and are in close apposition to the presynaptic marker synapsin1 (Overlay). The GIT1 clusters on the axons (Axonal) completely merge with the presynaptic marker SV2 and are in close apposition to the postsynaptic marker PSD-95 (Overlay). Note the colocalization of GIT1 clusters with the synaptic markers (arrowheads). Enlargements of individual synapses are shown in the right column. GFP-GIT1 is pseudocolored green, and the synaptic markers are pseudocolored red. Bar, 2 μm.
Figure Legend Snippet: GIT1 is expressed in cultured hippocampal neurons and enriched in synapses. (A) Western blot of a lysate from day 10 cultured hippocampal neurons. The blot was probed with a GIT1 antibody. A specific band at ∼95 kD confirms the presence of the GIT1 protein in these neurons. (B) Hippocampal neurons at 2–3 wk in culture were double immunostained for endogenous GIT1 (left column) and various synaptic proteins (right column). GIT1 colocalizes with the presynaptic marker SV2 (top). GIT1 shows colocalization with PSD-95 in some puncta (arrowheads), but some GIT1 puncta, especially those on the cell body, do not overlap with PSD-95 (middle, arrows). These puncta show colocalization with the inhibitory synapse marker GAD-6 (bottom). Enlargements of the boxed regions are shown in insets at the bottom right of each panel. Bar, 20 μm. (C) Hippocampal neurons were transfected with either GFP-GIT1 (top) or GIT1-FLAG (bottom) and immunostained for the presynaptic marker synapsin1 at 3 wk in culture. Both GFP-GIT1 and GIT1-FLAG colocalize with synapsin1 in dendritic spines and shafts (arrows). Bar, 20 μm. (D) Hippocampal neurons were transfected with GFP-GIT1 and immunostained for the appropriate synaptic markers at 2–3 wk in culture. The GIT1 clusters on the dendrites (Dendritic) almost completely merge with the postsynaptic marker PSD-95 and are in close apposition to the presynaptic marker synapsin1 (Overlay). The GIT1 clusters on the axons (Axonal) completely merge with the presynaptic marker SV2 and are in close apposition to the postsynaptic marker PSD-95 (Overlay). Note the colocalization of GIT1 clusters with the synaptic markers (arrowheads). Enlargements of individual synapses are shown in the right column. GFP-GIT1 is pseudocolored green, and the synaptic markers are pseudocolored red. Bar, 2 μm.

Techniques Used: Cell Culture, Western Blot, Marker, Transfection

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Cell Differentiation:

Article Title: GSK3β-mediated Ser156 phosphorylation modulates a BH3-like domain in BCL2L12 during TMZ-induced apoptosis and autophagy in glioma cells
Article Snippet: .. Cloning BCL2L12, BCL-XL, BCL2, BCL2 associated X (Bax), Beclin-1, induced myeloid leukemia cell differentiation protein MCL-1 (Mcl-1), and GSK3β were cloned into pACT2 and pAS2-1 vectors (Takara Bio Inc., Otsu, Japan) for yeast two-hybrid assay or into the pEGFP-C1 vector for overexpression. .. Polymerase chain reaction (PCR) technique was used to generate DNA fragments containing the desired gene sequences utilizing primers designed to contain the Xho I and Bam HI restriction enzyme recognition sites.

Real-time Polymerase Chain Reaction:

Article Title: Circular RNA AKT3 upregulates PIK3R1 to enhance cisplatin resistance in gastric cancer via miR-198 suppression
Article Snippet: .. For Quantitative real-time PCR (RT-PCR), 500 ng of treated RNA was directly reverse transcribed using Prime Script RT Master Mix (Takara, Japan) and either random or oligo(dT) primers. .. Reverse transcription of miRNA was performed using a New Poly(A) Tailing Kit (ThermoFisher Scientific, China). mRNA was reverse transcribed into cDNA with a PrimeScript RT Master Mix Kit (Takara, RR036A, Japan). cDNA was amplified using Universal SYBR Green Master Mix (4,913,914,001, Roche, Shanghai, China).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Circular RNA AKT3 upregulates PIK3R1 to enhance cisplatin resistance in gastric cancer via miR-198 suppression
Article Snippet: .. For Quantitative real-time PCR (RT-PCR), 500 ng of treated RNA was directly reverse transcribed using Prime Script RT Master Mix (Takara, Japan) and either random or oligo(dT) primers. .. Reverse transcription of miRNA was performed using a New Poly(A) Tailing Kit (ThermoFisher Scientific, China). mRNA was reverse transcribed into cDNA with a PrimeScript RT Master Mix Kit (Takara, RR036A, Japan). cDNA was amplified using Universal SYBR Green Master Mix (4,913,914,001, Roche, Shanghai, China).

Article Title: Development of a Conventional RT-PCR Assay for Rapid Detection of Porcine Deltacoronavirus with the Same Detection Limit as a SYBR Green-Based Real-Time RT-PCR Assay
Article Snippet: .. RT-PCR After isolating total RNA from samples, it was reverse transcribed into cDNA by following the manufacturer's instructions in PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara Biotechnology, Dalian, China). .. Using Premix Taq™ kit, the PCR assay was performed under the following conditions: 1 μ L each primer (10 μ m), 25 μ L premix, 1 μ L cDNA, and 22 μ L distilled water.

Expressing:

Article Title: Cell‐based high‐throughput screen for small molecule inhibitors of Bax translocation, et al. Cell‐based high‐throughput screen for small molecule inhibitors of Bax translocation
Article Snippet: .. 2.1 EGFP‐Bax expression vector The mouse Bax cDNA was PCR amplified from IMAGE clone 3968903 and cloned into mammalian expression vector pEGFP‐C1 (Clontech Laboratories, Inc.) via BglII and EcoRI sites. .. 2.2 Cell culture and transfection Certified Chinese Hamster Ovary (CHO) cells were maintained at 37°C, 6% CO2 in Dulbecco's Modified Eagle Medium (DMEM, 25 mmol/L HEPES) supplemented with 10% heat‐inactivated foetal bovine serum (Invitrogen Corp., 12483020), 2 mmol/L glutamine and 1% antibiotics (penicillin and streptomycin) (Invitrogen Corp., 10378016).

Article Title: Genetic variation affecting DNA methylation and the human imprinting disorder, Beckwith-Wiedemann syndrome
Article Snippet: .. Generation of DNMT1 plasmids The mammalian expression vector containing the long isoform of wild-type human DNMT1 with N-terminal GFP tag in pEGFP-C1 plasmid (Clontech) was obtained from Prof. Heinrich Leonhardt (Ludwig-Maximilians-University Biocentre, Munich). .. The pEGFP-C1-DNMT1 construct was transfected into competent DH5-alpha cells, and construct fidelity was assessed by sequencing plasmid DNA using primers designed to DNMT1 exons as described in the supplementary information (cDNMT1 sequencing primers).

Article Title: The Stress-Inducible Protein DRR1 Exerts Distinct Effects on Actin Dynamics
Article Snippet: .. For expression of DRR1 proteins N-terminally fused to EGFP or MBP, inserts of DRR1 wt and mutants were subcloned into the vector pEGFP-C1 (Clontech, Saint-Germain-en-Laye, France) or pMAL-CR1 (New England Biolabs, Ipswich, MA, USA), respectively. .. Details of the cloning strategies and primer sequences are available on request.

Y2H Assay:

Article Title: GSK3β-mediated Ser156 phosphorylation modulates a BH3-like domain in BCL2L12 during TMZ-induced apoptosis and autophagy in glioma cells
Article Snippet: .. Cloning BCL2L12, BCL-XL, BCL2, BCL2 associated X (Bax), Beclin-1, induced myeloid leukemia cell differentiation protein MCL-1 (Mcl-1), and GSK3β were cloned into pACT2 and pAS2-1 vectors (Takara Bio Inc., Otsu, Japan) for yeast two-hybrid assay or into the pEGFP-C1 vector for overexpression. .. Polymerase chain reaction (PCR) technique was used to generate DNA fragments containing the desired gene sequences utilizing primers designed to contain the Xho I and Bam HI restriction enzyme recognition sites.

Polymerase Chain Reaction:

Article Title: Cell‐based high‐throughput screen for small molecule inhibitors of Bax translocation, et al. Cell‐based high‐throughput screen for small molecule inhibitors of Bax translocation
Article Snippet: .. 2.1 EGFP‐Bax expression vector The mouse Bax cDNA was PCR amplified from IMAGE clone 3968903 and cloned into mammalian expression vector pEGFP‐C1 (Clontech Laboratories, Inc.) via BglII and EcoRI sites. .. 2.2 Cell culture and transfection Certified Chinese Hamster Ovary (CHO) cells were maintained at 37°C, 6% CO2 in Dulbecco's Modified Eagle Medium (DMEM, 25 mmol/L HEPES) supplemented with 10% heat‐inactivated foetal bovine serum (Invitrogen Corp., 12483020), 2 mmol/L glutamine and 1% antibiotics (penicillin and streptomycin) (Invitrogen Corp., 10378016).

Over Expression:

Article Title: GSK3β-mediated Ser156 phosphorylation modulates a BH3-like domain in BCL2L12 during TMZ-induced apoptosis and autophagy in glioma cells
Article Snippet: .. Cloning BCL2L12, BCL-XL, BCL2, BCL2 associated X (Bax), Beclin-1, induced myeloid leukemia cell differentiation protein MCL-1 (Mcl-1), and GSK3β were cloned into pACT2 and pAS2-1 vectors (Takara Bio Inc., Otsu, Japan) for yeast two-hybrid assay or into the pEGFP-C1 vector for overexpression. .. Polymerase chain reaction (PCR) technique was used to generate DNA fragments containing the desired gene sequences utilizing primers designed to contain the Xho I and Bam HI restriction enzyme recognition sites.

Plasmid Preparation:

Article Title: GSK3β-mediated Ser156 phosphorylation modulates a BH3-like domain in BCL2L12 during TMZ-induced apoptosis and autophagy in glioma cells
Article Snippet: .. Cloning BCL2L12, BCL-XL, BCL2, BCL2 associated X (Bax), Beclin-1, induced myeloid leukemia cell differentiation protein MCL-1 (Mcl-1), and GSK3β were cloned into pACT2 and pAS2-1 vectors (Takara Bio Inc., Otsu, Japan) for yeast two-hybrid assay or into the pEGFP-C1 vector for overexpression. .. Polymerase chain reaction (PCR) technique was used to generate DNA fragments containing the desired gene sequences utilizing primers designed to contain the Xho I and Bam HI restriction enzyme recognition sites.

Article Title: Cell‐based high‐throughput screen for small molecule inhibitors of Bax translocation, et al. Cell‐based high‐throughput screen for small molecule inhibitors of Bax translocation
Article Snippet: .. 2.1 EGFP‐Bax expression vector The mouse Bax cDNA was PCR amplified from IMAGE clone 3968903 and cloned into mammalian expression vector pEGFP‐C1 (Clontech Laboratories, Inc.) via BglII and EcoRI sites. .. 2.2 Cell culture and transfection Certified Chinese Hamster Ovary (CHO) cells were maintained at 37°C, 6% CO2 in Dulbecco's Modified Eagle Medium (DMEM, 25 mmol/L HEPES) supplemented with 10% heat‐inactivated foetal bovine serum (Invitrogen Corp., 12483020), 2 mmol/L glutamine and 1% antibiotics (penicillin and streptomycin) (Invitrogen Corp., 10378016).

Article Title: Genetic variation affecting DNA methylation and the human imprinting disorder, Beckwith-Wiedemann syndrome
Article Snippet: .. Generation of DNMT1 plasmids The mammalian expression vector containing the long isoform of wild-type human DNMT1 with N-terminal GFP tag in pEGFP-C1 plasmid (Clontech) was obtained from Prof. Heinrich Leonhardt (Ludwig-Maximilians-University Biocentre, Munich). .. The pEGFP-C1-DNMT1 construct was transfected into competent DH5-alpha cells, and construct fidelity was assessed by sequencing plasmid DNA using primers designed to DNMT1 exons as described in the supplementary information (cDNMT1 sequencing primers).

Article Title: The Stress-Inducible Protein DRR1 Exerts Distinct Effects on Actin Dynamics
Article Snippet: .. For expression of DRR1 proteins N-terminally fused to EGFP or MBP, inserts of DRR1 wt and mutants were subcloned into the vector pEGFP-C1 (Clontech, Saint-Germain-en-Laye, France) or pMAL-CR1 (New England Biolabs, Ipswich, MA, USA), respectively. .. Details of the cloning strategies and primer sequences are available on request.

Article Title: Src activation by Chk1 promotes actin patch formation and prevents chromatin bridge breakage in cytokinesis
Article Snippet: .. Plasmid pEGFP-vps4-K173Q encoding human VPS4 harboring the K173Q point mutation fused to EGFP into pEGFP-C1 vector (Takara Bio Inc.) was a gift from W. Sundquist (University of Utah, Salt Lake City, UT; ), and plasmid pEGFP-N1 coding for GFP under cytomegalovirus promoter was obtained from Takara Bio Inc. Plasmid pEGFP/Chk1 encoding human Chk1 fused to EGFP into pEGFP-N1 vector (Takara Bio Inc.) was from Addgene (22888; ). .. Sequencing of this plasmid showed that Chk1 exhibited mutation of aspartic acid-130 to alanine (D130A), and this mutation was reversed to obtain the WT pEGFP/Chk1 plasmid.

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  • 94
    TaKaRa pegfp c1
    Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected <t>pEGFP-C1</t> (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.
    Pegfp C1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 1379 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    TaKaRa plasmid cmv pegfp c1
    Expression of EGFP protein in cells transiently transfected with EGFP under the control of PABP3 promoter sequences. PABP3 genomic sequences extending from nt –498 to +30 were amplified by PCR, as described in Materials and Methods, and then inserted into a promoter-less EGFP vector <t>(pEGFP-1).</t> These constructs were transiently transfected into HeLa (black boxes) or NTERA-2 (grey boxes) cells. The numbers of cells counted were identical in the two transfection assays. EGFP fluorescence driven by each construct was normalised to that obtained with the <t>CMV</t> <t>promoter/pEGFP-C1</t> vector (fluorescence of 100%). Results are the means of triplicate determinations obtained in two independent experiments. CMV corresponds to the control construct of the CMV promoter upstream from EGFP. PC, P1, P2 and P3 refer to the different PABP3 promoter constructs upstream from the EGFP reporter gene as indicated on the left.
    Plasmid Cmv Pegfp C1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    TaKaRa egfp fused kctd5 encoding plasmid
    Retention of <t>KCTD5</t> in the Ni 2+ -NTA column during H. pylori infection. (A) HEK293T and (B) AGS cells were transfected with plasmids encoding <t>EGFP,</t> EGFP-KCTD5 and His-Ubi, lysates from these cells were processed for pull-down with Ni 2+ -NTA column, and the presence of KCTD5 was determined by immunoblot with antibody to KCTD5 (upper panel) and ubiquitinated proteins were shown as retention control in the column (lower panel). AGS transfected cells were infected by 8 h with H. pylori (MOI = 300).
    Egfp Fused Kctd5 Encoding Plasmid, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.

    Journal: PLoS ONE

    Article Title: Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids

    doi: 10.1371/journal.pone.0043283

    Figure Lengend Snippet: Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.

    Article Snippet: To get further insights into possible causes of inhibitory effects of pEGFP-C1, we re-examined deep sequencing data searching for any transcriptome features unique to pEGFP-C1.

    Techniques: Transfection, Expressing, Luciferase, Plasmid Preparation, Cotransfection, Flow Cytometry, Cytometry, Fluorescence, Cell Counting

    Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.

    Journal: PLoS ONE

    Article Title: Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids

    doi: 10.1371/journal.pone.0043283

    Figure Lengend Snippet: Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.

    Article Snippet: To get further insights into possible causes of inhibitory effects of pEGFP-C1, we re-examined deep sequencing data searching for any transcriptome features unique to pEGFP-C1.

    Techniques: Expressing, Luciferase, Derivative Assay, Sequencing, Transfection, Activity Assay

    Expression of EGFP protein in cells transiently transfected with EGFP under the control of PABP3 promoter sequences. PABP3 genomic sequences extending from nt –498 to +30 were amplified by PCR, as described in Materials and Methods, and then inserted into a promoter-less EGFP vector (pEGFP-1). These constructs were transiently transfected into HeLa (black boxes) or NTERA-2 (grey boxes) cells. The numbers of cells counted were identical in the two transfection assays. EGFP fluorescence driven by each construct was normalised to that obtained with the CMV promoter/pEGFP-C1 vector (fluorescence of 100%). Results are the means of triplicate determinations obtained in two independent experiments. CMV corresponds to the control construct of the CMV promoter upstream from EGFP. PC, P1, P2 and P3 refer to the different PABP3 promoter constructs upstream from the EGFP reporter gene as indicated on the left.

    Journal: Nucleic Acids Research

    Article Title: Human testis expresses a specific poly(A)-binding protein

    doi:

    Figure Lengend Snippet: Expression of EGFP protein in cells transiently transfected with EGFP under the control of PABP3 promoter sequences. PABP3 genomic sequences extending from nt –498 to +30 were amplified by PCR, as described in Materials and Methods, and then inserted into a promoter-less EGFP vector (pEGFP-1). These constructs were transiently transfected into HeLa (black boxes) or NTERA-2 (grey boxes) cells. The numbers of cells counted were identical in the two transfection assays. EGFP fluorescence driven by each construct was normalised to that obtained with the CMV promoter/pEGFP-C1 vector (fluorescence of 100%). Results are the means of triplicate determinations obtained in two independent experiments. CMV corresponds to the control construct of the CMV promoter upstream from EGFP. PC, P1, P2 and P3 refer to the different PABP3 promoter constructs upstream from the EGFP reporter gene as indicated on the left.

    Article Snippet: Plasmid CMV/pEGFP-C1 (Clontech), which contains the human cytomegalovirus (CMV) promoter, was used as a positive control in transfection experiments.

    Techniques: Expressing, Transfection, Genomic Sequencing, Amplification, Polymerase Chain Reaction, Plasmid Preparation, Construct, Fluorescence

    Retention of KCTD5 in the Ni 2+ -NTA column during H. pylori infection. (A) HEK293T and (B) AGS cells were transfected with plasmids encoding EGFP, EGFP-KCTD5 and His-Ubi, lysates from these cells were processed for pull-down with Ni 2+ -NTA column, and the presence of KCTD5 was determined by immunoblot with antibody to KCTD5 (upper panel) and ubiquitinated proteins were shown as retention control in the column (lower panel). AGS transfected cells were infected by 8 h with H. pylori (MOI = 300).

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: KCTD5 and Ubiquitin Proteasome Signaling Are Required for Helicobacter pylori Adherence

    doi: 10.3389/fcimb.2017.00450

    Figure Lengend Snippet: Retention of KCTD5 in the Ni 2+ -NTA column during H. pylori infection. (A) HEK293T and (B) AGS cells were transfected with plasmids encoding EGFP, EGFP-KCTD5 and His-Ubi, lysates from these cells were processed for pull-down with Ni 2+ -NTA column, and the presence of KCTD5 was determined by immunoblot with antibody to KCTD5 (upper panel) and ubiquitinated proteins were shown as retention control in the column (lower panel). AGS transfected cells were infected by 8 h with H. pylori (MOI = 300).

    Article Snippet: EGFP-fused KCTD5 encoding plasmid (pEGFP-C1-KCTD5) was generated by cloning the human KCTD5 cDNA from the pMaxKoz-HA-KCTD5 plasmid (Dementieva et al., ) (kindly donated by Dr. Steve Goldstein) into the XhoI/BamHI sites of the pEGFP-C1 plasmid (Catalog number: 6084, Clontech Laboratories, Mountain View, CA, USA).

    Techniques: Infection, Transfection