Structured Review

TaKaRa egfp
<t>STOML3-containing</t> vesicles are not associated with a classical endocytic vesicle pool. DRG neurons were co-transfected with STOML3-mCherry and either <t>EGFP</t> tagged Rab5 (Q79L mutant) ( a ), Rab14 ( b ) or LAMP1 ( c ), or immunostained for clathrin ( d ). Cells were examined using scanning confocal microscopy and no colocalization was observed with any of the markers (right column).
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Images

1) Product Images from "Regulation of ASIC channels by a stomatin/STOML3 complex located in a mobile vesicle pool in sensory neurons"

Article Title: Regulation of ASIC channels by a stomatin/STOML3 complex located in a mobile vesicle pool in sensory neurons

Journal: Open Biology

doi: 10.1098/rsob.120096

STOML3-containing vesicles are not associated with a classical endocytic vesicle pool. DRG neurons were co-transfected with STOML3-mCherry and either EGFP tagged Rab5 (Q79L mutant) ( a ), Rab14 ( b ) or LAMP1 ( c ), or immunostained for clathrin ( d ). Cells were examined using scanning confocal microscopy and no colocalization was observed with any of the markers (right column).
Figure Legend Snippet: STOML3-containing vesicles are not associated with a classical endocytic vesicle pool. DRG neurons were co-transfected with STOML3-mCherry and either EGFP tagged Rab5 (Q79L mutant) ( a ), Rab14 ( b ) or LAMP1 ( c ), or immunostained for clathrin ( d ). Cells were examined using scanning confocal microscopy and no colocalization was observed with any of the markers (right column).

Techniques Used: Transfection, Mutagenesis, Confocal Microscopy

STOML3 colocalizes with Rab11-positive vesicles. ( a ) STOML3-mCherry and Rab11-EGFP exhibit extensive overlap in axons and cell bodies of transfected sensory neurons. ( b ) Transfection of the dominant-negative mutant Rab11 S25N leads to accumulation of STOML3-positive vesicles in the cell body of sensory neurons. ( c ) The constitutively active Rab11 mutant Q70L-EGFP labels large axonal vesicles that are also positive for STOML3-mCherry.
Figure Legend Snippet: STOML3 colocalizes with Rab11-positive vesicles. ( a ) STOML3-mCherry and Rab11-EGFP exhibit extensive overlap in axons and cell bodies of transfected sensory neurons. ( b ) Transfection of the dominant-negative mutant Rab11 S25N leads to accumulation of STOML3-positive vesicles in the cell body of sensory neurons. ( c ) The constitutively active Rab11 mutant Q70L-EGFP labels large axonal vesicles that are also positive for STOML3-mCherry.

Techniques Used: Transfection, Dominant Negative Mutation, Mutagenesis

2) Product Images from "Identifying Activated T Cells in Reconstituted RAG Deficient Mice Using Retrovirally Transduced Pax5 Deficient Pro-B Cells"

Article Title: Identifying Activated T Cells in Reconstituted RAG Deficient Mice Using Retrovirally Transduced Pax5 Deficient Pro-B Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0005115

Gene-modified Pax5 −/− cells reconstitutes EGFP + T cell pool in RAG1 −/− mice. (a) Schematic drawing of the double-reporter retroviral construct used for transduction of Pax5 −/− pro-B cells. (b) Histological examination of a spleen section from a RAG1 −/− mouse reconstituted with retrovirally transduced Pax5 −/− cells that expressed EGFP constitutively. EGFP is mainly detected in the nuclei of cells. This experiment was performed twice.
Figure Legend Snippet: Gene-modified Pax5 −/− cells reconstitutes EGFP + T cell pool in RAG1 −/− mice. (a) Schematic drawing of the double-reporter retroviral construct used for transduction of Pax5 −/− pro-B cells. (b) Histological examination of a spleen section from a RAG1 −/− mouse reconstituted with retrovirally transduced Pax5 −/− cells that expressed EGFP constitutively. EGFP is mainly detected in the nuclei of cells. This experiment was performed twice.

Techniques Used: Modification, Mouse Assay, Construct, Transduction

3) Product Images from "Cervical Cancer Cells with Positive Sox2 Expression Exhibit the Properties of Cancer Stem Cells"

Article Title: Cervical Cancer Cells with Positive Sox2 Expression Exhibit the Properties of Cancer Stem Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0087092

EGFP-positive cells exhibit a greater capacity for differentiation than EGFP-negative cells. (A) Sox2 expression was detected by IHC in xenograft tissues formed by SiHa-EGFP+, SiHa-EGFP−, C33A-EGFP+ and C33A-EGFP− cells. (B) The percentages of EGFP+ cells in the first, second, and third passages in differentiation medium. Bars = SE. *, p
Figure Legend Snippet: EGFP-positive cells exhibit a greater capacity for differentiation than EGFP-negative cells. (A) Sox2 expression was detected by IHC in xenograft tissues formed by SiHa-EGFP+, SiHa-EGFP−, C33A-EGFP+ and C33A-EGFP− cells. (B) The percentages of EGFP+ cells in the first, second, and third passages in differentiation medium. Bars = SE. *, p

Techniques Used: Expressing, Immunohistochemistry

pSox2/EGFP reporter system construction. Schematic of the pSox2/EGFP reporter system. The hSox2 promoter and transcriptional elements including the 3'UTR, poly (A) tail, and 3′ enhancer were cloned into the pEGFP vector.
Figure Legend Snippet: pSox2/EGFP reporter system construction. Schematic of the pSox2/EGFP reporter system. The hSox2 promoter and transcriptional elements including the 3'UTR, poly (A) tail, and 3′ enhancer were cloned into the pEGFP vector.

Techniques Used: Clone Assay, Plasmid Preparation

Distinct molecular and biological properties of Sox2-positive and Sox2-negative cells. (A) Differential expression of several stem cell-related genes in SiHa-EGFP + and SiHa-EGFP − fractions validated by qPCR. (B) Detection of stem cell-related factors in sorted SiHa-EGFP + and SiHa-EGFP − cells by western blot. (C) Semi-quantitative analysis of stem cell-related factors relative to β-actin. (D) Stem cell-related gene expression in tumor xenografts was detected by immunohistochemistry. ALDH1 was detected by immunohistochemistry (E), western blot (F), and semi-quantitative analysis (G) in SiHa-EGFP + and SiHa-EGFP − tumors. β-actin was used as the loading control for RT-PCR and western blotting. Error bars represent S.D. (n = 3). * p
Figure Legend Snippet: Distinct molecular and biological properties of Sox2-positive and Sox2-negative cells. (A) Differential expression of several stem cell-related genes in SiHa-EGFP + and SiHa-EGFP − fractions validated by qPCR. (B) Detection of stem cell-related factors in sorted SiHa-EGFP + and SiHa-EGFP − cells by western blot. (C) Semi-quantitative analysis of stem cell-related factors relative to β-actin. (D) Stem cell-related gene expression in tumor xenografts was detected by immunohistochemistry. ALDH1 was detected by immunohistochemistry (E), western blot (F), and semi-quantitative analysis (G) in SiHa-EGFP + and SiHa-EGFP − tumors. β-actin was used as the loading control for RT-PCR and western blotting. Error bars represent S.D. (n = 3). * p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Reverse Transcription Polymerase Chain Reaction

Sorting living cervical cancer cells using the nuclear protein marker Sox2. Sox2 protein expression was detected by western blot (A) and immunohistochemistry (B) in the cervical cancer cell lines HeLa, SiHa, CaSki, and C33A. (C) The abundance of EGFP-positive tumor cells in cervical cancer cell lines transfected with the pSox2/EGFP reporter. (D) Immunohistochemical analysis of EGFP + and EGFP − cells. (E) Sox2 protein expression in a monolayer, tumorsphere cells, and sorted EGFP + and EGFP − cells.
Figure Legend Snippet: Sorting living cervical cancer cells using the nuclear protein marker Sox2. Sox2 protein expression was detected by western blot (A) and immunohistochemistry (B) in the cervical cancer cell lines HeLa, SiHa, CaSki, and C33A. (C) The abundance of EGFP-positive tumor cells in cervical cancer cell lines transfected with the pSox2/EGFP reporter. (D) Immunohistochemical analysis of EGFP + and EGFP − cells. (E) Sox2 protein expression in a monolayer, tumorsphere cells, and sorted EGFP + and EGFP − cells.

Techniques Used: Marker, Expressing, Western Blot, Immunohistochemistry, Transfection

Sox2-positive cells exhibit more EMT features but do not show altered proliferation in vitro . (A) Real-time PCR analysis of the mRNA levels of various EMT-related genes in SiHa-EGFP + and SiHa-EGFP − cells. EMT-related gene expression was measured by western blot (B) and immunochemistry (C) in SiHa-EGFP + and SiHa-EGFP − cells. (D) Immunochemistry for Sox2 and EMT-related genes in tumor xenografts. Bars = SE. *, p
Figure Legend Snippet: Sox2-positive cells exhibit more EMT features but do not show altered proliferation in vitro . (A) Real-time PCR analysis of the mRNA levels of various EMT-related genes in SiHa-EGFP + and SiHa-EGFP − cells. EMT-related gene expression was measured by western blot (B) and immunochemistry (C) in SiHa-EGFP + and SiHa-EGFP − cells. (D) Immunochemistry for Sox2 and EMT-related genes in tumor xenografts. Bars = SE. *, p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Western Blot

4) Product Images from "PiggyBac transgenic strategies in the developing chicken spinal cord"

Article Title: PiggyBac transgenic strategies in the developing chicken spinal cord

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkp686

Temporal control of PB transgene expression. ( A ) ERT2CreERT2 regulated transgene expression. Upon 4-OHT induction, excision of floxed DsRed leads to expression of EGFP. ( B ) A scheme of experimental design. EP: electroporation. ( C ) GFP expression is tightly regulated by 4-OHT induction during development. Bars are 100 µm.
Figure Legend Snippet: Temporal control of PB transgene expression. ( A ) ERT2CreERT2 regulated transgene expression. Upon 4-OHT induction, excision of floxed DsRed leads to expression of EGFP. ( B ) A scheme of experimental design. EP: electroporation. ( C ) GFP expression is tightly regulated by 4-OHT induction during development. Bars are 100 µm.

Techniques Used: Expressing, Electroporation

Labeling of astrocytes by a PB transgene. ( A ) Schematic of the mouse gfap gene and PB -gfap-EGFP transgene. ( B ) PB -gfap-EGFP specifically labels GFAP positive cells at late stages of spinal cord development. At E19 or E21, robust GFP signals are seen in regions where astrocytes reside. ( C ) High-magnification images showing that GFP is co-expressed in GFAP-positive astrocytes in both grey matter and white matter of the spinal cord. Note that GFP and endogenous GFAP signals are not completely overlapped in GFP-positive cells because of their different subcellular localizations. Bars are 50 µm in (B).
Figure Legend Snippet: Labeling of astrocytes by a PB transgene. ( A ) Schematic of the mouse gfap gene and PB -gfap-EGFP transgene. ( B ) PB -gfap-EGFP specifically labels GFAP positive cells at late stages of spinal cord development. At E19 or E21, robust GFP signals are seen in regions where astrocytes reside. ( C ) High-magnification images showing that GFP is co-expressed in GFAP-positive astrocytes in both grey matter and white matter of the spinal cord. Note that GFP and endogenous GFAP signals are not completely overlapped in GFP-positive cells because of their different subcellular localizations. Bars are 50 µm in (B).

Techniques Used: Labeling

PB-mediated labeling of V2b interneurons in the spinal cord. ( A ) A strategy to trace Scl -expressing cell lineage using PB transgenics. The − 7E3/Cre transgene that consists of mouse 5′Scl promoter sequences transiently express Cre in Scl -expressing progenitors. Cre -mediated excision leads to GFP expression from PB -loxP-luc-loxP-EGFP reporter. Scl -expressing cells are permanently labeled with GFP because the PB reporter is stably integrated in the progenitors. ( B ) Activation of GFP expression by −7E3/Cre transgene in the chicken spinal cord. ( C ) GATA3 partially overlaps GFP positive cells labeled by −7E3/Cre transgene. ( D ) Schematic illustration of the projection patterns of V2b ( Scl -expressing) interneurons in the spinal cord. The orientation of spinal cord and focal planes of confocal images of panel F a–e are marked. Turquoise: transverse section; Purple: longitudinal section. ( E ) A single Scl -expressing neuron labeled by GFP on a transverse spinal cord section. ( F ) The projection patterns of neuronal processes from GFP labeled V2b interneurons. a and b, transverse sections; c–e, longitudinal sections. Blue dotted line, midline. Bars are 100 µm in (F).
Figure Legend Snippet: PB-mediated labeling of V2b interneurons in the spinal cord. ( A ) A strategy to trace Scl -expressing cell lineage using PB transgenics. The − 7E3/Cre transgene that consists of mouse 5′Scl promoter sequences transiently express Cre in Scl -expressing progenitors. Cre -mediated excision leads to GFP expression from PB -loxP-luc-loxP-EGFP reporter. Scl -expressing cells are permanently labeled with GFP because the PB reporter is stably integrated in the progenitors. ( B ) Activation of GFP expression by −7E3/Cre transgene in the chicken spinal cord. ( C ) GATA3 partially overlaps GFP positive cells labeled by −7E3/Cre transgene. ( D ) Schematic illustration of the projection patterns of V2b ( Scl -expressing) interneurons in the spinal cord. The orientation of spinal cord and focal planes of confocal images of panel F a–e are marked. Turquoise: transverse section; Purple: longitudinal section. ( E ) A single Scl -expressing neuron labeled by GFP on a transverse spinal cord section. ( F ) The projection patterns of neuronal processes from GFP labeled V2b interneurons. a and b, transverse sections; c–e, longitudinal sections. Blue dotted line, midline. Bars are 100 µm in (F).

Techniques Used: Labeling, Expressing, Stable Transfection, Activation Assay

5) Product Images from "Expression of ?5 integrin rescues fibronectin responsiveness in NT2N CNS neuronal cells"

Article Title: Expression of ?5 integrin rescues fibronectin responsiveness in NT2N CNS neuronal cells

Journal: Journal of neuroscience research

doi: 10.1002/jnr.22171

The Murine Stem Cell Virus (MSCV) promoter drives transgene expression throughout NT2N cell differentiation. NT2 precursor cells were transduced with an MSCV-EGFP retroviral vector. After selection, the stably transduced, uncloned population was differentiated
Figure Legend Snippet: The Murine Stem Cell Virus (MSCV) promoter drives transgene expression throughout NT2N cell differentiation. NT2 precursor cells were transduced with an MSCV-EGFP retroviral vector. After selection, the stably transduced, uncloned population was differentiated

Techniques Used: Expressing, Cell Differentiation, Transduction, Plasmid Preparation, Selection, Stable Transfection

6) Product Images from "Syk Interacts with and Phosphorylates Nucleolin To Stabilize Bcl-xL mRNA and Promote Cell Survival"

Article Title: Syk Interacts with and Phosphorylates Nucleolin To Stabilize Bcl-xL mRNA and Promote Cell Survival

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00937-14

Syk expression protects MDA-MB-231 cells from oxidative stress-induced apoptosis and degradation of Bcl-x L mRNA. (A) MDA-MB-231-TR (TR) cells lacking Syk or MDA-MB-231-TRS (TRS) cells either induced (+) or not induced (−) with doxycycline (Tet) to express Syk-EGFP were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA (top) or by Western blotting to detect expressed Syk-EGFP (bottom). (B) Comparison of relative levels of Bcl-x L mRNA to Bcl-x S mRNA. Ratios were normalized to a value of 1.0 for Syk-deficient cells at time zero. Bars represent means ± SEMs from three replicate experiments. *, P
Figure Legend Snippet: Syk expression protects MDA-MB-231 cells from oxidative stress-induced apoptosis and degradation of Bcl-x L mRNA. (A) MDA-MB-231-TR (TR) cells lacking Syk or MDA-MB-231-TRS (TRS) cells either induced (+) or not induced (−) with doxycycline (Tet) to express Syk-EGFP were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA (top) or by Western blotting to detect expressed Syk-EGFP (bottom). (B) Comparison of relative levels of Bcl-x L mRNA to Bcl-x S mRNA. Ratios were normalized to a value of 1.0 for Syk-deficient cells at time zero. Bars represent means ± SEMs from three replicate experiments. *, P

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

Nucleolin is required for Syk-dependent stabilization of Bcl-x L mRNA. (A) Tet-responsive MDA-MB-231 cells were untreated (control [Ctrl]) or infected with one of a set of lentiviruses encoding shRNAs for nucleolin (shNCL). Nucleolin levels were measured by Western blotting. The level of GAPDH was measured as a loading control. Results from three different populations of infected cells are shown. (B) Tet-responsive MDA-MB-231 cells and two of the three sets of Tet-responsive cells carrying the nucleolin shRNA (shNCL2 and shNCL3) were either uninduced (−) or induced with doxycycline to express Syk-EGFP (+) and then treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA. (C) DG75 B cells were untreated (control) or infected with a lentivirus encoding shRNA directed against nucleolin. Nucleolin levels were measured by Western blotting. The level of GAPDH was measured as a loading control. (D) DG75 cells either infected (+) or not infected (−) with the lentivirus carrying the nucleolin shRNA were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA. (E) Tet-responsive MCF7 cells were untreated (control) or infected with a set of lentiviruses encoding shRNAs for nucleolin. Nucleolin levels were measured by Western blotting. Results from two different populations of infected cells are shown. (F) Tet-responsive MCF7 cells and the two sets of Tet-responsive cells carrying the nucleolin shRNA (shNCL1 and shNCL2) were either uninduced (−) or induced with doxycycline to express Syk-EGFP (+) and then treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA.
Figure Legend Snippet: Nucleolin is required for Syk-dependent stabilization of Bcl-x L mRNA. (A) Tet-responsive MDA-MB-231 cells were untreated (control [Ctrl]) or infected with one of a set of lentiviruses encoding shRNAs for nucleolin (shNCL). Nucleolin levels were measured by Western blotting. The level of GAPDH was measured as a loading control. Results from three different populations of infected cells are shown. (B) Tet-responsive MDA-MB-231 cells and two of the three sets of Tet-responsive cells carrying the nucleolin shRNA (shNCL2 and shNCL3) were either uninduced (−) or induced with doxycycline to express Syk-EGFP (+) and then treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA. (C) DG75 B cells were untreated (control) or infected with a lentivirus encoding shRNA directed against nucleolin. Nucleolin levels were measured by Western blotting. The level of GAPDH was measured as a loading control. (D) DG75 cells either infected (+) or not infected (−) with the lentivirus carrying the nucleolin shRNA were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA. (E) Tet-responsive MCF7 cells were untreated (control) or infected with a set of lentiviruses encoding shRNAs for nucleolin. Nucleolin levels were measured by Western blotting. Results from two different populations of infected cells are shown. (F) Tet-responsive MCF7 cells and the two sets of Tet-responsive cells carrying the nucleolin shRNA (shNCL1 and shNCL2) were either uninduced (−) or induced with doxycycline to express Syk-EGFP (+) and then treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA.

Techniques Used: Multiple Displacement Amplification, Infection, Western Blot, shRNA, Reverse Transcription Polymerase Chain Reaction

Syk interacts with nucleolin. (A) MDA-MB-231 cells expressing rtTA but not Syk or MDA-MB-231 cells with Tet-regulated expression of Syk-EGFP or Syk-EGFP(K396R) (Lenti-X Tet-On) pretreated with doxycycline (+) were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA (top) or by Western blotting with anti-Syk antibodies to detect Syk-EGFP or Syk-EGFP(K396R) (bottom). (B) Tet-responsive MDA-MB-231 cells not induced (−) or induced with doxycycline to express Syk-EGFP (+) were treated with 5 mM H 2 O 2 for the indicated times. Syk-EGFP was immunoprecipitated (IP) from cell lysates with GFP-nanotrap beads. Anti-GFP immune complexes were separated by SDS-PAGE and analyzed by Western blotting (WB) with antibodies against NCL, γ-tubulin, or GFP (to detect Syk-EGFP). Whole-cell lysates (WCL) were analyzed by Western blotting with antibodies against phosphotyrosine (pTyr) (bottom). The migration position of the 50-kDa molecular mass marker is indicated. (C) Syk-EGFP was immunoprecipitated with GFP-nanotrap beads from lysates of Tet-responsive MDA-MB-231 cells induced to express Syk-EGFP. Immune complexes and whole-cell lysates were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL or γ-tubulin. (D) Proteins were immunoprecipitated with GFP-nanotrap beads from lysates of Tet-responsive MDA-MB-231 cells induced to express Syk-EGFP (Syk) or Syk-EGFP(K396R) (KD) and treated with (+) or without (−) 5 mM H 2 O 2 . Immune complexes and whole-cell lysates were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL (top) and, to detect Syk-GFP, antibodies against GFP (bottom). (E) Proteins were immunoprecipitated with GFP-nanotrap beads from lysates of Tet-responsive MDA-MB-231 cells induced to express EGFP (lane GFP), Syk-EGFP (lane Syk), Syk-EGFP(Y342F/Y346F) (lane 2F), Syk-EGFP(Y317F/Y342F/Y346F) (lane 3F), Syk-EGFP(Y317F) (lane 317), Syk-EGFP(Y342F) (lane 342), or Syk-EGFP(Y346F) (lane 346) and treated with (+) or without (−) 5 mM H 2 O 2 . Immune complexes and whole-cell lysates were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL (top) or GFP (bottom).
Figure Legend Snippet: Syk interacts with nucleolin. (A) MDA-MB-231 cells expressing rtTA but not Syk or MDA-MB-231 cells with Tet-regulated expression of Syk-EGFP or Syk-EGFP(K396R) (Lenti-X Tet-On) pretreated with doxycycline (+) were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA (top) or by Western blotting with anti-Syk antibodies to detect Syk-EGFP or Syk-EGFP(K396R) (bottom). (B) Tet-responsive MDA-MB-231 cells not induced (−) or induced with doxycycline to express Syk-EGFP (+) were treated with 5 mM H 2 O 2 for the indicated times. Syk-EGFP was immunoprecipitated (IP) from cell lysates with GFP-nanotrap beads. Anti-GFP immune complexes were separated by SDS-PAGE and analyzed by Western blotting (WB) with antibodies against NCL, γ-tubulin, or GFP (to detect Syk-EGFP). Whole-cell lysates (WCL) were analyzed by Western blotting with antibodies against phosphotyrosine (pTyr) (bottom). The migration position of the 50-kDa molecular mass marker is indicated. (C) Syk-EGFP was immunoprecipitated with GFP-nanotrap beads from lysates of Tet-responsive MDA-MB-231 cells induced to express Syk-EGFP. Immune complexes and whole-cell lysates were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL or γ-tubulin. (D) Proteins were immunoprecipitated with GFP-nanotrap beads from lysates of Tet-responsive MDA-MB-231 cells induced to express Syk-EGFP (Syk) or Syk-EGFP(K396R) (KD) and treated with (+) or without (−) 5 mM H 2 O 2 . Immune complexes and whole-cell lysates were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL (top) and, to detect Syk-GFP, antibodies against GFP (bottom). (E) Proteins were immunoprecipitated with GFP-nanotrap beads from lysates of Tet-responsive MDA-MB-231 cells induced to express EGFP (lane GFP), Syk-EGFP (lane Syk), Syk-EGFP(Y342F/Y346F) (lane 2F), Syk-EGFP(Y317F/Y342F/Y346F) (lane 3F), Syk-EGFP(Y317F) (lane 317), Syk-EGFP(Y342F) (lane 342), or Syk-EGFP(Y346F) (lane 346) and treated with (+) or without (−) 5 mM H 2 O 2 . Immune complexes and whole-cell lysates were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL (top) or GFP (bottom).

Techniques Used: Multiple Displacement Amplification, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunoprecipitation, SDS Page, Migration, Marker

Syk expression protects MCF7 cells from oxidative stress-induced apoptosis and degradation of Bcl-x L mRNA. (A) MCF7-BD cells lacking Syk (−) or stably expressing Syk-EGFP (+) were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by SDS-PAGE and Western blotting with antibodies against PARP (top). The cleaved form of PARP is indicated by the arrow. The expression of Syk-EGFP was visualized by Western blotting of cell lysates (bottom). (B) MCF7-BD cells lacking Syk (−) or stably expressing Syk-EGFP (+) were exposed to 5 mM H 2 O 2 for 30 min (pulse) and then moved to fresh medium for the indicated total incubation times or treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA (top) or by Western blotting to detect expressed Syk-EGFP (bottom). (C) Comparison of relative levels of Bcl-x L mRNA. Changes in the ratio of Bcl-x L mRNA to Bcl-x S mRNA were normalized to their relative levels of expression in Syk-deficient cells at time zero, which was set equal to a value of 1.0. Bars represent means ± SEMs from three replicate experiments. Significant differences between pairs were determined using an unpaired, two-tailed Student's t test. *, P
Figure Legend Snippet: Syk expression protects MCF7 cells from oxidative stress-induced apoptosis and degradation of Bcl-x L mRNA. (A) MCF7-BD cells lacking Syk (−) or stably expressing Syk-EGFP (+) were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by SDS-PAGE and Western blotting with antibodies against PARP (top). The cleaved form of PARP is indicated by the arrow. The expression of Syk-EGFP was visualized by Western blotting of cell lysates (bottom). (B) MCF7-BD cells lacking Syk (−) or stably expressing Syk-EGFP (+) were exposed to 5 mM H 2 O 2 for 30 min (pulse) and then moved to fresh medium for the indicated total incubation times or treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA (top) or by Western blotting to detect expressed Syk-EGFP (bottom). (C) Comparison of relative levels of Bcl-x L mRNA. Changes in the ratio of Bcl-x L mRNA to Bcl-x S mRNA were normalized to their relative levels of expression in Syk-deficient cells at time zero, which was set equal to a value of 1.0. Bars represent means ± SEMs from three replicate experiments. Significant differences between pairs were determined using an unpaired, two-tailed Student's t test. *, P

Techniques Used: Expressing, Stable Transfection, SDS Page, Western Blot, Incubation, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Nucleolin is required for Syk-dependent protection of cells from stress-induced apoptosis. (A) Tet-responsive MCF7 cells or Tet-responsive cells carrying the nucleolin shRNA were either uninduced or induced with doxycycline to express Syk-EGFP and then treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (B) DG75 cells or DG75 cells expressing the shRNA targeting either Syk (Syk-shRNA) or nucleolin (NCL-shRNA) were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (C) Tet-responsive MCF7 cells or Tet-responsive cells carrying the nucleolin shRNA were either uninduced or induced with doxycycline to express Syk-EGFP and then treated with 1 μg/ml doxorubicin (Dox) for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (D) DG75 cells or DG75 cells expressing either the shRNA targeting Syk (Syk-shRNA) or nucleolin (NCL-shRNA) were treated with 1 μg/ml doxorubicin for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (E) The degree of PARP cleavage was quantified from Western blots of lysates of MCF7 cells lacking Syk (no Syk), expressing Syk-EGFP (SykGFP), or expressing Syk-EGFP and shRNA for nucleolin (shNCL) and treated for 24 h with 5 mM H 2 O 2 (left) or 1 μg/ml doxorubicin (right). The data represent means ± SEMs from three replicate experiments. **, P
Figure Legend Snippet: Nucleolin is required for Syk-dependent protection of cells from stress-induced apoptosis. (A) Tet-responsive MCF7 cells or Tet-responsive cells carrying the nucleolin shRNA were either uninduced or induced with doxycycline to express Syk-EGFP and then treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (B) DG75 cells or DG75 cells expressing the shRNA targeting either Syk (Syk-shRNA) or nucleolin (NCL-shRNA) were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (C) Tet-responsive MCF7 cells or Tet-responsive cells carrying the nucleolin shRNA were either uninduced or induced with doxycycline to express Syk-EGFP and then treated with 1 μg/ml doxorubicin (Dox) for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (D) DG75 cells or DG75 cells expressing either the shRNA targeting Syk (Syk-shRNA) or nucleolin (NCL-shRNA) were treated with 1 μg/ml doxorubicin for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (E) The degree of PARP cleavage was quantified from Western blots of lysates of MCF7 cells lacking Syk (no Syk), expressing Syk-EGFP (SykGFP), or expressing Syk-EGFP and shRNA for nucleolin (shNCL) and treated for 24 h with 5 mM H 2 O 2 (left) or 1 μg/ml doxorubicin (right). The data represent means ± SEMs from three replicate experiments. **, P

Techniques Used: shRNA, SDS Page, Expressing, Western Blot

Syk phosphorylates nucleolin and promotes its binding to Bcl-x L mRNA. (A) Tet-responsive MDA-MB-231 cells pretreated without or with doxycycline to induce Syk-EGFP were treated without or with 5 mM H 2 O 2 for 15 min. Tyrosine-phosphorylated proteins were immunoprecipitated from cell lysates with antibodies against phosphotyrosine. Immune complexes (top) and whole-cell lysates (WCL; bottom) were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (B) DG75 B cells were pretreated with 50 μM piceatannol (PIC; +) or dimethyl sulfoxide carrier alone (−) and then treated without or with 5 mM H 2 O 2 for 15 min. Tyrosine-phosphorylated proteins were immunoprecipitated from cell lysates with antibodies against phosphotyrosine. Immune complexes (top) and whole-cell lysates (bottom) were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (C) DG75 cells were pretreated with the indicated concentrations of R406 and then treated without or with 5 mM H 2 O 2 for 15 min. Tyrosine-phosphorylated proteins were immunoprecipitated from cell lysates with antibodies against phosphotyrosine. Immune complexes (top) and whole-cell lysates (bottom) were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (D) MDA-MB-231-TR (TR) or MDA-MB-231-TRS cells induced to express Syk-EGFP (TRS) were treated without or with 5 mM H 2 O 2 for 15 min. Nucleolin was immunoprecipitated, and the resulting immune complexes were probed by Western blotting for phosphotyrosine (top) or NCL (bottom). (E) DG75 B cells (−) or DG75 cells stably expressing shRNA targeted against Syk (+) were treated without or with 5 mM H 2 O 2 for 15 min. Nucleolin was immunoprecipitated, and the resulting immune complexes were probed by Western blotting for phosphotyrosine (top) or NCL (bottom). (F) Syk-EGFP (Syk) or Syk-EGFP(K396R) (KD) was immunoprecipitated from the corresponding doxycycline-induced lines of MDA-MB-231 cells using GFP-nanotrap beads. The resulting immune complexes were incubated with buffer containing (+) or lacking (−) ATP. The immune complexes and whole-cell lysates were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (G) Nucleolin was immunoprecipitated from Tet-responsive MDA-MB-231 cells either uninduced (−) or induced (+) with doxycycline to express Syk-EGFP and either treated with 5 mM H 2 O 2 for 3 h or not treated. Immune complexes were examined for the presence of Bcl-x L mRNA by RT-PCR (top) and nucleolin by Western blotting (middle). The expression of Syk-EGFP was determined by Western blotting of whole-cell lysates with antibodies against Syk (bottom). (H) The relative amount of Bcl-x L mRNA associated with nucleolin, analyzed as described in the legend to panel G, was quantified. The data represent means ± SEMs from three replicate experiments. The level of mRNA bound to nucleolin in Syk-EGFP-expressing cells not treated with H 2 O 2 was set equal to a value of 1.0.
Figure Legend Snippet: Syk phosphorylates nucleolin and promotes its binding to Bcl-x L mRNA. (A) Tet-responsive MDA-MB-231 cells pretreated without or with doxycycline to induce Syk-EGFP were treated without or with 5 mM H 2 O 2 for 15 min. Tyrosine-phosphorylated proteins were immunoprecipitated from cell lysates with antibodies against phosphotyrosine. Immune complexes (top) and whole-cell lysates (WCL; bottom) were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (B) DG75 B cells were pretreated with 50 μM piceatannol (PIC; +) or dimethyl sulfoxide carrier alone (−) and then treated without or with 5 mM H 2 O 2 for 15 min. Tyrosine-phosphorylated proteins were immunoprecipitated from cell lysates with antibodies against phosphotyrosine. Immune complexes (top) and whole-cell lysates (bottom) were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (C) DG75 cells were pretreated with the indicated concentrations of R406 and then treated without or with 5 mM H 2 O 2 for 15 min. Tyrosine-phosphorylated proteins were immunoprecipitated from cell lysates with antibodies against phosphotyrosine. Immune complexes (top) and whole-cell lysates (bottom) were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (D) MDA-MB-231-TR (TR) or MDA-MB-231-TRS cells induced to express Syk-EGFP (TRS) were treated without or with 5 mM H 2 O 2 for 15 min. Nucleolin was immunoprecipitated, and the resulting immune complexes were probed by Western blotting for phosphotyrosine (top) or NCL (bottom). (E) DG75 B cells (−) or DG75 cells stably expressing shRNA targeted against Syk (+) were treated without or with 5 mM H 2 O 2 for 15 min. Nucleolin was immunoprecipitated, and the resulting immune complexes were probed by Western blotting for phosphotyrosine (top) or NCL (bottom). (F) Syk-EGFP (Syk) or Syk-EGFP(K396R) (KD) was immunoprecipitated from the corresponding doxycycline-induced lines of MDA-MB-231 cells using GFP-nanotrap beads. The resulting immune complexes were incubated with buffer containing (+) or lacking (−) ATP. The immune complexes and whole-cell lysates were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (G) Nucleolin was immunoprecipitated from Tet-responsive MDA-MB-231 cells either uninduced (−) or induced (+) with doxycycline to express Syk-EGFP and either treated with 5 mM H 2 O 2 for 3 h or not treated. Immune complexes were examined for the presence of Bcl-x L mRNA by RT-PCR (top) and nucleolin by Western blotting (middle). The expression of Syk-EGFP was determined by Western blotting of whole-cell lysates with antibodies against Syk (bottom). (H) The relative amount of Bcl-x L mRNA associated with nucleolin, analyzed as described in the legend to panel G, was quantified. The data represent means ± SEMs from three replicate experiments. The level of mRNA bound to nucleolin in Syk-EGFP-expressing cells not treated with H 2 O 2 was set equal to a value of 1.0.

Techniques Used: Binding Assay, Multiple Displacement Amplification, Immunoprecipitation, SDS Page, Western Blot, Stable Transfection, Expressing, shRNA, Incubation, Reverse Transcription Polymerase Chain Reaction

7) Product Images from "Map7/7D1 and Dvl form a feedback loop that facilitates microtubule remodeling and Wnt5a signaling"

Article Title: Map7/7D1 and Dvl form a feedback loop that facilitates microtubule remodeling and Wnt5a signaling

Journal: EMBO Reports

doi: 10.15252/embr.201745471

Map7/7D1 bind to Dvl, a key mediator of Wnt5a signaling Lysates from HeLa cells transfected with control vector or pcDNA3.1‐hMAP7‐V5His 6 were immunoprecipitated with an anti‐V5 antibody, and the immunoprecipitates were probed with anti‐Dvl2 and anti‐V5 antibodies. Lysates from HeLa cells were subjected to immunoprecipitation with control IgG or an anti‐Dvl2 antibody and analyzed by immunoblotting with an anti‐Map7/7D1 or anti‐Dvl2 antibody. Lysates from HeLa cells co‐expressing various deletion derivatives of hMap7‐V5His 6 with EGFP‐mDvl2 were immunoprecipitated with an anti‐GFP antibody, and the immunoprecipitates were probed with anti‐V5 and anti‐GFP antibodies. Lysates from HeLa cells co‐expressing various derivatives of EGFP‐mDvl2 with hMap7‐V5His 6 were immunoprecipitated with an anti‐V5 antibody, and the immunoprecipitates were probed with anti‐GFP and anti‐V5 antibodies. MBP‐hMap7 1‐265ΔCC1 (30 pmol) conjugated to amylose resin was incubated with purified GST, GST‐hDvl1 DIX , or GST‐hDvl1 DEP (30 pmol of each), and the bound proteins were analyzed by immunoblotting with anti‐GST and anti‐MBP antibodies. The positions of GST, GST‐hDvl1 DIX , and GST‐hDvl1 DEP were revealed by loading 1.5 pmol (5%) of each purified protein. Depletion efficiency of si WNT5A or si DVLs . Lysates derived from the indicated cells were probed with anti‐Dvl2 and anti‐Wnt5a antibodies. The blot was reprobed for γ‐tubulin as a loading control (left). Effects of si WNT5A or si DVLs on the protein levels of Map7 and Map7D1 were also analyzed (right). The blot was reprobed for γ‐tubulin as a loading control. Graph shows the relative mRNA levels of MAP7 and MAP7D1 in the indicated cells 72 h after siRNA transfection. Expression levels of MAP7 and MAP7D1 transcripts were quantified by normalization to the GAPDH expression. Data are from three independent experiments and represent average ± SD. Source data are available online for this figure.
Figure Legend Snippet: Map7/7D1 bind to Dvl, a key mediator of Wnt5a signaling Lysates from HeLa cells transfected with control vector or pcDNA3.1‐hMAP7‐V5His 6 were immunoprecipitated with an anti‐V5 antibody, and the immunoprecipitates were probed with anti‐Dvl2 and anti‐V5 antibodies. Lysates from HeLa cells were subjected to immunoprecipitation with control IgG or an anti‐Dvl2 antibody and analyzed by immunoblotting with an anti‐Map7/7D1 or anti‐Dvl2 antibody. Lysates from HeLa cells co‐expressing various deletion derivatives of hMap7‐V5His 6 with EGFP‐mDvl2 were immunoprecipitated with an anti‐GFP antibody, and the immunoprecipitates were probed with anti‐V5 and anti‐GFP antibodies. Lysates from HeLa cells co‐expressing various derivatives of EGFP‐mDvl2 with hMap7‐V5His 6 were immunoprecipitated with an anti‐V5 antibody, and the immunoprecipitates were probed with anti‐GFP and anti‐V5 antibodies. MBP‐hMap7 1‐265ΔCC1 (30 pmol) conjugated to amylose resin was incubated with purified GST, GST‐hDvl1 DIX , or GST‐hDvl1 DEP (30 pmol of each), and the bound proteins were analyzed by immunoblotting with anti‐GST and anti‐MBP antibodies. The positions of GST, GST‐hDvl1 DIX , and GST‐hDvl1 DEP were revealed by loading 1.5 pmol (5%) of each purified protein. Depletion efficiency of si WNT5A or si DVLs . Lysates derived from the indicated cells were probed with anti‐Dvl2 and anti‐Wnt5a antibodies. The blot was reprobed for γ‐tubulin as a loading control (left). Effects of si WNT5A or si DVLs on the protein levels of Map7 and Map7D1 were also analyzed (right). The blot was reprobed for γ‐tubulin as a loading control. Graph shows the relative mRNA levels of MAP7 and MAP7D1 in the indicated cells 72 h after siRNA transfection. Expression levels of MAP7 and MAP7D1 transcripts were quantified by normalization to the GAPDH expression. Data are from three independent experiments and represent average ± SD. Source data are available online for this figure.

Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Expressing, Incubation, Purification, Derivative Assay

8) Product Images from "The LTB4–BLT1 axis regulates the polarized trafficking of chemoattractant GPCRs during neutrophil chemotaxis"

Article Title: The LTB4–BLT1 axis regulates the polarized trafficking of chemoattractant GPCRs during neutrophil chemotaxis

Journal: Journal of Cell Science

doi: 10.1242/jcs.217422

C5aR1 internalizes in a clathrin-independent manner and is trafficked to lysosomes. (A) Graph depicting the extent of internalization of C5aR1–EGFP in dPLBs expressing C5aR1–EGFP treated with the indicated doses of C5a or fMLF (10 nM) for 10 min and fixed. Quantification of the extent of C5aR1 internalization is shown relative to untreated control (UT) with n =2 independent experiments and represented as mean±s.d. (B) Representative confocal images of vehicle [0.2% (v/v) DMSO] or inhibitor (denoted with an ‘i’ after the protein targeted) (5 µM PitStop2; 100 nM Lat A; 100 µM ZCL278)-treated C5aR1–EGFP dPLBs (30 min) stimulated with C5a (100 ng/ml) for 15 min, and fixed and co-stained with phalloidin (red) and DAPI (blue). Scale bar: 4 µm. Images are representative of three independent experiments. (C) Graph depicting the extent of internalization of C5aR1 in vehicle or inhibitor-treated C5aR1-EGFP dPLBs (as in panel B and with 5 µM MitMAB), represented as the percentage with respect to vehicle control with n =3 independent experiments. Findings are represented as mean±s.e.m. **** P ≤0.0001 (one-way ANOVA with Dunnett's multiple comparisons test). (D,E) Representative multiple intensity projections of Z -stacks for C5aR1–EGFP (green) dPLBs pre-treated with Alexa Fluor 555-conjugated CTxB (1 µg/ml; red; D) or Alexa Fluor 568-conjugated transferrin (5 µg/ml; red; E) for 5 min, and stimulated with C5a (100 ng/ml) for 5 min, fixed and stained with DAPI (blue). Magenta arrowheads represent colocalization on the plasma membrane. Scale bar: 2 µm. Images are representative of two independent experiments. (F) Representative multiple intensity projections of Z-stacks of dPLBs expressing FPR1–EGFP and C5aR1–EGFP treated with fNLFNYK (10 nM) or C5a (100 ng/ml) for the indicated time points, and fixed and co-stained for EEA1 (red; 5 min post stimulation), Rab11 (red; 15 min post stimulation) and LysoTracker (red; 30 min post stimulation). Magenta arrowheads represents colocalization. Scale bar: 10 µm. Images are representative of two independent experiments.
Figure Legend Snippet: C5aR1 internalizes in a clathrin-independent manner and is trafficked to lysosomes. (A) Graph depicting the extent of internalization of C5aR1–EGFP in dPLBs expressing C5aR1–EGFP treated with the indicated doses of C5a or fMLF (10 nM) for 10 min and fixed. Quantification of the extent of C5aR1 internalization is shown relative to untreated control (UT) with n =2 independent experiments and represented as mean±s.d. (B) Representative confocal images of vehicle [0.2% (v/v) DMSO] or inhibitor (denoted with an ‘i’ after the protein targeted) (5 µM PitStop2; 100 nM Lat A; 100 µM ZCL278)-treated C5aR1–EGFP dPLBs (30 min) stimulated with C5a (100 ng/ml) for 15 min, and fixed and co-stained with phalloidin (red) and DAPI (blue). Scale bar: 4 µm. Images are representative of three independent experiments. (C) Graph depicting the extent of internalization of C5aR1 in vehicle or inhibitor-treated C5aR1-EGFP dPLBs (as in panel B and with 5 µM MitMAB), represented as the percentage with respect to vehicle control with n =3 independent experiments. Findings are represented as mean±s.e.m. **** P ≤0.0001 (one-way ANOVA with Dunnett's multiple comparisons test). (D,E) Representative multiple intensity projections of Z -stacks for C5aR1–EGFP (green) dPLBs pre-treated with Alexa Fluor 555-conjugated CTxB (1 µg/ml; red; D) or Alexa Fluor 568-conjugated transferrin (5 µg/ml; red; E) for 5 min, and stimulated with C5a (100 ng/ml) for 5 min, fixed and stained with DAPI (blue). Magenta arrowheads represent colocalization on the plasma membrane. Scale bar: 2 µm. Images are representative of two independent experiments. (F) Representative multiple intensity projections of Z-stacks of dPLBs expressing FPR1–EGFP and C5aR1–EGFP treated with fNLFNYK (10 nM) or C5a (100 ng/ml) for the indicated time points, and fixed and co-stained for EEA1 (red; 5 min post stimulation), Rab11 (red; 15 min post stimulation) and LysoTracker (red; 30 min post stimulation). Magenta arrowheads represents colocalization. Scale bar: 10 µm. Images are representative of two independent experiments.

Techniques Used: Expressing, Staining

9) Product Images from "Adaptor Protein Crk Is Required for Ephrin-B1-induced Membrane Ruffling and Focal Complex Assembly of Human Aortic Endothelial Cells V⃞"

Article Title: Adaptor Protein Crk Is Required for Ephrin-B1-induced Membrane Ruffling and Focal Complex Assembly of Human Aortic Endothelial Cells V⃞

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E02-04-0181

CrkI is translocated to nascent focal complexes upon ephrin-B1 stimulation and is involved in the development of focal adhesion. HAECs cultured on a collagen-coated glass-base dish were transfected with pCA-DsRed-CrkI, starved for 8 h, and stimulated with preclustered 1 μg/ml soluble ephrin-B1/Fc. EGFP-actin and DsRed-CrkI were imaged on an IX-70 inverted microscope ( Olympus ). A series of DsRed and EGFP images were collected by MetaMorph 4.6 software. Before, before the stimulation; 15, 40, and 90 min, time for stimulation. Bar, 10 μm. (A) Arrowheads indicate typical accumulation of DsRed-CrkI to nascent focal complexes (top). The GFP image of the same cell transfected with pEGFP-actin was merged with the DsRed-CrkI image (bottom). Arrows indicate an example of focal adhesions connecting actin stress fiber (also see Video 2A2). (B) The boxed region in the lower panel of A was enlarged. An arrow indicates a focal complex detached from the substratum and floating in membrane ruffles. The arrowhead indicates the development of a focal complex to a focal adhesion (also see Video 2B).
Figure Legend Snippet: CrkI is translocated to nascent focal complexes upon ephrin-B1 stimulation and is involved in the development of focal adhesion. HAECs cultured on a collagen-coated glass-base dish were transfected with pCA-DsRed-CrkI, starved for 8 h, and stimulated with preclustered 1 μg/ml soluble ephrin-B1/Fc. EGFP-actin and DsRed-CrkI were imaged on an IX-70 inverted microscope ( Olympus ). A series of DsRed and EGFP images were collected by MetaMorph 4.6 software. Before, before the stimulation; 15, 40, and 90 min, time for stimulation. Bar, 10 μm. (A) Arrowheads indicate typical accumulation of DsRed-CrkI to nascent focal complexes (top). The GFP image of the same cell transfected with pEGFP-actin was merged with the DsRed-CrkI image (bottom). Arrows indicate an example of focal adhesions connecting actin stress fiber (also see Video 2A2). (B) The boxed region in the lower panel of A was enlarged. An arrow indicates a focal complex detached from the substratum and floating in membrane ruffles. The arrowhead indicates the development of a focal complex to a focal adhesion (also see Video 2B).

Techniques Used: Cell Culture, Transfection, Inverted Microscopy, Software

10) Product Images from "Synaptic Regulation of Microtubule Dynamics in Dendritic Spines by Calcium, F-Actin, and Drebrin"

Article Title: Synaptic Regulation of Microtubule Dynamics in Dendritic Spines by Calcium, F-Actin, and Drebrin

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.0661-13.2013

Drebrin A knockdown inhibits MT entry into spines. A , Representative drebrin immunofluorescent images from neurons transfected with EGFP, EB3-tdTomato (data not shown), and either drebrin siRNA (top) or nontargeting siRNA (bottom). B , Representative images of spines from neurons transfected with EGFP (green), EB3-tdTomato (red), and either drebrin siRNA (top, Db siRNA) or nontargeting siRNA (bottom, nt siRNA). Scale bar, 1 μm. C , Comparison of drebrin intensity in neurons transfected with EGFP, EB3-tdTomato, and either nontargeting siRNA or drebrin-targeting siRNAs [ t test, **** p
Figure Legend Snippet: Drebrin A knockdown inhibits MT entry into spines. A , Representative drebrin immunofluorescent images from neurons transfected with EGFP, EB3-tdTomato (data not shown), and either drebrin siRNA (top) or nontargeting siRNA (bottom). B , Representative images of spines from neurons transfected with EGFP (green), EB3-tdTomato (red), and either drebrin siRNA (top, Db siRNA) or nontargeting siRNA (bottom, nt siRNA). Scale bar, 1 μm. C , Comparison of drebrin intensity in neurons transfected with EGFP, EB3-tdTomato, and either nontargeting siRNA or drebrin-targeting siRNAs [ t test, **** p

Techniques Used: Transfection

MTs polymerize into spines from local sites. A , Maximum projection of EB3-tdTomato comets across a period of 36 s. Images were acquired at 1.2 s intervals (2.4 s interval used for projection). White lines trace the neuron's shape in the TIRF plane (obtained with EGFP volume marker, shown in inset). Red lines track comets that entered a spine; blue and yellow lines track comets that remained in the dendrite shaft (blue, anterograde; yellow, retrograde movement). Lines were obtained by tracking comets at each frame (1.2 s intervals). Scale bar, 1 μm. B , C , Distribution of distances traversed ( B ) and numbers of spines passed ( C ) by EB3 comets that entered spines (red bars; n = 141 comets, 72 spines, 8 neurons from 2 preparations) versus comets that did not enter spines (black bars; n = 127 comets, 67 spines, 8 neurons from 2 preparations). Distributions for spine-entering EB3 comets were well described by a two-phase exponential decay function (red lines; R 2 = 0.9898 for distance traversed, 0.9942 for number of spines passed). Distributions for EB3 comets that did not enter spines were well described by a biexponential (rise and decay) function (black lines; R 2 = 0.8879 for distance traversed, 0.7346 for number of spines passed). Insets compare mean ± SEM of each distribution. t test, *** p
Figure Legend Snippet: MTs polymerize into spines from local sites. A , Maximum projection of EB3-tdTomato comets across a period of 36 s. Images were acquired at 1.2 s intervals (2.4 s interval used for projection). White lines trace the neuron's shape in the TIRF plane (obtained with EGFP volume marker, shown in inset). Red lines track comets that entered a spine; blue and yellow lines track comets that remained in the dendrite shaft (blue, anterograde; yellow, retrograde movement). Lines were obtained by tracking comets at each frame (1.2 s intervals). Scale bar, 1 μm. B , C , Distribution of distances traversed ( B ) and numbers of spines passed ( C ) by EB3 comets that entered spines (red bars; n = 141 comets, 72 spines, 8 neurons from 2 preparations) versus comets that did not enter spines (black bars; n = 127 comets, 67 spines, 8 neurons from 2 preparations). Distributions for spine-entering EB3 comets were well described by a two-phase exponential decay function (red lines; R 2 = 0.9898 for distance traversed, 0.9942 for number of spines passed). Distributions for EB3 comets that did not enter spines were well described by a biexponential (rise and decay) function (black lines; R 2 = 0.8879 for distance traversed, 0.7346 for number of spines passed). Insets compare mean ± SEM of each distribution. t test, *** p

Techniques Used: Marker

MT invasions are calcium-dependent. A , Sequential frames from a neuron transfected with DsRed2 and EB3-EGFP taken before calcium chelation (Baseline) showing EB3 puncta moving into spines. Scale bar, 1 μm. B , Sequential frames from the same neuron as in A taken before (left) and after (right) calcium chelation with BAPTA-AM. White lines indicate EB3 puncta moving in the dendritic shaft. Arrowheads indicate an EB3 punctum in a spine (the larger spine from A ). Scale bar, 2 μm. C , Representative kymographs from spines invaded by EB3 puncta before (left) and after (right) incubation with BAPTA-AM. Same neuron as in A . D , Representative kymographs from 10 μm segments of dendrite shaft before (left) and after (right) incubation with BAPTA-AM. Same neuron as in A and B . E , Comparison of MT spine invasion frequency during baseline (Bsln) and after incubation with BAPTA-AM ( n = 88 spines, 4 neurons, 3 preparations). Only spines invaded some time during imaging (before or after BAPTA-AM) are depicted (paired t test, *** p
Figure Legend Snippet: MT invasions are calcium-dependent. A , Sequential frames from a neuron transfected with DsRed2 and EB3-EGFP taken before calcium chelation (Baseline) showing EB3 puncta moving into spines. Scale bar, 1 μm. B , Sequential frames from the same neuron as in A taken before (left) and after (right) calcium chelation with BAPTA-AM. White lines indicate EB3 puncta moving in the dendritic shaft. Arrowheads indicate an EB3 punctum in a spine (the larger spine from A ). Scale bar, 2 μm. C , Representative kymographs from spines invaded by EB3 puncta before (left) and after (right) incubation with BAPTA-AM. Same neuron as in A . D , Representative kymographs from 10 μm segments of dendrite shaft before (left) and after (right) incubation with BAPTA-AM. Same neuron as in A and B . E , Comparison of MT spine invasion frequency during baseline (Bsln) and after incubation with BAPTA-AM ( n = 88 spines, 4 neurons, 3 preparations). Only spines invaded some time during imaging (before or after BAPTA-AM) are depicted (paired t test, *** p

Techniques Used: Transfection, Incubation, Imaging

11) Product Images from "Asymmetrically dividing Drosophila neuroblasts utilize two spatially and temporally independent cytokinesis pathways"

Article Title: Asymmetrically dividing Drosophila neuroblasts utilize two spatially and temporally independent cytokinesis pathways

Journal: Nature Communications

doi: 10.1038/ncomms7551

Survivin’s localization is independent of the polarity pathway. ( a ) Schematic representation of the genetic spindle rotation experiment to uncouple the orientation of the mitotic spindle in relation to the neuroblast intrinsic polarity axis (apical, blue; basal, red). ( b ) Image sequence of a representative mud 4 mutant neuroblast expressing Survivin::GFP (top row) and Sqh::mCherry (Myo; second row), dividing symmetrically and forming a polar lobe (06:30; orange arrowhead). Blue arrowheads highlight Survivin localization at the spindle-induced furrow. ( c ) Quantification of Survivin appearance at polarity-dependent cleavage furrow (polar lobe). Number of cells scored is highlighted in bars. ( d ) Survivin was ectopically localized at the apical neuroblast cortex (green); intensity measurements were performed along the dashed yellow line from apical to basal. ( e ) Third instar larval neuroblast expressing ALD-Survivin::EGFP (top row; green) and Sqh::mCherry (bottom row; white). Intensity measurements (green, ALD-Survivin::EGFP; grey, sqh::mCherry) are shown below. ( f ) Quantification of ectopic furrowing and Myosin distribution. Number of cells scored is highlighted in bars. Ap, apical; Ba, basal. Time in min:s; scale bar, 5 μm.
Figure Legend Snippet: Survivin’s localization is independent of the polarity pathway. ( a ) Schematic representation of the genetic spindle rotation experiment to uncouple the orientation of the mitotic spindle in relation to the neuroblast intrinsic polarity axis (apical, blue; basal, red). ( b ) Image sequence of a representative mud 4 mutant neuroblast expressing Survivin::GFP (top row) and Sqh::mCherry (Myo; second row), dividing symmetrically and forming a polar lobe (06:30; orange arrowhead). Blue arrowheads highlight Survivin localization at the spindle-induced furrow. ( c ) Quantification of Survivin appearance at polarity-dependent cleavage furrow (polar lobe). Number of cells scored is highlighted in bars. ( d ) Survivin was ectopically localized at the apical neuroblast cortex (green); intensity measurements were performed along the dashed yellow line from apical to basal. ( e ) Third instar larval neuroblast expressing ALD-Survivin::EGFP (top row; green) and Sqh::mCherry (bottom row; white). Intensity measurements (green, ALD-Survivin::EGFP; grey, sqh::mCherry) are shown below. ( f ) Quantification of ectopic furrowing and Myosin distribution. Number of cells scored is highlighted in bars. Ap, apical; Ba, basal. Time in min:s; scale bar, 5 μm.

Techniques Used: Sequencing, Mutagenesis, Expressing

12) Product Images from "A double helical motif in OCIAD2 is essential for its localization, interactions and STAT3 activation"

Article Title: A double helical motif in OCIAD2 is essential for its localization, interactions and STAT3 activation

Journal: Scientific Reports

doi: 10.1038/s41598-018-25667-3

OCIAD2 localizes to endosomes and mitochondria. ( A – G ) Confocal images and co-localization plots of OCIAD2_FL-EGFP transfected HEK293 cells showing localization of OCIAD2 with various markers detected by immunofluorescence staining (red). ( A ) endogenous OCIAD2; ( B ) cytochrome oxidase subunit IV (CoxIV); ( C ) early endosome, Rab5; ( D ) late endosome, Rab7; ( E ) recycling endosome, Rab11; ( F ) lysosome, Lysotracker; ( G ) Golgi, GM130. Insets (A, B and C) show magnified view of the boxed region. Scale bars = 5 µm in C, D, E and 10 µm in A, B, F and G.
Figure Legend Snippet: OCIAD2 localizes to endosomes and mitochondria. ( A – G ) Confocal images and co-localization plots of OCIAD2_FL-EGFP transfected HEK293 cells showing localization of OCIAD2 with various markers detected by immunofluorescence staining (red). ( A ) endogenous OCIAD2; ( B ) cytochrome oxidase subunit IV (CoxIV); ( C ) early endosome, Rab5; ( D ) late endosome, Rab7; ( E ) recycling endosome, Rab11; ( F ) lysosome, Lysotracker; ( G ) Golgi, GM130. Insets (A, B and C) show magnified view of the boxed region. Scale bars = 5 µm in C, D, E and 10 µm in A, B, F and G.

Techniques Used: Transfection, Immunofluorescence, Staining

The double helical motif of OCIAD2 interacts with STAT3 and promotes its activation. ( A ) Western Blotting for detection of STAT3 and pSTAT3 (Y705) levels in HEK293 cells transfected with various OCIAD2 constructs as indicated. UT: untransfected, V: empty vector transfected; FL: OCIAD2_FL-EGFP; N: OCIAD2_N-EGFP; Hph: OCIAD2_Hph-dsRed and C: OCIAD2_C-dsRed. Graph represents pSTAT3/STAT3 ratio obtained upon overexpression of various OCIAD2 constructs relative to control (shown as a dotted line). n = 4 ( B ) Validation of transfection of FLAG-STAT3 construct into HEK293 cells by Western Blotting. ( C ) FLAG-STAT3 transfected HEK293 cell lysate subjected to immunoprecipitation with anti-FLAG antibody and assessed for interaction with OCIAD1 and OCIAD2. ( D – G ) HEK293 cell lysates expressing various OCIAD2 constructs (FL, N, Hph and C) were subjected to immunoprecipitation (IP) followed by immunoblotting (IB) to probe for interaction with STAT3. Protein size markers (in kDa) are indicated to the left. Data for STAT3/pSTAT3 Western Blotting are representative of three independent experiments and * p
Figure Legend Snippet: The double helical motif of OCIAD2 interacts with STAT3 and promotes its activation. ( A ) Western Blotting for detection of STAT3 and pSTAT3 (Y705) levels in HEK293 cells transfected with various OCIAD2 constructs as indicated. UT: untransfected, V: empty vector transfected; FL: OCIAD2_FL-EGFP; N: OCIAD2_N-EGFP; Hph: OCIAD2_Hph-dsRed and C: OCIAD2_C-dsRed. Graph represents pSTAT3/STAT3 ratio obtained upon overexpression of various OCIAD2 constructs relative to control (shown as a dotted line). n = 4 ( B ) Validation of transfection of FLAG-STAT3 construct into HEK293 cells by Western Blotting. ( C ) FLAG-STAT3 transfected HEK293 cell lysate subjected to immunoprecipitation with anti-FLAG antibody and assessed for interaction with OCIAD1 and OCIAD2. ( D – G ) HEK293 cell lysates expressing various OCIAD2 constructs (FL, N, Hph and C) were subjected to immunoprecipitation (IP) followed by immunoblotting (IB) to probe for interaction with STAT3. Protein size markers (in kDa) are indicated to the left. Data for STAT3/pSTAT3 Western Blotting are representative of three independent experiments and * p

Techniques Used: Activation Assay, Western Blot, Transfection, Construct, Plasmid Preparation, Over Expression, Immunoprecipitation, Expressing

The double helical motif of OCIAD2 interacts with STAT3 and promotes its activation. ( A ) Western Blotting for detection of STAT3 and pSTAT3 (Y705) levels in HEK293 cells transfected with various OCIAD2 constructs as indicated. UT: untransfected, V: empty vector transfected; FL: OCIAD2_FL-EGFP; N: OCIAD2_N-EGFP; Hph: OCIAD2_Hph-dsRed and C: OCIAD2_C-dsRed. Graph represents pSTAT3/STAT3 ratio obtained upon overexpression of various OCIAD2 constructs relative to control (shown as a dotted line). n = 4 ( B ) Validation of transfection of FLAG-STAT3 construct into HEK293 cells by Western Blotting. ( C ) FLAG-STAT3 transfected HEK293 cell lysate subjected to immunoprecipitation with anti-FLAG antibody and assessed for interaction with OCIAD1 and OCIAD2. ( D – G ) HEK293 cell lysates expressing various OCIAD2 constructs (FL, N, Hph and C) were subjected to immunoprecipitation (IP) followed by immunoblotting (IB) to probe for interaction with STAT3. Protein size markers (in kDa) are indicated to the left. Data for STAT3/pSTAT3 Western Blotting are representative of three independent experiments and * p
Figure Legend Snippet: The double helical motif of OCIAD2 interacts with STAT3 and promotes its activation. ( A ) Western Blotting for detection of STAT3 and pSTAT3 (Y705) levels in HEK293 cells transfected with various OCIAD2 constructs as indicated. UT: untransfected, V: empty vector transfected; FL: OCIAD2_FL-EGFP; N: OCIAD2_N-EGFP; Hph: OCIAD2_Hph-dsRed and C: OCIAD2_C-dsRed. Graph represents pSTAT3/STAT3 ratio obtained upon overexpression of various OCIAD2 constructs relative to control (shown as a dotted line). n = 4 ( B ) Validation of transfection of FLAG-STAT3 construct into HEK293 cells by Western Blotting. ( C ) FLAG-STAT3 transfected HEK293 cell lysate subjected to immunoprecipitation with anti-FLAG antibody and assessed for interaction with OCIAD1 and OCIAD2. ( D – G ) HEK293 cell lysates expressing various OCIAD2 constructs (FL, N, Hph and C) were subjected to immunoprecipitation (IP) followed by immunoblotting (IB) to probe for interaction with STAT3. Protein size markers (in kDa) are indicated to the left. Data for STAT3/pSTAT3 Western Blotting are representative of three independent experiments and * p

Techniques Used: Activation Assay, Western Blot, Transfection, Construct, Plasmid Preparation, Over Expression, Immunoprecipitation, Expressing

13) Product Images from "Dynamic Smad-mediated BMP signaling revealed through transgenic zebrafish"

Article Title: Dynamic Smad-mediated BMP signaling revealed through transgenic zebrafish

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

doi: 10.1002/dvdy.22567

BRE:eGFP in adult fish. (A) eGFP expression viewed in whole fish. (B) Dissected kidney showing eGFP expression (C) Higher magnification of proximal and (C) distal regions (white brackets).
Figure Legend Snippet: BRE:eGFP in adult fish. (A) eGFP expression viewed in whole fish. (B) Dissected kidney showing eGFP expression (C) Higher magnification of proximal and (C) distal regions (white brackets).

Techniques Used: Fluorescence In Situ Hybridization, Expressing

BRE:eGFP expression in adult dorsal Müller glia. (A) Central transverse cryosection of an adult BRE:eGFP eye. Note that the lens was not imaged as part of the montage. Müller glia (mg); optic nerve head (onh). Region showing eGFP-positive
Figure Legend Snippet: BRE:eGFP expression in adult dorsal Müller glia. (A) Central transverse cryosection of an adult BRE:eGFP eye. Note that the lens was not imaged as part of the montage. Müller glia (mg); optic nerve head (onh). Region showing eGFP-positive

Techniques Used: Expressing

Adult ocular BRE:eGFP expression revealed in cryosections. (A) BRE:eGFP expression was obvious in the lens and dorsal ciliary margin, but not in the central retina (r). (B) Transmitted light image of A. (C) BRE:eGFP expression in the ciliary marginal
Figure Legend Snippet: Adult ocular BRE:eGFP expression revealed in cryosections. (A) BRE:eGFP expression was obvious in the lens and dorsal ciliary margin, but not in the central retina (r). (B) Transmitted light image of A. (C) BRE:eGFP expression in the ciliary marginal

Techniques Used: Expressing

BRE:eGFP expression in adult tissues revealed through cryosections. (A) BRE:eGFP expression in the atrium and ventricle of the heart (dotted line) and in the gill filaments. (B) BRE:eGFP expression in the vasculature of the brain. Higher magnification
Figure Legend Snippet: BRE:eGFP expression in adult tissues revealed through cryosections. (A) BRE:eGFP expression in the atrium and ventricle of the heart (dotted line) and in the gill filaments. (B) BRE:eGFP expression in the vasculature of the brain. Higher magnification

Techniques Used: Expressing

Expression of eGFP, d2GFP or KO2-PEST driven by the BRE promoter lines during larval development. BRE:eGFP was expressed in the dorsal developing eye (e), somites (s), tail (t), heart (h) and pineal (p) during the first 5 days post fertilization (A, C,
Figure Legend Snippet: Expression of eGFP, d2GFP or KO2-PEST driven by the BRE promoter lines during larval development. BRE:eGFP was expressed in the dorsal developing eye (e), somites (s), tail (t), heart (h) and pineal (p) during the first 5 days post fertilization (A, C,

Techniques Used: Expressing

Diverse tissue expression of eGFP or d2GFP in BRE transgenic lines. (A) Expression of eGFP in cranial vasculature of 2 dpf BRE:eGFP larvae. (B) Similar expression of d2GFP in 2 dpf cranial vasculature of BRE:d2GFP larvae. (C) Expression of d2GFP in intersomitic
Figure Legend Snippet: Diverse tissue expression of eGFP or d2GFP in BRE transgenic lines. (A) Expression of eGFP in cranial vasculature of 2 dpf BRE:eGFP larvae. (B) Similar expression of d2GFP in 2 dpf cranial vasculature of BRE:d2GFP larvae. (C) Expression of d2GFP in intersomitic

Techniques Used: Expressing, Transgenic Assay

Expression of eGFP in regenerating fins of BRE:eGFP adult fish. (A–F) BRE:eGFP expression in adult caudal fins before and during regeneration. Times post-amputation are indicated above each image. Note that BRE:eGFP expression is increased in
Figure Legend Snippet: Expression of eGFP in regenerating fins of BRE:eGFP adult fish. (A–F) BRE:eGFP expression in adult caudal fins before and during regeneration. Times post-amputation are indicated above each image. Note that BRE:eGFP expression is increased in

Techniques Used: Expressing, Fluorescence In Situ Hybridization

14) Product Images from "Characterization of the Oligomeric Structure of the Ca2+-activated Cl− Channel Ano1/TMEM16A *"

Article Title: Characterization of the Oligomeric Structure of the Ca2+-activated Cl− Channel Ano1/TMEM16A *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.174847

Ano1-eGFP and Ano1-mCherry co-immunoprecipitate. A , HEK293 cells were transfected with Lipofectamine alone, or with Ano1-mCherry and Ano1-eGFP, A2B-R-eGFP, and eGFP. Input represents 5% of total protein used in co-immunoprecipitation. B , Western blot
Figure Legend Snippet: Ano1-eGFP and Ano1-mCherry co-immunoprecipitate. A , HEK293 cells were transfected with Lipofectamine alone, or with Ano1-mCherry and Ano1-eGFP, A2B-R-eGFP, and eGFP. Input represents 5% of total protein used in co-immunoprecipitation. B , Western blot

Techniques Used: Transfection, Immunoprecipitation, Western Blot

eGFP linked to mCherry with 5 glycines (eGFP-5G-mCherry) undergoes near-maximal FRET. A , typical confocal micrographs of eGFP-5G-mCherry ( green , donor; red , acceptor) before and after photobleaching of the acceptor fluorophore. B , graph showing typical
Figure Legend Snippet: eGFP linked to mCherry with 5 glycines (eGFP-5G-mCherry) undergoes near-maximal FRET. A , typical confocal micrographs of eGFP-5G-mCherry ( green , donor; red , acceptor) before and after photobleaching of the acceptor fluorophore. B , graph showing typical

Techniques Used:

Ano1-eGFP and Ano1-mCherry FRET are not affected by changes in intracellular Ca 2+ . A , typical Fura-2 emission ratio observed following exposure to 2 μ m thapsigargin ( n = 10). B , mean FRET (%E) from Ano1 in the plasma membrane of HEK293 cells after
Figure Legend Snippet: Ano1-eGFP and Ano1-mCherry FRET are not affected by changes in intracellular Ca 2+ . A , typical Fura-2 emission ratio observed following exposure to 2 μ m thapsigargin ( n = 10). B , mean FRET (%E) from Ano1 in the plasma membrane of HEK293 cells after

Techniques Used:

Ano1-eGFP and Ano1-mCherry undergo specific FRET in the plasma membrane. A , typical confocal micrographs of Ano1-eGFP ( green , donor) and Ano1-mCherry ( red , acceptor) before and after photobleaching of Ano1-mCherry. B , graph showing typical acceptor photobleaching
Figure Legend Snippet: Ano1-eGFP and Ano1-mCherry undergo specific FRET in the plasma membrane. A , typical confocal micrographs of Ano1-eGFP ( green , donor) and Ano1-mCherry ( red , acceptor) before and after photobleaching of Ano1-mCherry. B , graph showing typical acceptor photobleaching

Techniques Used:

Determination of Ano1 subunit stoichiometry by chemical cross-linking and non-denaturing gel electrophoresis. A , determination of possible oligomeric structures of Ano1-eGFP using chemical cross-linking. Cell lysates were treated with increasing concentrations
Figure Legend Snippet: Determination of Ano1 subunit stoichiometry by chemical cross-linking and non-denaturing gel electrophoresis. A , determination of possible oligomeric structures of Ano1-eGFP using chemical cross-linking. Cell lysates were treated with increasing concentrations

Techniques Used: Nucleic Acid Electrophoresis

Ano1-eGFP and Ano1-mCherry FRET are not dependent on an intact actin cytoskeleton. Left , confocal micrographs of Ano1-mCherry in HEK293 cells ( red ) before and after disruption of actin cytoskeleton (phalloidin/green) with 1 μ m cytochalasin D for
Figure Legend Snippet: Ano1-eGFP and Ano1-mCherry FRET are not dependent on an intact actin cytoskeleton. Left , confocal micrographs of Ano1-mCherry in HEK293 cells ( red ) before and after disruption of actin cytoskeleton (phalloidin/green) with 1 μ m cytochalasin D for

Techniques Used:

15) Product Images from "Large-Scale Identification and Characterization of Heterodera avenae Putative Effectors Suppressing or Inducing Cell Death in Nicotiana benthamiana"

Article Title: Large-Scale Identification and Characterization of Heterodera avenae Putative Effectors Suppressing or Inducing Cell Death in Nicotiana benthamiana

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2017.02062

Candidate Heterodera avenae effectors (example isotig18549) suppress cell death triggered by other candidate H. avenae effectors (example isotig12969) in Nicotiana benthamiana . (A) Assay of the suppression of isotig12969-triggered cell death in N. benthamiana by isotig18549. The results of the verification of gene expression of isotig18549 and isotig12969 by western blotting are shown below. (B) Necrosis index of isotig18549 and control eGFP followed by isotig12969. Each column shows the mean and standard deviation.
Figure Legend Snippet: Candidate Heterodera avenae effectors (example isotig18549) suppress cell death triggered by other candidate H. avenae effectors (example isotig12969) in Nicotiana benthamiana . (A) Assay of the suppression of isotig12969-triggered cell death in N. benthamiana by isotig18549. The results of the verification of gene expression of isotig18549 and isotig12969 by western blotting are shown below. (B) Necrosis index of isotig18549 and control eGFP followed by isotig12969. Each column shows the mean and standard deviation.

Techniques Used: Expressing, Western Blot, Standard Deviation

Symptoms of systemic transient expression of Heterodera avenae effectors in Nicotiana benthamiana . (A) Untreated wild plant. (B) Empty vector control. (C) eGFP control. (D) Severe necrosis with wilting and even withering (example isotig15576). (E) Moderate necrosis (example isotig19600). (F) Aggravation of PVX symptoms (example isotig15773). (G) No obvious difference compared to the eGFP control (example isotig14561). (H) Stunting indicated by a significant decrease in average plant height after infiltration with isotig18549, isotig13069, isotig18925, or isotig19369 compared to the eGFP control ( P
Figure Legend Snippet: Symptoms of systemic transient expression of Heterodera avenae effectors in Nicotiana benthamiana . (A) Untreated wild plant. (B) Empty vector control. (C) eGFP control. (D) Severe necrosis with wilting and even withering (example isotig15576). (E) Moderate necrosis (example isotig19600). (F) Aggravation of PVX symptoms (example isotig15773). (G) No obvious difference compared to the eGFP control (example isotig14561). (H) Stunting indicated by a significant decrease in average plant height after infiltration with isotig18549, isotig13069, isotig18925, or isotig19369 compared to the eGFP control ( P

Techniques Used: Expressing, Plasmid Preparation

RNA-silencing suppression assay of candidate Heterodera avenae effectors in Nicotiana benthamiana (example isotig18549). (A) Negative control: N. benthamiana leaves were infiltrated with a mixture of Agrobacterium tumefaciens cells containing the empty pGD vector and pGD-eGFP showing no green fluorescence. (B) Positive control: N. benthamiana leaves were infiltrated with a mixture of A. tumefaciens cells containing pGD-p19 and pGD-eGFP showing green fluorescence. (C) Example isotig18549: N. benthamiana leaves were infiltrated with a mixture of A. tumefaciens cells containing pGD-isotig18549 and pGD-eGFP showing no green fluorescence.
Figure Legend Snippet: RNA-silencing suppression assay of candidate Heterodera avenae effectors in Nicotiana benthamiana (example isotig18549). (A) Negative control: N. benthamiana leaves were infiltrated with a mixture of Agrobacterium tumefaciens cells containing the empty pGD vector and pGD-eGFP showing no green fluorescence. (B) Positive control: N. benthamiana leaves were infiltrated with a mixture of A. tumefaciens cells containing pGD-p19 and pGD-eGFP showing green fluorescence. (C) Example isotig18549: N. benthamiana leaves were infiltrated with a mixture of A. tumefaciens cells containing pGD-isotig18549 and pGD-eGFP showing no green fluorescence.

Techniques Used: Suppression Assay, Negative Control, Plasmid Preparation, Fluorescence, Positive Control

Effect of Heterodera avenae candidate effectors on Nicotiana benthamiana PCD. (A) Number and proportion of putative effector genes that induce PCD, suppress BAX-triggered cell death (BT-PCD) or have no effect on leaves of N. benthamiana . (B) Putative effectors that trigger cell death and chlorosis symptoms in N. benthamiana compared to eGFP as the negative control. (C) Suppression of BT-PCD in N. benthamiana by effectors (example isotig18549). N. benthamiana leaves were infiltrated with buffer or with Agrobacterium tumefaciens cells carrying isotig18549 or the negative control eGFP gene; infiltration was either performed alone or followed 24 h later by infiltration with A. tumefaciens cells carrying a mouse Bax gene. Western blotting confirmed the expression of BAX. (D) Necrosis indices of the infiltration spots of the example gene isotig18549 and control eGFP followed by Bax . Each column shows the mean and standard deviation. The columns with asterisks show a statistically significant reduction of the necrosis index of isotig18549 compared with that of eGFP ( P
Figure Legend Snippet: Effect of Heterodera avenae candidate effectors on Nicotiana benthamiana PCD. (A) Number and proportion of putative effector genes that induce PCD, suppress BAX-triggered cell death (BT-PCD) or have no effect on leaves of N. benthamiana . (B) Putative effectors that trigger cell death and chlorosis symptoms in N. benthamiana compared to eGFP as the negative control. (C) Suppression of BT-PCD in N. benthamiana by effectors (example isotig18549). N. benthamiana leaves were infiltrated with buffer or with Agrobacterium tumefaciens cells carrying isotig18549 or the negative control eGFP gene; infiltration was either performed alone or followed 24 h later by infiltration with A. tumefaciens cells carrying a mouse Bax gene. Western blotting confirmed the expression of BAX. (D) Necrosis indices of the infiltration spots of the example gene isotig18549 and control eGFP followed by Bax . Each column shows the mean and standard deviation. The columns with asterisks show a statistically significant reduction of the necrosis index of isotig18549 compared with that of eGFP ( P

Techniques Used: Negative Control, Western Blot, Expressing, Standard Deviation

Assay of the suppression of PTI (triggered by psojNIP) and ETI (triggered by Avr3a/R3a or Rbp-1/Gpa2) by Heterodera avenae candidate effectors in Nicotiana benthamiana . (A,C) Visualization of the phenotype of example isotig18549, which suppressed PTI triggered by psojNIP and ETI triggered by Avr3a/R3a. Western blotting confirmed the expression of psojNIP. (E) Visualization of the phenotypes of necrosis suppression (example isotig18549) and no suppression (example isotig15186) of ETI triggered by Rbp-1/Gpa2). N. benthamiana leaves were infiltrated with buffer or with Agrobacterium tumefaciens cells carrying the effector genes isotig18549 or isotig15186 or the negative control ( eGFP or empty vector PMD1) either alone or followed 24 h later by A. tumefaciens cells carrying the psojNIP, Avr3a / R3a or Rbp-1 / Gpa2 genes. (B,D,F) Necrosis indices of the infiltration spots of the 10 selected effector genes and controls ( eGFP or empty vector PMD1) followed by infiltration with vectors carrying the psojNIP , Avr3a / R3a or Rbp-1 / Gpa2 genes. Each column shows the mean and standard deviation. The columns with asterisks show a statistically significant reduction of the necrosis index compared with the control ( P
Figure Legend Snippet: Assay of the suppression of PTI (triggered by psojNIP) and ETI (triggered by Avr3a/R3a or Rbp-1/Gpa2) by Heterodera avenae candidate effectors in Nicotiana benthamiana . (A,C) Visualization of the phenotype of example isotig18549, which suppressed PTI triggered by psojNIP and ETI triggered by Avr3a/R3a. Western blotting confirmed the expression of psojNIP. (E) Visualization of the phenotypes of necrosis suppression (example isotig18549) and no suppression (example isotig15186) of ETI triggered by Rbp-1/Gpa2). N. benthamiana leaves were infiltrated with buffer or with Agrobacterium tumefaciens cells carrying the effector genes isotig18549 or isotig15186 or the negative control ( eGFP or empty vector PMD1) either alone or followed 24 h later by A. tumefaciens cells carrying the psojNIP, Avr3a / R3a or Rbp-1 / Gpa2 genes. (B,D,F) Necrosis indices of the infiltration spots of the 10 selected effector genes and controls ( eGFP or empty vector PMD1) followed by infiltration with vectors carrying the psojNIP , Avr3a / R3a or Rbp-1 / Gpa2 genes. Each column shows the mean and standard deviation. The columns with asterisks show a statistically significant reduction of the necrosis index compared with the control ( P

Techniques Used: Western Blot, Expressing, Negative Control, Plasmid Preparation, Standard Deviation

16) Product Images from "CHIP as a membrane-shuttling proteostasis sensor"

Article Title: CHIP as a membrane-shuttling proteostasis sensor

Journal: eLife

doi: 10.7554/eLife.29388

Cellular reorganization by chaperone-free CHIP. ( a ) Murine embryonic fibroblasts (MEF) lacking CHIP (CHIP K.O.) were engineered using CRISPR/Cas9 system. GAPDH was used as loading control. ( b ) HSF1 translocation into nucleus after 60 min at 43°C was analyzed by means of nuclei isolation as detailed in Materials and methods. Lamin B1 and GAPDH were used as markers of nuclei and cytosol, respectively. One representative out of three independent experiments is shown. ( c ) Phospholipase D inhibitors do not affect steady-state levels of transiently transfected EGFP-CHIP-K30A in MEFs as determined by western blotting. Samples were normalized to have the same protein concentration before loading on gel. One representative out of three independent experiments is shown. ( d ) Phospholipase D inhibitors do not affect membrane localization of transiently transfected farnesylated EGFP in MEFs. One representative out of three independent experiments is shown. Scale bar 20 μm. ( e ) Inhibition of type III phosphatidylinositol-4 kinases (PI4KIII) by wortmannin causes an acute undocking of EGFP-CHIP-K30A from cellular membranes (mean ± SD). ***p
Figure Legend Snippet: Cellular reorganization by chaperone-free CHIP. ( a ) Murine embryonic fibroblasts (MEF) lacking CHIP (CHIP K.O.) were engineered using CRISPR/Cas9 system. GAPDH was used as loading control. ( b ) HSF1 translocation into nucleus after 60 min at 43°C was analyzed by means of nuclei isolation as detailed in Materials and methods. Lamin B1 and GAPDH were used as markers of nuclei and cytosol, respectively. One representative out of three independent experiments is shown. ( c ) Phospholipase D inhibitors do not affect steady-state levels of transiently transfected EGFP-CHIP-K30A in MEFs as determined by western blotting. Samples were normalized to have the same protein concentration before loading on gel. One representative out of three independent experiments is shown. ( d ) Phospholipase D inhibitors do not affect membrane localization of transiently transfected farnesylated EGFP in MEFs. One representative out of three independent experiments is shown. Scale bar 20 μm. ( e ) Inhibition of type III phosphatidylinositol-4 kinases (PI4KIII) by wortmannin causes an acute undocking of EGFP-CHIP-K30A from cellular membranes (mean ± SD). ***p

Techniques Used: Chromatin Immunoprecipitation, CRISPR, Translocation Assay, Isolation, Transfection, Western Blot, Protein Concentration, Inhibition

17) Product Images from "The Specialized Roles in Carotenogenesis and Apocarotenogenesis of the Phytoene Synthase Gene Family in Saffron"

Article Title: The Specialized Roles in Carotenogenesis and Apocarotenogenesis of the Phytoene Synthase Gene Family in Saffron

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2019.00249

Three dimensional models and location of the saffron phytoene synthase enzymes. (A) Three-dimensional models of the CsPSY enzymes. The models for CsPSY1a, CsPSY1b, CsPSY2, and CsPSY3 were created using the PPM Server ( http://opm.phar.umich.edu/server.php ). The α-helices, and loops are depicted as pink and blue, respectively. Blue dots indicate the membrane surface. (B) Subcellular localization of GFP fusion proteins of CsPSY1a, CsPSY1b, CsPSY2, and CsPSY3 in agro-infiltrated tobacco leaves after 5 days as detected with confocal laser scanning microscopy and enhanced green fluorescent protein (eGFP) expression. Chlorophyll auto-fluorescence in red (left panel), eGFP fluorescence is shown in green (middle panel) and a merged overlay of the eGFP/chlorophyll fluorescence (right panel) is shown in yellow.
Figure Legend Snippet: Three dimensional models and location of the saffron phytoene synthase enzymes. (A) Three-dimensional models of the CsPSY enzymes. The models for CsPSY1a, CsPSY1b, CsPSY2, and CsPSY3 were created using the PPM Server ( http://opm.phar.umich.edu/server.php ). The α-helices, and loops are depicted as pink and blue, respectively. Blue dots indicate the membrane surface. (B) Subcellular localization of GFP fusion proteins of CsPSY1a, CsPSY1b, CsPSY2, and CsPSY3 in agro-infiltrated tobacco leaves after 5 days as detected with confocal laser scanning microscopy and enhanced green fluorescent protein (eGFP) expression. Chlorophyll auto-fluorescence in red (left panel), eGFP fluorescence is shown in green (middle panel) and a merged overlay of the eGFP/chlorophyll fluorescence (right panel) is shown in yellow.

Techniques Used: Confocal Laser Scanning Microscopy, Expressing, Fluorescence

18) Product Images from "A nucleolar TAR decoy inhibitor of HIV-1 replication"

Article Title: A nucleolar TAR decoy inhibitor of HIV-1 replication

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

doi: 10.1073/pnas.212229599

Intracellular localization of U16TAR and colocalization with Tat-EGFP. The 293 cells were grown on coverslips and transiently transfected with 10 μg of the U6+1/U16TAR plasmid and/or 4 μg of pLEGFP-C1/Tat plasmid. After 48 h the cells were fixed in 4% para -formaldehyde dissolved in 1× PBS and in situ hybridizations were carried out. ( A ) The 293 cells transiently transfected with the U6+1/U16TAR plasmid. Hybridization was performed by using a U16TAR RNA-specific probe conjugated with the CY3 fluorophore (red fluorescence, Upper Left ) and a U3 snoRNA-specific probe (for a nucleolar control), conjugated with Oregon green fluorophore (green fluorescence, Upper Right ). The yellow signal depicts overlapping of the two hybridization signals and confirms the U16TAR nucleolar localization ( Lower Left ). The blue staining nuclei (4′-6′-diamino-2-phenyindole, DAPI) are indicated ( Lower Right ). In some of the 293 cells a fraction of the U16TAR signal (red) does not overlap with the U3 snoRNA (green) signal (white arrow, Lower Left ), suggesting a small amount of localization at a site other than the nucleolus when U16TAR is overexpressed (data not shown). ( B ) The 293 cells transiently transfected with the pLEGFP-C1/Tat plasmid. The Tat EGFP green fluorescence was detected in the nucleus of 293 cells ( Left ) (confirmed by DAPI staining, Right ). ( C ) The 293 cells transiently transfected with 4 μg of pLEGFP-C1/Tat plasmid. Hybridization was performed by using a U3 snoRNA-specific probe conjugated with the CY3 fluorophore (red fluorescence, Upper Right ). Tat EGFP is detectable via the green fluorescence ( Upper Left ). The yellow signal depicts overlapping signals of the green fluorescence from the Tat EGFP fusion protein and the red fluorescence of the U3 snoRNA hybridization ( Lower Left ). ( D ) The 293 cells transiently transfected with pLEGFP-C1/Tat and U6+1/U16TAR plasmids. Hybridization was performed by using a U16TAR-specific probe conjugated with CY3 (red fluorescence, Upper Right ). Tat EGFP is detected by the green fluorescence ( Upper Left ). The yellow signal in the nucleoli indicates an overlap between the red fluorescence of the U16TAR probe and the green fluorescence of the Tat EGFP fusion protein ( Lower Left ).
Figure Legend Snippet: Intracellular localization of U16TAR and colocalization with Tat-EGFP. The 293 cells were grown on coverslips and transiently transfected with 10 μg of the U6+1/U16TAR plasmid and/or 4 μg of pLEGFP-C1/Tat plasmid. After 48 h the cells were fixed in 4% para -formaldehyde dissolved in 1× PBS and in situ hybridizations were carried out. ( A ) The 293 cells transiently transfected with the U6+1/U16TAR plasmid. Hybridization was performed by using a U16TAR RNA-specific probe conjugated with the CY3 fluorophore (red fluorescence, Upper Left ) and a U3 snoRNA-specific probe (for a nucleolar control), conjugated with Oregon green fluorophore (green fluorescence, Upper Right ). The yellow signal depicts overlapping of the two hybridization signals and confirms the U16TAR nucleolar localization ( Lower Left ). The blue staining nuclei (4′-6′-diamino-2-phenyindole, DAPI) are indicated ( Lower Right ). In some of the 293 cells a fraction of the U16TAR signal (red) does not overlap with the U3 snoRNA (green) signal (white arrow, Lower Left ), suggesting a small amount of localization at a site other than the nucleolus when U16TAR is overexpressed (data not shown). ( B ) The 293 cells transiently transfected with the pLEGFP-C1/Tat plasmid. The Tat EGFP green fluorescence was detected in the nucleus of 293 cells ( Left ) (confirmed by DAPI staining, Right ). ( C ) The 293 cells transiently transfected with 4 μg of pLEGFP-C1/Tat plasmid. Hybridization was performed by using a U3 snoRNA-specific probe conjugated with the CY3 fluorophore (red fluorescence, Upper Right ). Tat EGFP is detectable via the green fluorescence ( Upper Left ). The yellow signal depicts overlapping signals of the green fluorescence from the Tat EGFP fusion protein and the red fluorescence of the U3 snoRNA hybridization ( Lower Left ). ( D ) The 293 cells transiently transfected with pLEGFP-C1/Tat and U6+1/U16TAR plasmids. Hybridization was performed by using a U16TAR-specific probe conjugated with CY3 (red fluorescence, Upper Right ). Tat EGFP is detected by the green fluorescence ( Upper Left ). The yellow signal in the nucleoli indicates an overlap between the red fluorescence of the U16TAR probe and the green fluorescence of the Tat EGFP fusion protein ( Lower Left ).

Techniques Used: Transfection, Plasmid Preparation, In Situ, Hybridization, Fluorescence, Staining

19) Product Images from "Real-Time Analysis of Endogenous Wnt Signalling in 3D Mesenchymal Stromal Cells"

Article Title: Real-Time Analysis of Endogenous Wnt Signalling in 3D Mesenchymal Stromal Cells

Journal: Stem Cells International

doi: 10.1155/2016/7132529

Wnt pathway activation is observed reproducibly in 3D MSC culture and during osteogenic differentiation but not during adipogenic differentiation. (a) Lightsheet microscopy of cell tracker red stained human Y201 Wnt EGFP MSCs showing sporadic activation of Wnt signalling increasing with time in culture, with Wnt3a-treated controls (right panels). Scale bar = 100 μ m. (b) Confocal microscopy of murine C3H10T1/2 MSC 3D cultures showing Wnt signalling is active in basal conditions and during osteogenic differentiation but not adipogenic differentiation. Scale bar = 100 μ m. (c) Immunofluorescence of sectioned spheroids stained for EGFP demonstrates location of EGFP fluorescence in 3D culture. Scale bar = 100 μ m. Adipogenic differentiation causes spheroids to enlarge in size; a lower magnification image of the whole spheroid is included (top right, merge panel). (d) Adipogenic C3H10T1/2 MSC spheroids show inverse relationship between adipogenesis and Wnt signalling by Oil Red O staining.
Figure Legend Snippet: Wnt pathway activation is observed reproducibly in 3D MSC culture and during osteogenic differentiation but not during adipogenic differentiation. (a) Lightsheet microscopy of cell tracker red stained human Y201 Wnt EGFP MSCs showing sporadic activation of Wnt signalling increasing with time in culture, with Wnt3a-treated controls (right panels). Scale bar = 100 μ m. (b) Confocal microscopy of murine C3H10T1/2 MSC 3D cultures showing Wnt signalling is active in basal conditions and during osteogenic differentiation but not adipogenic differentiation. Scale bar = 100 μ m. (c) Immunofluorescence of sectioned spheroids stained for EGFP demonstrates location of EGFP fluorescence in 3D culture. Scale bar = 100 μ m. Adipogenic differentiation causes spheroids to enlarge in size; a lower magnification image of the whole spheroid is included (top right, merge panel). (d) Adipogenic C3H10T1/2 MSC spheroids show inverse relationship between adipogenesis and Wnt signalling by Oil Red O staining.

Techniques Used: Activation Assay, Microscopy, Staining, Confocal Microscopy, Immunofluorescence, Fluorescence

Stimulation using Wnt3a results in observable fluorescence in human Y201 MSC-EGFP reporter lines. (a) Overview of activated canonical Wnt signalling pathway. (b) Fluorescence microscopy of human Y201 MSC-EGFP reporter cells treated with varying concentrations of Wnt3a with or without a Wnt pathway inhibitor, IWR-1. Scale bar = 200 μ m. (c) Flow cytometry scatter plot histograms of Wnt3a-treated Y201 Wnt EGFP reporters versus untreated, after removal of dead cells and debris by gating. Shift in population away from autofluorescence indicates positive EGFP expression (lower right quadrant).
Figure Legend Snippet: Stimulation using Wnt3a results in observable fluorescence in human Y201 MSC-EGFP reporter lines. (a) Overview of activated canonical Wnt signalling pathway. (b) Fluorescence microscopy of human Y201 MSC-EGFP reporter cells treated with varying concentrations of Wnt3a with or without a Wnt pathway inhibitor, IWR-1. Scale bar = 200 μ m. (c) Flow cytometry scatter plot histograms of Wnt3a-treated Y201 Wnt EGFP reporters versus untreated, after removal of dead cells and debris by gating. Shift in population away from autofluorescence indicates positive EGFP expression (lower right quadrant).

Techniques Used: Fluorescence, Microscopy, Flow Cytometry, Cytometry, Expressing

Endogenous Wnt signalling is not activated during 2D MSC differentiation. (a) Percentage of positively expressing human Y201 MSC-EGFP reporter cells during osteogenic differentiation as measured by flow cytometry, ∗∗∗∗ p
Figure Legend Snippet: Endogenous Wnt signalling is not activated during 2D MSC differentiation. (a) Percentage of positively expressing human Y201 MSC-EGFP reporter cells during osteogenic differentiation as measured by flow cytometry, ∗∗∗∗ p

Techniques Used: Expressing, Flow Cytometry, Cytometry

20) Product Images from "Molecular analysis of muskelin identifies a conserved discoidin-like domain that contributes to protein self-association"

Article Title: Molecular analysis of muskelin identifies a conserved discoidin-like domain that contributes to protein self-association

Journal: Biochemical Journal

doi: 10.1042/BJ20040253

Roles of the discoidin-like domain and the β-propeller in particle formation in cells ( A , B ) Cos-7 cells expressing EGFP–MK ( A ) or EGFP–MK488A/V495A ( B ) were treated with 100 μM PSI for 12 h before fixation. Large muskelin aggregates were formed in the cells. ( C )–( E ) Localization of muskelin mutants. Confocal XY sections of Cos-7 cells transiently expressing EGFP–MKDD ( C ), EGFP–MKKC ( D ) or EGFP–MK-Y488A/V495A ( E ) are shown. None of these proteins formed particles. Bars=10 μm. ( F ) Expression of EGFP–MK in Cos-7 cells relative to endogenous muskelin in SMCs. The indicated protein loads of whole-cell extracts were resolved on an SDS/10%-polyacrylamide gel, transferred to a PVDF membrane and probed with anti-muskelin antibodies. Two ECL® exposure times were taken for quantification of expression using Scion Image (NIH). Pixel intensity per area was quantified at each exposure time (EGFP–MK signal from a 10 μg protein load compared with muskelin signal from 40 and 20 μg protein loads at 20 min exposure time) and mean values calculated.
Figure Legend Snippet: Roles of the discoidin-like domain and the β-propeller in particle formation in cells ( A , B ) Cos-7 cells expressing EGFP–MK ( A ) or EGFP–MK488A/V495A ( B ) were treated with 100 μM PSI for 12 h before fixation. Large muskelin aggregates were formed in the cells. ( C )–( E ) Localization of muskelin mutants. Confocal XY sections of Cos-7 cells transiently expressing EGFP–MKDD ( C ), EGFP–MKKC ( D ) or EGFP–MK-Y488A/V495A ( E ) are shown. None of these proteins formed particles. Bars=10 μm. ( F ) Expression of EGFP–MK in Cos-7 cells relative to endogenous muskelin in SMCs. The indicated protein loads of whole-cell extracts were resolved on an SDS/10%-polyacrylamide gel, transferred to a PVDF membrane and probed with anti-muskelin antibodies. Two ECL® exposure times were taken for quantification of expression using Scion Image (NIH). Pixel intensity per area was quantified at each exposure time (EGFP–MK signal from a 10 μg protein load compared with muskelin signal from 40 and 20 μg protein loads at 20 min exposure time) and mean values calculated.

Techniques Used: Expressing

Expression and localization of muskelin in cells ( A ) Western blot of an SDS/10%-polyacrylamide gel loaded with 40 μg of 1% Triton X-100 cell extracts from SMCs, Cos-7 cells and 293T cells, probed with anti-muskelin antibodies. ( B ) Immunoblot showing relative expression of EGFP–MK proteins, detected by anti-GFP antibody. Protein designations are as in ( A ). Molecular mass markers are in kDa. ( C )–( I ) Confocal images of the distribution of transiently expressed EGFP–MK ( C , D ), MK–EGFP ( G ) or MK–V5His 6 ( H ) in Cos-7 cells, or EGFP–MK in SMCs ( F ). Panels ( C ) and ( D ) show a Cos-7 cell co-stained for F-actin. ( E ) Distribution of EGFP in Cos-7 cells. ( I ) Endogenous muskelin of C2C12 cells (as XY section from Z stack). Arrows in panels ( C ) and ( F )–( I ) indicate the muskelin particles of various sizes observed in the cells. Bars=10 μm.
Figure Legend Snippet: Expression and localization of muskelin in cells ( A ) Western blot of an SDS/10%-polyacrylamide gel loaded with 40 μg of 1% Triton X-100 cell extracts from SMCs, Cos-7 cells and 293T cells, probed with anti-muskelin antibodies. ( B ) Immunoblot showing relative expression of EGFP–MK proteins, detected by anti-GFP antibody. Protein designations are as in ( A ). Molecular mass markers are in kDa. ( C )–( I ) Confocal images of the distribution of transiently expressed EGFP–MK ( C , D ), MK–EGFP ( G ) or MK–V5His 6 ( H ) in Cos-7 cells, or EGFP–MK in SMCs ( F ). Panels ( C ) and ( D ) show a Cos-7 cell co-stained for F-actin. ( E ) Distribution of EGFP in Cos-7 cells. ( I ) Endogenous muskelin of C2C12 cells (as XY section from Z stack). Arrows in panels ( C ) and ( F )–( I ) indicate the muskelin particles of various sizes observed in the cells. Bars=10 μm.

Techniques Used: Expressing, Western Blot, Staining

Biochemical analysis of muskelin self-association ( A ) Schematic diagram of muskelin constructs, their designations and the various N- and C-terminal tags utilized. HA, haemagglutinin; DD, discoidin-like domain; L, LisH motif; H, CTLH motif; K, kelch repeats; C, C-terminal region. ( B ) Co-immunoprecipitation of muskelin with EGFP–MK. Lysates from 293T cells expressing EGFP or EGFP–MK were mixed 1:1 (v/v) with SMC lysates and immunoprecipitated (IP) for GFP. Bound proteins were resolved on 10% (w/v) polyacrylamide gels, transferred to a PVDF membrane and probed with antibodies to muskelin. Expression of the transiently expressed proteins was monitored by immunoblot (IB) of cell lysates with anti-GFP antibody (CL). ( C ) Specific pull-down (PD) of EGFP–MK with MK–V5His 6 . Lysates of 293T cells transiently expressing MK–V5His 6 were mixed 1:1 (v/v) with lysates from cells expressing EGFP or EGFP–MK and incubated with nickel beads. Bead-bound proteins were resolved on 10% (w/v) polyacrylamide gels, transferred to a PVDF membrane and probed with antibodies to polyhistidine or GFP. Expression of the transiently expressed proteins was monitored by immunoblotting (IB) of cell lysates (CL). ( D ) Mapping requirements for pull-down (PD) by GST–MKDD. Aliquots of 400 μg of detergent lysates of 293T cells transiently transfected with the indicated EGFP–MK proteins were incubated with either GST–MKDD or GST-loaded glutathione–agarose beads. Aliquots of 45 μg of the lysates were also taken to monitor protein levels (CL). The bound proteins were resolved on SDS/10%-polyacrylamide gels, transferred to a PVDF membrane and probed with antibody to GFP. Only wild-type EGFP–MK and EGFP–MKKC bound to GST–MKDD, and there was no binding to GST. The results are representative of three independent experiments.
Figure Legend Snippet: Biochemical analysis of muskelin self-association ( A ) Schematic diagram of muskelin constructs, their designations and the various N- and C-terminal tags utilized. HA, haemagglutinin; DD, discoidin-like domain; L, LisH motif; H, CTLH motif; K, kelch repeats; C, C-terminal region. ( B ) Co-immunoprecipitation of muskelin with EGFP–MK. Lysates from 293T cells expressing EGFP or EGFP–MK were mixed 1:1 (v/v) with SMC lysates and immunoprecipitated (IP) for GFP. Bound proteins were resolved on 10% (w/v) polyacrylamide gels, transferred to a PVDF membrane and probed with antibodies to muskelin. Expression of the transiently expressed proteins was monitored by immunoblot (IB) of cell lysates with anti-GFP antibody (CL). ( C ) Specific pull-down (PD) of EGFP–MK with MK–V5His 6 . Lysates of 293T cells transiently expressing MK–V5His 6 were mixed 1:1 (v/v) with lysates from cells expressing EGFP or EGFP–MK and incubated with nickel beads. Bead-bound proteins were resolved on 10% (w/v) polyacrylamide gels, transferred to a PVDF membrane and probed with antibodies to polyhistidine or GFP. Expression of the transiently expressed proteins was monitored by immunoblotting (IB) of cell lysates (CL). ( D ) Mapping requirements for pull-down (PD) by GST–MKDD. Aliquots of 400 μg of detergent lysates of 293T cells transiently transfected with the indicated EGFP–MK proteins were incubated with either GST–MKDD or GST-loaded glutathione–agarose beads. Aliquots of 45 μg of the lysates were also taken to monitor protein levels (CL). The bound proteins were resolved on SDS/10%-polyacrylamide gels, transferred to a PVDF membrane and probed with antibody to GFP. Only wild-type EGFP–MK and EGFP–MKKC bound to GST–MKDD, and there was no binding to GST. The results are representative of three independent experiments.

Techniques Used: Construct, Immunoprecipitation, Expressing, Incubation, Transfection, Binding Assay

21) 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

Downregulated in renal cell carcinoma 1 (DRR1) features an actin binding site at each terminus. ( A ; 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 ; 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, Immunoprecipitation, SDS Page, Western Blot, Sedimentation, Recombinant, Mutagenesis, Staining, Polyacrylamide Gel Electrophoresis

22) Product Images from "The Growth Factor Granulin Interacts with Cyclin T1 and Modulates P-TEFb-Dependent Transcription"

Article Title: The Growth Factor Granulin Interacts with Cyclin T1 and Modulates P-TEFb-Dependent Transcription

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.5.1688-1702.2003

Intracellular localization of granulins and cyclin T1. (A) EGFP-fused granulins and RFP-fused cyclin T1 were expressed individually in COS7 cells (panels 1 to 5). The dotted nuclear distribution of cyclin T1 is seen in panel 4, whereas the overexposed image from another cell shows the presence of cyclin T1 in the nucleoplasm but not in the nucleoli (panel 5). In panel 6, untagged GEP was coexpressed with RFP-fused cyclin T1. Monochromatic images are shown in each case. (B) Distribution of cyclin T1 and the granulins in cells coexpressing RFP-fused cyclin T1 with EGFP-tagged GEP, GrnCDE, or GrnDE (panels 7 to 9, respectively). Monochromatic images for EGFP and for RFP and the merged images are displayed from left to right. Arrows and white dots show the position of the nucleus in individual cells.
Figure Legend Snippet: Intracellular localization of granulins and cyclin T1. (A) EGFP-fused granulins and RFP-fused cyclin T1 were expressed individually in COS7 cells (panels 1 to 5). The dotted nuclear distribution of cyclin T1 is seen in panel 4, whereas the overexposed image from another cell shows the presence of cyclin T1 in the nucleoplasm but not in the nucleoli (panel 5). In panel 6, untagged GEP was coexpressed with RFP-fused cyclin T1. Monochromatic images are shown in each case. (B) Distribution of cyclin T1 and the granulins in cells coexpressing RFP-fused cyclin T1 with EGFP-tagged GEP, GrnCDE, or GrnDE (panels 7 to 9, respectively). Monochromatic images for EGFP and for RFP and the merged images are displayed from left to right. Arrows and white dots show the position of the nucleus in individual cells.

Techniques Used:

Intracellular localization of granulins and cyclin T1. (A) EGFP-fused granulins and RFP-fused cyclin T1 were expressed individually in COS7 cells (panels 1 to 5). The dotted nuclear distribution of cyclin T1 is seen in panel 4, whereas the overexposed image from another cell shows the presence of cyclin T1 in the nucleoplasm but not in the nucleoli (panel 5). In panel 6, untagged GEP was coexpressed with RFP-fused cyclin T1. Monochromatic images are shown in each case. (B) Distribution of cyclin T1 and the granulins in cells coexpressing RFP-fused cyclin T1 with EGFP-tagged GEP, GrnCDE, or GrnDE (panels 7 to 9, respectively). Monochromatic images for EGFP and for RFP and the merged images are displayed from left to right. Arrows and white dots show the position of the nucleus in individual cells.
Figure Legend Snippet: Intracellular localization of granulins and cyclin T1. (A) EGFP-fused granulins and RFP-fused cyclin T1 were expressed individually in COS7 cells (panels 1 to 5). The dotted nuclear distribution of cyclin T1 is seen in panel 4, whereas the overexposed image from another cell shows the presence of cyclin T1 in the nucleoplasm but not in the nucleoli (panel 5). In panel 6, untagged GEP was coexpressed with RFP-fused cyclin T1. Monochromatic images are shown in each case. (B) Distribution of cyclin T1 and the granulins in cells coexpressing RFP-fused cyclin T1 with EGFP-tagged GEP, GrnCDE, or GrnDE (panels 7 to 9, respectively). Monochromatic images for EGFP and for RFP and the merged images are displayed from left to right. Arrows and white dots show the position of the nucleus in individual cells.

Techniques Used:

23) Product Images from "LTP-triggered cholesterol redistribution activates Cdc42 and drives AMPA receptor synaptic delivery"

Article Title: LTP-triggered cholesterol redistribution activates Cdc42 and drives AMPA receptor synaptic delivery

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201407122

Cholesterol reduction from intracellular membranes after cLTP induction. (A) Quantification of cholesterol content by LC/MS from hippocampal slices immediately after cLTP induction (cLTP), 20 min recovery after cLTP induction (Rec), or from untreated slices (baseline [Bsln]). Cholesterol (Chol) content was normalized to major phospholipids (phosphocholine [PC] plus sphingomyelin [SM]) and plotted relative to baseline values. Experiments were performed with or without the NMDAR antagonist AP5, as indicated. Symbols connected with lines represent time course experiments performed in parallel. Different lines represent independent experiments. Quantifications were performed from plasma membrane fraction (left) or from microsomal fractions (right). Statistical significance was evaluated with the Kruskal–Wallis test. (B) Quantification of Bodipy-cholesterol fluorescence from hippocampal slices undergoing cLTP induction (blue-shaded area), with or without AP5, as indicated. Fluorescence values are normalized to the mean fluorescence before cLTP induction. n represents the number of cells. Representative images of Bodipy-cholesterol–labeled neurons are shown on the left. (C) Mean values of Bodipy-cholesterol fluorescence immediately at the end of the cLTP induction period (cLTP) and after 10-min recovery, from the time course shown in B. The p-value was determined with the Mann–Whitney test. n represents the number of cells. (D) Diffuse and clustered mCherry-D4 fluorescence was quantified (see Materials and methods) from hippocampal slices undergoing cLTP induction (blue-shaded area). Fluorescence values are normalized to the mean fluorescence before cLTP induction. n represents the number of cells. Left shows representative images of a CA1 neuron expressing mCherry-D4 (top) and EGFP (bottom), before (Baseline) and after 15-min cLTP induction. (E) Mean values of mCherry-D4 diffuse or clustered fluorescence immediately before (Baseline), at the end of the cLTP induction period (cLTP), or after 10 min of recovery, from the time course shown in D. The p-value was determined with the Mann–Whitney test. n represent the number of cells. Error bars show means ± SEM. Bars, 10 µm.
Figure Legend Snippet: Cholesterol reduction from intracellular membranes after cLTP induction. (A) Quantification of cholesterol content by LC/MS from hippocampal slices immediately after cLTP induction (cLTP), 20 min recovery after cLTP induction (Rec), or from untreated slices (baseline [Bsln]). Cholesterol (Chol) content was normalized to major phospholipids (phosphocholine [PC] plus sphingomyelin [SM]) and plotted relative to baseline values. Experiments were performed with or without the NMDAR antagonist AP5, as indicated. Symbols connected with lines represent time course experiments performed in parallel. Different lines represent independent experiments. Quantifications were performed from plasma membrane fraction (left) or from microsomal fractions (right). Statistical significance was evaluated with the Kruskal–Wallis test. (B) Quantification of Bodipy-cholesterol fluorescence from hippocampal slices undergoing cLTP induction (blue-shaded area), with or without AP5, as indicated. Fluorescence values are normalized to the mean fluorescence before cLTP induction. n represents the number of cells. Representative images of Bodipy-cholesterol–labeled neurons are shown on the left. (C) Mean values of Bodipy-cholesterol fluorescence immediately at the end of the cLTP induction period (cLTP) and after 10-min recovery, from the time course shown in B. The p-value was determined with the Mann–Whitney test. n represents the number of cells. (D) Diffuse and clustered mCherry-D4 fluorescence was quantified (see Materials and methods) from hippocampal slices undergoing cLTP induction (blue-shaded area). Fluorescence values are normalized to the mean fluorescence before cLTP induction. n represents the number of cells. Left shows representative images of a CA1 neuron expressing mCherry-D4 (top) and EGFP (bottom), before (Baseline) and after 15-min cLTP induction. (E) Mean values of mCherry-D4 diffuse or clustered fluorescence immediately before (Baseline), at the end of the cLTP induction period (cLTP), or after 10 min of recovery, from the time course shown in D. The p-value was determined with the Mann–Whitney test. n represent the number of cells. Error bars show means ± SEM. Bars, 10 µm.

Techniques Used: Liquid Chromatography with Mass Spectroscopy, Fluorescence, Labeling, MANN-WHITNEY, Expressing

24) Product Images from "Characterization of marine diatom-infecting virus promoters in the model diatom Phaeodactylum tricornutum"

Article Title: Characterization of marine diatom-infecting virus promoters in the model diatom Phaeodactylum tricornutum

Journal: Scientific Reports

doi: 10.1038/srep18708

Relative activities of various promoters including DIV promoters in transformants. Ten independent transformants for each promoter were analysed. The promoter activity levels were determined by dividing egfp mRNA levels by Sh ble mRNA levels. The circles indicate the mean triplicate measurements of the independent transformants. The diamonds denote the average values of the six transformants from Cfnr pro. or of the ten transformants from the other promoters. Pro.-less denotes cases in which no promoter was linked to the egfp gene in the transformation vector (negative control). PtfcpA pro. shows the positive control in the transformation vector egfp gene driven by PtfcpA pro. Asterisks indicate the presence of a statistically significant difference derived from the PtfcpA pro. transformants (** P
Figure Legend Snippet: Relative activities of various promoters including DIV promoters in transformants. Ten independent transformants for each promoter were analysed. The promoter activity levels were determined by dividing egfp mRNA levels by Sh ble mRNA levels. The circles indicate the mean triplicate measurements of the independent transformants. The diamonds denote the average values of the six transformants from Cfnr pro. or of the ten transformants from the other promoters. Pro.-less denotes cases in which no promoter was linked to the egfp gene in the transformation vector (negative control). PtfcpA pro. shows the positive control in the transformation vector egfp gene driven by PtfcpA pro. Asterisks indicate the presence of a statistically significant difference derived from the PtfcpA pro. transformants (** P

Techniques Used: Activity Assay, Transformation Assay, Plasmid Preparation, Negative Control, Positive Control, Derivative Assay

Schematic diagram for the evaluation of promoter activity. ( a ) Outline of the construction of transformation vectors and transformations. After predicting putative ORF positions 32 , upstream regions of the ORFs were determined as potential promoter regions. Potential promoter regions amplified by PCR were used to construct the transformation vectors. The double-cassette vector containing the reporter gene egfp driven by each tested promoter and the antibiotic-resistant gene Sh ble driven by the promoter region of the fucoxanthin chlorophyll a / c -binding protein (FCP) A-1A gene derived from Cyl. fusiformis (termed CffcpA pro.) were constructed. ( b ) Assessment of promoter activity. Promoter activity was determined by averaging the ratios of egfp mRNA transcript levels to those of Sh ble mRNA transcripts in ten transformants to minimize the effects of copy numbers on the expression of transgenes. These transformants were also used to investigate eGFP protein expression patterns. CffcpA ter.: terminator region of the FCP A-1A gene derived from Cyl. fusiformis . The structure of the ClorDNA genome was modified from Tomaru et al. 32 . *For the transformation vector of the nitrate reductase gene promoter, we used pNICgfp 18 ( Supplementary Fig. 5a ).
Figure Legend Snippet: Schematic diagram for the evaluation of promoter activity. ( a ) Outline of the construction of transformation vectors and transformations. After predicting putative ORF positions 32 , upstream regions of the ORFs were determined as potential promoter regions. Potential promoter regions amplified by PCR were used to construct the transformation vectors. The double-cassette vector containing the reporter gene egfp driven by each tested promoter and the antibiotic-resistant gene Sh ble driven by the promoter region of the fucoxanthin chlorophyll a / c -binding protein (FCP) A-1A gene derived from Cyl. fusiformis (termed CffcpA pro.) were constructed. ( b ) Assessment of promoter activity. Promoter activity was determined by averaging the ratios of egfp mRNA transcript levels to those of Sh ble mRNA transcripts in ten transformants to minimize the effects of copy numbers on the expression of transgenes. These transformants were also used to investigate eGFP protein expression patterns. CffcpA ter.: terminator region of the FCP A-1A gene derived from Cyl. fusiformis . The structure of the ClorDNA genome was modified from Tomaru et al. 32 . *For the transformation vector of the nitrate reductase gene promoter, we used pNICgfp 18 ( Supplementary Fig. 5a ).

Techniques Used: Activity Assay, Transformation Assay, Amplification, Polymerase Chain Reaction, Construct, Plasmid Preparation, Binding Assay, Derivative Assay, Expressing, Modification

Analyses of viral promoter activities in different culture conditions. ( a ) Growth of two transformants with ClP1-driven egfp in f/10 medium and f/2 medium. ( b ) Relative abundances of egfp mRNA determined by dividing egfp mRNA transcript levels by those of ribosomal protein small subunit 30S gene ( rps ; internal control gene) mRNA transcripts in the transformant cells incubated in f/10 and f/2 media and harvested at various growth phases and at different times during a light/dark period. The arrows show cell collection points for the qRT-PCR analysis.
Figure Legend Snippet: Analyses of viral promoter activities in different culture conditions. ( a ) Growth of two transformants with ClP1-driven egfp in f/10 medium and f/2 medium. ( b ) Relative abundances of egfp mRNA determined by dividing egfp mRNA transcript levels by those of ribosomal protein small subunit 30S gene ( rps ; internal control gene) mRNA transcripts in the transformant cells incubated in f/10 and f/2 media and harvested at various growth phases and at different times during a light/dark period. The arrows show cell collection points for the qRT-PCR analysis.

Techniques Used: Incubation, Quantitative RT-PCR

25) Product Images from "Detection by Epitope-defined Monoclonal Antibodies of Werner DNA Helicases in the Nucleoplasm and Their Upregulation by Cell Transformation and Immortalization "

Article Title: Detection by Epitope-defined Monoclonal Antibodies of Werner DNA Helicases in the Nucleoplasm and Their Upregulation by Cell Transformation and Immortalization

Journal: The Journal of Cell Biology

doi:

Determination of subnuclear distribution of WRN helicase by immunocytochemical staining and expression of a full-size WRN cDNA. (I) Subnuclear distribution of endogenous WRN helicase was examined by indirect immunocytochemical staining with mAb 4H12 for fibroblast cells from normal donors and WS patient no. 15501, and for Tera2 and WI38/SV cells. For controls, the nucleoli of normal fibroblast, WS patient no. 15501 and Tera2 cells were stained separately by mAb, anti human nucleolus clone AE3 (Leinco Technology Inc.), specific for human nucleolus. The nuclei of these cells were also stained by DAPI. (A and B) Fibroblast cells (interphase) from normal individual stained by DAPI and 4H12 antibody, respectively. (C and D) Fibroblast cells (interphase) from normal individual stained by DAPI and anti-nucleolus antibody, respectively. (E and F) Fibroblast cells (interphase) from WS patient no. 15501 containing homomutations 4/4 stained by DAPI and 4H12 antibody, respectively. (G and H) Fibroblast cells (interphase) from WS patient no. 15501 containing homomutations 4/4 stained by DAPI and anti-nucleolus antibody, respectively. (I and J) Tera2 cells (interphase) stained by DAPI and 4H12 antibody respectively. (K and L) Tera2 cells (interphase) stained by DAPI and anti-nucleolus antibody, respectively. (M and N) Tera2 cells chromatins from the metaphase stained by DAPI and 4H12 antibody, respectively. (O and P) WI38/SV cells (interphase) stained by DAPI and 4H12 antibody, respectively. (Q and R) Mouse colon 26 cells stained by DAPI and rat mAb to mouse WRN protein, respectively. (II) Subnuclear distribution in HeLa cells of WRN helicase synthesized as fusion protein with EGFP. (A) The DAPI stained HeLa cell nuclei. (B) The EGFP-human WRN helicase expressed in HeLa cells.
Figure Legend Snippet: Determination of subnuclear distribution of WRN helicase by immunocytochemical staining and expression of a full-size WRN cDNA. (I) Subnuclear distribution of endogenous WRN helicase was examined by indirect immunocytochemical staining with mAb 4H12 for fibroblast cells from normal donors and WS patient no. 15501, and for Tera2 and WI38/SV cells. For controls, the nucleoli of normal fibroblast, WS patient no. 15501 and Tera2 cells were stained separately by mAb, anti human nucleolus clone AE3 (Leinco Technology Inc.), specific for human nucleolus. The nuclei of these cells were also stained by DAPI. (A and B) Fibroblast cells (interphase) from normal individual stained by DAPI and 4H12 antibody, respectively. (C and D) Fibroblast cells (interphase) from normal individual stained by DAPI and anti-nucleolus antibody, respectively. (E and F) Fibroblast cells (interphase) from WS patient no. 15501 containing homomutations 4/4 stained by DAPI and 4H12 antibody, respectively. (G and H) Fibroblast cells (interphase) from WS patient no. 15501 containing homomutations 4/4 stained by DAPI and anti-nucleolus antibody, respectively. (I and J) Tera2 cells (interphase) stained by DAPI and 4H12 antibody respectively. (K and L) Tera2 cells (interphase) stained by DAPI and anti-nucleolus antibody, respectively. (M and N) Tera2 cells chromatins from the metaphase stained by DAPI and 4H12 antibody, respectively. (O and P) WI38/SV cells (interphase) stained by DAPI and 4H12 antibody, respectively. (Q and R) Mouse colon 26 cells stained by DAPI and rat mAb to mouse WRN protein, respectively. (II) Subnuclear distribution in HeLa cells of WRN helicase synthesized as fusion protein with EGFP. (A) The DAPI stained HeLa cell nuclei. (B) The EGFP-human WRN helicase expressed in HeLa cells.

Techniques Used: Staining, Expressing, Synthesized

Determination of the epitopes of mAbs 4H12 and 8H3 that recognize COOH- and NH 2 -terminal region of WRN helicase. (A) The epitope of mAb 4H12 was determined using the parts of WRN helicase, C59 (COOH-terminal 59 aa) and C49 (COOH-terminal 49 aa), expressed as a fusion protein with EGFP in B16F10 mouse melanoma cells. The cells were fixed and tested for immunostaining by mAb 4H12, which was conjugated with fluorescent reagent Texas red (right). DAPI stains DNA and shows the location of the nucleus (left) and EGFP (middle) shows the locations of expressed EGFP fusion proteins. C59 contains the nuclear localization signal NKRRCF at aa residues 1,369–1,402 ( Matsumoto et al., 1997a ; Matsumoto et al., 1998a ) and localizes in the nucleus, while C49 lacks the nuclear localization signal and does not migrate to the nucleus. (B) The epitope of mAb 8H3 was determined as for 4H12. The NH 2 -terminal fragment N368 (NH 2 -terminal 368 aa) was expressed in B16F10 cells as an EGFP-fused form and was tested for immunostaining with Texas red–conjugated mAb 8H3. The EGFP-N368 fusion protein does not have NLS and localizes in the cytoplasm of B16F10 cells. (C) Further epitope analysis of mAb 8H3 was made by Western blot analysis. Here, a full-size WRN helicase (lane 2) and a C1201-WRN helicase lacking NH 2 -terminal 231 aa synthesized by a baculovirus system (lane 1) were resolved by SDS-PAGE in 7% polyacrylamide gel and stained by Coomassie brilliant blue. They were blotted to filter and were tested for immunoreactivity with mAb 8H3 (lanes 3 and 4). (D) Schematic representation of WRN helicase molecule and the epitope sites for mAbs.
Figure Legend Snippet: Determination of the epitopes of mAbs 4H12 and 8H3 that recognize COOH- and NH 2 -terminal region of WRN helicase. (A) The epitope of mAb 4H12 was determined using the parts of WRN helicase, C59 (COOH-terminal 59 aa) and C49 (COOH-terminal 49 aa), expressed as a fusion protein with EGFP in B16F10 mouse melanoma cells. The cells were fixed and tested for immunostaining by mAb 4H12, which was conjugated with fluorescent reagent Texas red (right). DAPI stains DNA and shows the location of the nucleus (left) and EGFP (middle) shows the locations of expressed EGFP fusion proteins. C59 contains the nuclear localization signal NKRRCF at aa residues 1,369–1,402 ( Matsumoto et al., 1997a ; Matsumoto et al., 1998a ) and localizes in the nucleus, while C49 lacks the nuclear localization signal and does not migrate to the nucleus. (B) The epitope of mAb 8H3 was determined as for 4H12. The NH 2 -terminal fragment N368 (NH 2 -terminal 368 aa) was expressed in B16F10 cells as an EGFP-fused form and was tested for immunostaining with Texas red–conjugated mAb 8H3. The EGFP-N368 fusion protein does not have NLS and localizes in the cytoplasm of B16F10 cells. (C) Further epitope analysis of mAb 8H3 was made by Western blot analysis. Here, a full-size WRN helicase (lane 2) and a C1201-WRN helicase lacking NH 2 -terminal 231 aa synthesized by a baculovirus system (lane 1) were resolved by SDS-PAGE in 7% polyacrylamide gel and stained by Coomassie brilliant blue. They were blotted to filter and were tested for immunoreactivity with mAb 8H3 (lanes 3 and 4). (D) Schematic representation of WRN helicase molecule and the epitope sites for mAbs.

Techniques Used: Immunostaining, Western Blot, Synthesized, SDS Page, Staining

26) Product Images from "BARP suppresses voltage-gated calcium channel activity and Ca2+-evoked exocytosis"

Article Title: BARP suppresses voltage-gated calcium channel activity and Ca2+-evoked exocytosis

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201304101

BARP down-regulates voltage-gated Ca 2+ channel activity without affecting its cell surface expression. (A–C) BARP down-regulates VGCC activity. Effect of BARP on activity of P/Q (A)- or N (B)-type Ca 2+ channels reconstituted in BHK cells or on VGCCs in tsA201 cells (C). (A and B) BHK cells stably expressing Ca v 2.1 (P/Q type) or Ca v 2.2 (N type) together with Ca v β1a and Ca v α2δ were transfected with a vector carrying WT or mutated BARP and EGFP cDNAs, and cells expressing EGFP were selected for electrophysiology. (C) tsA201 cells were cotransfected with a vector carrying WT or mutated BARP and mCherry cDNAs, a vector containing Ca v 1.2, and an internal ribosomal entry site followed by Ca v β3 and EGFP cDNAs, and cells expressing mCherry and EGFP were selected for electrophysiology. Current–voltage (I–V) relationships of the different Ca 2+ channels in the different cells expressing the different BARP proteins (a and b) as well as the traces for P/Q (c)- and N ( Fig. S5 A )-type channel recordings are shown. Three independent experiments were performed each, and the data were combined to obtain the indicated n values. Shown are the means ± SEM. Paired Student’s t test at 20 mV. For P/Q-type channels, control versus BARP WT or domain II mutated (P
Figure Legend Snippet: BARP down-regulates voltage-gated Ca 2+ channel activity without affecting its cell surface expression. (A–C) BARP down-regulates VGCC activity. Effect of BARP on activity of P/Q (A)- or N (B)-type Ca 2+ channels reconstituted in BHK cells or on VGCCs in tsA201 cells (C). (A and B) BHK cells stably expressing Ca v 2.1 (P/Q type) or Ca v 2.2 (N type) together with Ca v β1a and Ca v α2δ were transfected with a vector carrying WT or mutated BARP and EGFP cDNAs, and cells expressing EGFP were selected for electrophysiology. (C) tsA201 cells were cotransfected with a vector carrying WT or mutated BARP and mCherry cDNAs, a vector containing Ca v 1.2, and an internal ribosomal entry site followed by Ca v β3 and EGFP cDNAs, and cells expressing mCherry and EGFP were selected for electrophysiology. Current–voltage (I–V) relationships of the different Ca 2+ channels in the different cells expressing the different BARP proteins (a and b) as well as the traces for P/Q (c)- and N ( Fig. S5 A )-type channel recordings are shown. Three independent experiments were performed each, and the data were combined to obtain the indicated n values. Shown are the means ± SEM. Paired Student’s t test at 20 mV. For P/Q-type channels, control versus BARP WT or domain II mutated (P

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

27) Product Images from "The biogenesis and characterization of mammalian microRNAs of mirtron origin"

Article Title: The biogenesis and characterization of mammalian microRNAs of mirtron origin

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkr722

miR-877 and miR-1224 are functional mammalian introns. (A ) Putative mirtrons were studied by inserting appropriate sequences as introns within an eGFP transcript. ( B ) Predicted mirtron alignments for miR-877 and miR-1224 sequences incorporated into the pMirt vector. Variants with modifications made to splicing regulatory sequences are indicated by dashed boxes. US = unspliceable, DUS = double unspliceable, BP = Branch-point, Scr = Scrambled. Blue and red nucleotides represent the guide sequence and modified nucleotides, respectively. ( C ) Representative fluorescent microscopy images of different mirt-miR-877 and mirt-miR-1224 variants 48 h after transfection in HEK-293 cells. NAD represents a control intron of comparable length to miR-877 and miR-1224 with no hairpin-forming potential. ( D ) Upper panel—Quantification of eGFP fluorescence following expression of mirt-miR-877 and mirt-miR-1224 variants in HEK-293 cells. Values represent mean fluorescent ±SD from n = 3. * P
Figure Legend Snippet: miR-877 and miR-1224 are functional mammalian introns. (A ) Putative mirtrons were studied by inserting appropriate sequences as introns within an eGFP transcript. ( B ) Predicted mirtron alignments for miR-877 and miR-1224 sequences incorporated into the pMirt vector. Variants with modifications made to splicing regulatory sequences are indicated by dashed boxes. US = unspliceable, DUS = double unspliceable, BP = Branch-point, Scr = Scrambled. Blue and red nucleotides represent the guide sequence and modified nucleotides, respectively. ( C ) Representative fluorescent microscopy images of different mirt-miR-877 and mirt-miR-1224 variants 48 h after transfection in HEK-293 cells. NAD represents a control intron of comparable length to miR-877 and miR-1224 with no hairpin-forming potential. ( D ) Upper panel—Quantification of eGFP fluorescence following expression of mirt-miR-877 and mirt-miR-1224 variants in HEK-293 cells. Values represent mean fluorescent ±SD from n = 3. * P

Techniques Used: Functional Assay, Plasmid Preparation, Sequencing, Modification, Microscopy, Transfection, Fluorescence, Expressing

28) Product Images from "Dominant-Negative Myosin Va Impairs Retrograde but Not Anterograde Axonal Transport of Large Dense Core Vesicles"

Article Title: Dominant-Negative Myosin Va Impairs Retrograde but Not Anterograde Axonal Transport of Large Dense Core Vesicles

Journal: Cellular and molecular neurobiology

doi: 10.1007/s10571-009-9459-2

Subcellular localization of fluorescently labeled NPY expressed in cultured hippocampal neurons. Cells were transfected with a plasmid encoding NPY-mRFP or NPY-EGFP at 11 DIV and fixed and imaged with a confocal microscope ( a–c ) or a wide field
Figure Legend Snippet: Subcellular localization of fluorescently labeled NPY expressed in cultured hippocampal neurons. Cells were transfected with a plasmid encoding NPY-mRFP or NPY-EGFP at 11 DIV and fixed and imaged with a confocal microscope ( a–c ) or a wide field

Techniques Used: Labeling, Cell Culture, Transfection, Plasmid Preparation, Microscopy

Dynamics of LDCVs in neurons. Cells were double-transfected with a plasmid encoding NPY-mRFP and one encoding EGFP as a cytoplasmic marker at 11 DIV. After 24 h, time-lapse recordings were taken at 1 s intervals for 100 s. a1 Kymograph of dendritic LDCVs.
Figure Legend Snippet: Dynamics of LDCVs in neurons. Cells were double-transfected with a plasmid encoding NPY-mRFP and one encoding EGFP as a cytoplasmic marker at 11 DIV. After 24 h, time-lapse recordings were taken at 1 s intervals for 100 s. a1 Kymograph of dendritic LDCVs.

Techniques Used: Transfection, Plasmid Preparation, Marker

Colocalization of NPY-EGFP with endogenous myosin Va. a Immunofluorescence with an antibody directed against myosin Va reveals extensive punctate staining in dendrites and axons of cultured hippocampal neurons at 12 DIV. b1 Punctate structures of endogenous
Figure Legend Snippet: Colocalization of NPY-EGFP with endogenous myosin Va. a Immunofluorescence with an antibody directed against myosin Va reveals extensive punctate staining in dendrites and axons of cultured hippocampal neurons at 12 DIV. b1 Punctate structures of endogenous

Techniques Used: Immunofluorescence, Staining, Cell Culture

Cotransport of myosin Va-tail with LDCVs. a dual-color axon kymograph. Neurons expressing myosin Va-tail-mCherry and NPY-EGFP were imaged for 100 s at 1 frame per second. A kymograph was made from the axon-showing myosin Va-tail-mCherry-labeled vesicles
Figure Legend Snippet: Cotransport of myosin Va-tail with LDCVs. a dual-color axon kymograph. Neurons expressing myosin Va-tail-mCherry and NPY-EGFP were imaged for 100 s at 1 frame per second. A kymograph was made from the axon-showing myosin Va-tail-mCherry-labeled vesicles

Techniques Used: Expressing, Labeling

29) Product Images from "Improvement of the transient expression system for production of recombinant proteins in plants"

Article Title: Improvement of the transient expression system for production of recombinant proteins in plants

Journal: Scientific Reports

doi: 10.1038/s41598-018-23024-y

Effect of HSP terminator on transient GFP expression at 3 days post-agroinfiltration. Total soluble proteins were extracted from agroinfiltrated plant leaves with pBYR2HS-EGFP or pBYR2fp-EGFP. Coomassie Brilliant Blue (CBB) staining and immunoblot analysis with anti-GFP antibodies were performed by using agroinfiltrated leaves of N. benthamiana ( A ), lettuce ( B ), eggplants ( C ), tomatoes (D) , hot peppers, and roses ( E ). The numbers at the top of the gels indicate different samples taken from different leaves from different plants. NT indicates non-transfected plants. The amount of protein was measured according to band intensity from CBB staining gel using ImageJ software. Arrowheads indicate bands corresponding to GFP protein. The band clearly seen at 55 kDa in a CBB staining gel is corresponding to large subunit of Rubisco. Data represent the means ± SD ( n = 3 to 4). Significance was determined using unpaired Student’s t tests (* p
Figure Legend Snippet: Effect of HSP terminator on transient GFP expression at 3 days post-agroinfiltration. Total soluble proteins were extracted from agroinfiltrated plant leaves with pBYR2HS-EGFP or pBYR2fp-EGFP. Coomassie Brilliant Blue (CBB) staining and immunoblot analysis with anti-GFP antibodies were performed by using agroinfiltrated leaves of N. benthamiana ( A ), lettuce ( B ), eggplants ( C ), tomatoes (D) , hot peppers, and roses ( E ). The numbers at the top of the gels indicate different samples taken from different leaves from different plants. NT indicates non-transfected plants. The amount of protein was measured according to band intensity from CBB staining gel using ImageJ software. Arrowheads indicate bands corresponding to GFP protein. The band clearly seen at 55 kDa in a CBB staining gel is corresponding to large subunit of Rubisco. Data represent the means ± SD ( n = 3 to 4). Significance was determined using unpaired Student’s t tests (* p

Techniques Used: Expressing, Staining, Transfection, Software

Effect of single or double terminator on transient EGFP expression at 3 days post-agroinfiltration. GFP emission from leaves of N. benthamiana agroinfiltrated with several kinds of plasmids (Fig. 1 ) was observed with an ultraviolet-absorbing filter, Fujifilm SC-52 ( A – E ). Total soluble proteins were extracted from agroinfiltrated N. benthamiana leaves with pBYR2HS-EGFP, pBYR2H-EGFP, or pBYR2fp-EGFP. Coomassie Brilliant Blue staining were performed ( F ) and the amount of protein was measured ( G ). Total soluble proteins were extracted from agroinfiltrated N. benthamiana leaves with pBYR2HS-EGFP, pBYR2HH-EGFP, pBYR2EE-EGFP, pBYR2TN-EGFP, pBYR2HT-EGFP, and pBYR2HTS-EGFP. Coomassie Brilliant Blue staining was performed ( H ) and the amount of protein was measured ( I ). The numbers at the top of the gels indicate different samples taken from different leaves from different plants. Data represent the means ± SD ( n = 3 to 4). Significance was determined using unpaired Student’s t tests (* p
Figure Legend Snippet: Effect of single or double terminator on transient EGFP expression at 3 days post-agroinfiltration. GFP emission from leaves of N. benthamiana agroinfiltrated with several kinds of plasmids (Fig. 1 ) was observed with an ultraviolet-absorbing filter, Fujifilm SC-52 ( A – E ). Total soluble proteins were extracted from agroinfiltrated N. benthamiana leaves with pBYR2HS-EGFP, pBYR2H-EGFP, or pBYR2fp-EGFP. Coomassie Brilliant Blue staining were performed ( F ) and the amount of protein was measured ( G ). Total soluble proteins were extracted from agroinfiltrated N. benthamiana leaves with pBYR2HS-EGFP, pBYR2HH-EGFP, pBYR2EE-EGFP, pBYR2TN-EGFP, pBYR2HT-EGFP, and pBYR2HTS-EGFP. Coomassie Brilliant Blue staining was performed ( H ) and the amount of protein was measured ( I ). The numbers at the top of the gels indicate different samples taken from different leaves from different plants. Data represent the means ± SD ( n = 3 to 4). Significance was determined using unpaired Student’s t tests (* p

Techniques Used: Expressing, Staining

Schematic diagram of the T-DNA region of the plasmids pBYR2fp-EGFP, pBYR2HS-EGFP, pBYR2EE-EGFP, pBYR2HH-EGFP, pBYR2H-EGFP, pBYR2TN-EGFP, pBYR2HT-EGFP, pBYR2HTS-EGFP, and pBYR2T-EGFP. 35S-p x 2, CaMV 35 S promoter with double-enhanced element; AtADH5′, 5′-untranslated region (UTR) of Arabidopsis thaliana alcohol dehydrogenase gene; TMV Ω, 5′-leader sequence of tobacco mosaic virus; EGFP, enhanced green fluorescence protein; HSPter, terminator of heat shock protein gene; Ext3′, tobacco extension gene 3′ element; 35Ster, terminator of CaMV 35S; NOSter, NOS terminator; LIR, long intergenic region of bean yellow dwarf virus (BeYDV) genome; SIR, short intergenic region of BeYDV genome; C1/C2, BeYDV ORFs C1 and C2 encoding for replication initiation protein (Rep) and RepA, respectively; LB and RB, the left and right borders of the T-DNA region, respectively; Nos-p and Nos-t, NOS promoter and terminator, respectively; and p19, a gene-silencing suppressor gene from tomato bushy stunt virus.
Figure Legend Snippet: Schematic diagram of the T-DNA region of the plasmids pBYR2fp-EGFP, pBYR2HS-EGFP, pBYR2EE-EGFP, pBYR2HH-EGFP, pBYR2H-EGFP, pBYR2TN-EGFP, pBYR2HT-EGFP, pBYR2HTS-EGFP, and pBYR2T-EGFP. 35S-p x 2, CaMV 35 S promoter with double-enhanced element; AtADH5′, 5′-untranslated region (UTR) of Arabidopsis thaliana alcohol dehydrogenase gene; TMV Ω, 5′-leader sequence of tobacco mosaic virus; EGFP, enhanced green fluorescence protein; HSPter, terminator of heat shock protein gene; Ext3′, tobacco extension gene 3′ element; 35Ster, terminator of CaMV 35S; NOSter, NOS terminator; LIR, long intergenic region of bean yellow dwarf virus (BeYDV) genome; SIR, short intergenic region of BeYDV genome; C1/C2, BeYDV ORFs C1 and C2 encoding for replication initiation protein (Rep) and RepA, respectively; LB and RB, the left and right borders of the T-DNA region, respectively; Nos-p and Nos-t, NOS promoter and terminator, respectively; and p19, a gene-silencing suppressor gene from tomato bushy stunt virus.

Techniques Used: Sequencing, Fluorescence

Introduction of HSP terminator improved transient expression of EGFP. Agrobacterium tumefaciens harboring pBYR2HS-EGFP and pBYR2fp-EGFP were transfected into Nicotiana benthamiana ( A ), lettuce Lactuca sativa var. crispa ( B ), eggplant Solanum melongena cv. ‘Dewakonasu’ ( C ), tomato fruits Solanum lycopersicum cv. ‘M82’ ( D ), tomato leaves Solanum lycopersicum cv. ‘Micro-Tom’ ( E ), hot pepper Capsicum frutescens cv. ‘Shima-togarashi’ ( F ), melon Cucumis melo cv. ‘Earl’s Favorite Harukei No.3′ ( G ), orchid Phalaenopsis Aphrodite ( H ), and a rose Rosa sp. ‘Bonheur’ ( I ). These plants were incubated for 3 days after agroinfiltration. Then, after blue-light excitation, GFP emission was observed with an ultraviolet-absorbing filter Fujifilm SC-52. Bars indicate a 1-cm length.
Figure Legend Snippet: Introduction of HSP terminator improved transient expression of EGFP. Agrobacterium tumefaciens harboring pBYR2HS-EGFP and pBYR2fp-EGFP were transfected into Nicotiana benthamiana ( A ), lettuce Lactuca sativa var. crispa ( B ), eggplant Solanum melongena cv. ‘Dewakonasu’ ( C ), tomato fruits Solanum lycopersicum cv. ‘M82’ ( D ), tomato leaves Solanum lycopersicum cv. ‘Micro-Tom’ ( E ), hot pepper Capsicum frutescens cv. ‘Shima-togarashi’ ( F ), melon Cucumis melo cv. ‘Earl’s Favorite Harukei No.3′ ( G ), orchid Phalaenopsis Aphrodite ( H ), and a rose Rosa sp. ‘Bonheur’ ( I ). These plants were incubated for 3 days after agroinfiltration. Then, after blue-light excitation, GFP emission was observed with an ultraviolet-absorbing filter Fujifilm SC-52. Bars indicate a 1-cm length.

Techniques Used: Expressing, Transfection, Incubation

30) Product Images from "Quantification of protein mobility and associated reshuffling of cytoplasm during chemical fixation"

Article Title: Quantification of protein mobility and associated reshuffling of cytoplasm during chemical fixation

Journal: Scientific Reports

doi: 10.1038/s41598-018-36112-w

Membrane blebbing and associated loss of cytosolic protein in HeLa cells upon fixation with glutaraldehyde (GA) and formaldehyde (FA). ( A ) Shown are representative fluorescence microscopy images of HeLa cells transfected with cytoplasmic EGFP (green) and stained with the membrane marker DiIC12 (magenta) 9.5 min after application of 4% formaldehyde (FA) and 9 min after application of 2% glutaraldehyde (GA). See Figs S5 and S7 for complete time series. White arrowheads point to plasma membrane blebs that develop during fixation and are filled with (FA) or devoid of (GA) cytoplasmic EGFP. Scale bar: 10 μm; ( B ) Depicted is the fraction of HeLa cells that showed membrane blebs after fixation with the indicated concentrations of FA and GA as identified by DiIC12-staining (compare Figs S5 – S7 ). n = 6 experiments (8–12 cells). ( C ) Depicted is the mean fluorescence intensity of cytoplasmic EGFP in the plasma membrane blebs normalised to the mean fluorescence intensity of the corresponding cell body for each blebbing cell (orange circles) and the mean +/− s.d. (black lines). The solid red line and the dashed red line indicates mean and the mean + s.d. of the background fluorescence intensity around the cells. ***p
Figure Legend Snippet: Membrane blebbing and associated loss of cytosolic protein in HeLa cells upon fixation with glutaraldehyde (GA) and formaldehyde (FA). ( A ) Shown are representative fluorescence microscopy images of HeLa cells transfected with cytoplasmic EGFP (green) and stained with the membrane marker DiIC12 (magenta) 9.5 min after application of 4% formaldehyde (FA) and 9 min after application of 2% glutaraldehyde (GA). See Figs S5 and S7 for complete time series. White arrowheads point to plasma membrane blebs that develop during fixation and are filled with (FA) or devoid of (GA) cytoplasmic EGFP. Scale bar: 10 μm; ( B ) Depicted is the fraction of HeLa cells that showed membrane blebs after fixation with the indicated concentrations of FA and GA as identified by DiIC12-staining (compare Figs S5 – S7 ). n = 6 experiments (8–12 cells). ( C ) Depicted is the mean fluorescence intensity of cytoplasmic EGFP in the plasma membrane blebs normalised to the mean fluorescence intensity of the corresponding cell body for each blebbing cell (orange circles) and the mean +/− s.d. (black lines). The solid red line and the dashed red line indicates mean and the mean + s.d. of the background fluorescence intensity around the cells. ***p

Techniques Used: Fluorescence, Microscopy, Transfection, Staining, Marker

Fixation time of cytoplasmic protein and development of autofluorescence by aldehyde-fixation in HeLa cells. ( A ) Shown is a representative FRAP experiment during fixation with 2% glutaraldehyde (GA). A circular area with 2 μm diameter (white arrow) was bleached in a HeLa cell, which expressed cytosolic mCitrine, at the indicated time points after changing the medium to 2% GA. Bleaching was achieved by scanning the corresponding area with 5 laser lines of a white light laser at 100% transmission and a 405-nm laser diode at 100% intensity. The upper row shows fluorescent micrographs directly before each bleaching. The lower row shows images directly after bleaching. Scale Bar: 10 μm; ( B ) Fixation time for cultured HeLa cells as determined by consecutive bleaching and fluorescence recovery of mCitrine during chemical fixation using the indicated aldehyde concentrations. Depicted are the time points after which no further diffusion of mCitrine was observed except for 4% FA, 0% GA ( #) , where diffusion was measurable during the whole course of the 20-min experiment after fixation and also in separate experiments after more than 60 min (Figs S1 and S2 ). ( C ) Increase in autofluorescence of HeLa cells upon fixation with the indicated concentrations of aldehydes in 3 different fluorescence channels corresponding to blue (DAPI; excitation (ex.) 360–370 nm/emission (em.) 420–470 nm), green (EGFP; ex. 460–480 nm/em. 495–540 nm) and red (RFP; ex. 535–550 nm/em. 570–625 nm) fluorescence measured by widefield microscopy normalised to living cells. Data is shown for single cells (coloured symbols) and mean (black lines). All autofluorescence measurements in EGFP and RFP channels are significantly higher (p
Figure Legend Snippet: Fixation time of cytoplasmic protein and development of autofluorescence by aldehyde-fixation in HeLa cells. ( A ) Shown is a representative FRAP experiment during fixation with 2% glutaraldehyde (GA). A circular area with 2 μm diameter (white arrow) was bleached in a HeLa cell, which expressed cytosolic mCitrine, at the indicated time points after changing the medium to 2% GA. Bleaching was achieved by scanning the corresponding area with 5 laser lines of a white light laser at 100% transmission and a 405-nm laser diode at 100% intensity. The upper row shows fluorescent micrographs directly before each bleaching. The lower row shows images directly after bleaching. Scale Bar: 10 μm; ( B ) Fixation time for cultured HeLa cells as determined by consecutive bleaching and fluorescence recovery of mCitrine during chemical fixation using the indicated aldehyde concentrations. Depicted are the time points after which no further diffusion of mCitrine was observed except for 4% FA, 0% GA ( #) , where diffusion was measurable during the whole course of the 20-min experiment after fixation and also in separate experiments after more than 60 min (Figs S1 and S2 ). ( C ) Increase in autofluorescence of HeLa cells upon fixation with the indicated concentrations of aldehydes in 3 different fluorescence channels corresponding to blue (DAPI; excitation (ex.) 360–370 nm/emission (em.) 420–470 nm), green (EGFP; ex. 460–480 nm/em. 495–540 nm) and red (RFP; ex. 535–550 nm/em. 570–625 nm) fluorescence measured by widefield microscopy normalised to living cells. Data is shown for single cells (coloured symbols) and mean (black lines). All autofluorescence measurements in EGFP and RFP channels are significantly higher (p

Techniques Used: Transmission Assay, Cell Culture, Fluorescence, Diffusion-based Assay, Microscopy

31) Product Images from "Structural basis for the fast maturation of Arthropoda green fluorescent protein"

Article Title: Structural basis for the fast maturation of Arthropoda green fluorescent protein

Journal: EMBO Reports

doi: 10.1038/sj.embor.7400787

Comparison of TurboGFP and enhanced green fluorescent protein maturation speed in developing Xenopus laevis embryos. At the stage of two blastomeres, embryos were microinjected with TurboGFP-C1 and pEGFP-C1 vectors. Living embryos were photographed from the animal pole side at the early and mid-gastrula stages. EGFP, enhanced green fluorescent protein.
Figure Legend Snippet: Comparison of TurboGFP and enhanced green fluorescent protein maturation speed in developing Xenopus laevis embryos. At the stage of two blastomeres, embryos were microinjected with TurboGFP-C1 and pEGFP-C1 vectors. Living embryos were photographed from the animal pole side at the early and mid-gastrula stages. EGFP, enhanced green fluorescent protein.

Techniques Used:

32) Product Images from "Genetically targeted single-channel optical recording reveals multiple Orai1 gating states and oscillations in calcium influx"

Article Title: Genetically targeted single-channel optical recording reveals multiple Orai1 gating states and oscillations in calcium influx

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

doi: 10.1073/pnas.1523410113

Some EGFP–Orai1 channels exhibit restricted diffusion in the presence of CAD. EGFP–Orai1 and mCherry–CAD were cotransfected into HEK 293A cells and imaged using TIRF microscopy. ( A ) Single-particle track of a single EGFP–Orai1
Figure Legend Snippet: Some EGFP–Orai1 channels exhibit restricted diffusion in the presence of CAD. EGFP–Orai1 and mCherry–CAD were cotransfected into HEK 293A cells and imaged using TIRF microscopy. ( A ) Single-particle track of a single EGFP–Orai1

Techniques Used: Diffusion-based Assay, Microscopy

33) Product Images from "High-Throughput Nano-Scale Characterization of Membrane Proteins Using Fluorescence-Detection Size-Exclusion Chromatography"

Article Title: High-Throughput Nano-Scale Characterization of Membrane Proteins Using Fluorescence-Detection Size-Exclusion Chromatography

Journal: Methods in molecular biology (Clifton, N.J.)

doi: 10.1007/978-1-4939-9624-7_17

HTP expression and homogeneity screening of membrane proteins transiently transfected in human cells using F-SEC. GFP fluorescence ( λ ex = 488 nm, λ em = 509 nm) was monitored while performing size-exclusion chromatography on 18 βDDM-solubilized POI membrane protein orthologs. The SEC peak heights provide an implication to the level of expression, while the trace symmetry indicates the quality of each protein screened. Analysis of the above results shows many sharp, monodisperse peaks, with highly variable levels of expression. Red, yellow, and green bars indicate three estimated classes of expressers; red being poor, yellow as moderate, and green as high. Note the aggregated (void) and proteolyzed (eGFP) peak intensities compared to POI-eGFP fusion peaks when analyzing results and deciding on targets
Figure Legend Snippet: HTP expression and homogeneity screening of membrane proteins transiently transfected in human cells using F-SEC. GFP fluorescence ( λ ex = 488 nm, λ em = 509 nm) was monitored while performing size-exclusion chromatography on 18 βDDM-solubilized POI membrane protein orthologs. The SEC peak heights provide an implication to the level of expression, while the trace symmetry indicates the quality of each protein screened. Analysis of the above results shows many sharp, monodisperse peaks, with highly variable levels of expression. Red, yellow, and green bars indicate three estimated classes of expressers; red being poor, yellow as moderate, and green as high. Note the aggregated (void) and proteolyzed (eGFP) peak intensities compared to POI-eGFP fusion peaks when analyzing results and deciding on targets

Techniques Used: Expressing, Transfection, Size-exclusion Chromatography, Fluorescence

In vivo POI expression levels from transient transfection of HEK-293T cells in a 24-well culture plate. Overlay of bright field and eGFP fluorescence images using fluorescence microscopy of 18 POI membrane protein orthologs, with exposure times normalized to that of the brightest sample. The negative control represents cells transfected with a non-eGFP vector containing a CMV promoter, while the vector control was transfected with pEGFP-N3 mod , which contains eGFP but no POI. Transfection efficiencies can be quantified by calculating the number of fluorescent cells per total cells, while approximates of total cell expression can be ascertained by quantifying the number and fluorescence intensity per cell
Figure Legend Snippet: In vivo POI expression levels from transient transfection of HEK-293T cells in a 24-well culture plate. Overlay of bright field and eGFP fluorescence images using fluorescence microscopy of 18 POI membrane protein orthologs, with exposure times normalized to that of the brightest sample. The negative control represents cells transfected with a non-eGFP vector containing a CMV promoter, while the vector control was transfected with pEGFP-N3 mod , which contains eGFP but no POI. Transfection efficiencies can be quantified by calculating the number of fluorescent cells per total cells, while approximates of total cell expression can be ascertained by quantifying the number and fluorescence intensity per cell

Techniques Used: In Vivo, Expressing, Transfection, Fluorescence, Microscopy, Negative Control, Plasmid Preparation

34) Product Images from "A53T Mutant Alpha-Synuclein Induces Tau-Dependent Postsynaptic Impairment Independently of Neurodegenerative Changes"

Article Title: A53T Mutant Alpha-Synuclein Induces Tau-Dependent Postsynaptic Impairment Independently of Neurodegenerative Changes

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.0344-18.2018

A53T αS-induced postsynaptic deficits are cell autonomous. A , Wide-field fluorescence photomicrographs from cultured rat hippocampal DAPI-stained neurons expressing eGFP-tagged WT and A53T αS plasmids via calcium-phosphate transfection.
Figure Legend Snippet: A53T αS-induced postsynaptic deficits are cell autonomous. A , Wide-field fluorescence photomicrographs from cultured rat hippocampal DAPI-stained neurons expressing eGFP-tagged WT and A53T αS plasmids via calcium-phosphate transfection.

Techniques Used: Fluorescence, Cell Culture, Staining, Expressing, Transfection

Stable and consistent expression of eGFP-fused human αS across constructs. A , Contour plots of flow cytometry gating parameters from the nontransfected group. B , Contour plots of two populations of cells in the nontransfected group: eGFP-negative,
Figure Legend Snippet: Stable and consistent expression of eGFP-fused human αS across constructs. A , Contour plots of flow cytometry gating parameters from the nontransfected group. B , Contour plots of two populations of cells in the nontransfected group: eGFP-negative,

Techniques Used: Expressing, Construct, Flow Cytometry, Cytometry

A53T αS at two expression levels induces phosphorylation-dependent mislocalization of tau to dendritic spines. Neurons were cultured from TgNg, H5, and G2-3 hippocampi and transfected with DsRed to visualize cellular architecture and eGFP-fused
Figure Legend Snippet: A53T αS at two expression levels induces phosphorylation-dependent mislocalization of tau to dendritic spines. Neurons were cultured from TgNg, H5, and G2-3 hippocampi and transfected with DsRed to visualize cellular architecture and eGFP-fused

Techniques Used: Expressing, Cell Culture, Transfection

35) Product Images from "Overexpression of SMPX in Adult Skeletal Muscle Does not Change Skeletal Muscle Fiber Type or Size"

Article Title: Overexpression of SMPX in Adult Skeletal Muscle Does not Change Skeletal Muscle Fiber Type or Size

Journal: PLoS ONE

doi: 10.1371/journal.pone.0099232

Muscle fiber type and cross sectional area (CSA) in mouse EDL. Single fibers from muscles transfected with either pCMS-EGFP (left leg sham control, only expressing EGFP) or pCMS-EGFP- Smpx (expressing SMPX and EGFP). A) CSA with n = 226/254 cells for sham/SMPX. Values are means +/- SD. B) Fiber type composition of the same fibers as in A.
Figure Legend Snippet: Muscle fiber type and cross sectional area (CSA) in mouse EDL. Single fibers from muscles transfected with either pCMS-EGFP (left leg sham control, only expressing EGFP) or pCMS-EGFP- Smpx (expressing SMPX and EGFP). A) CSA with n = 226/254 cells for sham/SMPX. Values are means +/- SD. B) Fiber type composition of the same fibers as in A.

Techniques Used: Transfection, Expressing

Expression of SMPX plasmids, in vitro and in vivo. A) Western blot of SMPX-EGFP expression in HEK-293 cells stained with antibodies against EGFP. Lane 1: Negative control. Lane 2: EGFP only. Lane 3-6: SMPX-EGFP. B) In vivo fluorescence image of EDL muscle expressing pCMS-EGFP- Smpx . Scalebar is 500 microns. Inset: High magnification of single fibers. Scalebar is 50 microns. C) β-gal (left) and myosin heavy chain type 2A (right) stain on neighboring cross-sections from the same muscle as in B). β-gal expressing fibers marked with asterisks. Scale bar is 50 microns.
Figure Legend Snippet: Expression of SMPX plasmids, in vitro and in vivo. A) Western blot of SMPX-EGFP expression in HEK-293 cells stained with antibodies against EGFP. Lane 1: Negative control. Lane 2: EGFP only. Lane 3-6: SMPX-EGFP. B) In vivo fluorescence image of EDL muscle expressing pCMS-EGFP- Smpx . Scalebar is 500 microns. Inset: High magnification of single fibers. Scalebar is 50 microns. C) β-gal (left) and myosin heavy chain type 2A (right) stain on neighboring cross-sections from the same muscle as in B). β-gal expressing fibers marked with asterisks. Scale bar is 50 microns.

Techniques Used: Expressing, In Vitro, In Vivo, Western Blot, Staining, Negative Control, Fluorescence

SMPX-EGFP was excluded from all myonuclei. A) C2C12 myoblasts expressing either EGFP or SMPX-EGFP stained with Hoechst 33342 to visualize nuclei. B) In vitro confocal image of dissected single rat EDL muscle fibers expressing EGFP or SMPX-EGFP stained with DAPI to visualize nuclei. C) Confocal images of EDL muscle fibers in situ, after no treatment (CON) or functional overload for 18 hours (LOAD), expressing SMPX-EGFP stained with Hoechst 33342 to visualize nuclei. Myonuclei are labeled with arrowheads. Scale bar is 10 microns.
Figure Legend Snippet: SMPX-EGFP was excluded from all myonuclei. A) C2C12 myoblasts expressing either EGFP or SMPX-EGFP stained with Hoechst 33342 to visualize nuclei. B) In vitro confocal image of dissected single rat EDL muscle fibers expressing EGFP or SMPX-EGFP stained with DAPI to visualize nuclei. C) Confocal images of EDL muscle fibers in situ, after no treatment (CON) or functional overload for 18 hours (LOAD), expressing SMPX-EGFP stained with Hoechst 33342 to visualize nuclei. Myonuclei are labeled with arrowheads. Scale bar is 10 microns.

Techniques Used: Expressing, Staining, In Vitro, In Situ, Functional Assay, Labeling

SMPX-EGFP was localized in bands flanking the z-disc. A) In situ confocal image of EGFP or SMPX-EGFP expression from an isolated EDL rat muscle in Ringer-solution. Scale bar is 10 microns. B) In vitro confocal image of EGFP or SMPX-EGFP stained with Alexa Fluor 680 Phalloidin (red) to visualize actin filaments. Scale bar is 10 microns (inset 1 micron).
Figure Legend Snippet: SMPX-EGFP was localized in bands flanking the z-disc. A) In situ confocal image of EGFP or SMPX-EGFP expression from an isolated EDL rat muscle in Ringer-solution. Scale bar is 10 microns. B) In vitro confocal image of EGFP or SMPX-EGFP stained with Alexa Fluor 680 Phalloidin (red) to visualize actin filaments. Scale bar is 10 microns (inset 1 micron).

Techniques Used: In Situ, Expressing, Isolation, In Vitro, Staining

36) Product Images from "Covalent Attachment of Proteins to Solid Supports and Surfaces via Sortase-Mediated Ligation"

Article Title: Covalent Attachment of Proteins to Solid Supports and Surfaces via Sortase-Mediated Ligation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0001164

(a,b) Ligation of fluorescent proteins to Affi-Gel resin. Left to right, negative control, BFP-, EGFP-, and DsRed-LPETGG-His 6 were ligated to Affi-Gel 102 Resin modified with oligoglycine. The negative control reaction contained EGFP-LPETGG-His 6 and no Sortase. After washing with buffer pictures were taken with a white light (a) and UV transilluminator (b, 312 nm). (c–f) Ligation of EGFP to a glass surface. Microscope coverslips were modified with triethoxy(aminopropyl) silane and oligoglycine before incubation with EGFP-LPETGG-His 6 . The slides were washed with Sortase buffer containing 1% SDS and photographed using the FITC filter set on an Axiovert 200 microscope. (c) Glycine modified surface with EGFP-LPETGG-His 6 but no Sortase, (d) amino modified surface (i.e. without glycine) with EGFP-LPETGG-His 6 and Sortase, (e) Glycine modified surface with EGFP-LPETGG-His 6 and Sortase with same exposure settings as negative controls, (f) same as (e) with five-fold reduced exposure time.
Figure Legend Snippet: (a,b) Ligation of fluorescent proteins to Affi-Gel resin. Left to right, negative control, BFP-, EGFP-, and DsRed-LPETGG-His 6 were ligated to Affi-Gel 102 Resin modified with oligoglycine. The negative control reaction contained EGFP-LPETGG-His 6 and no Sortase. After washing with buffer pictures were taken with a white light (a) and UV transilluminator (b, 312 nm). (c–f) Ligation of EGFP to a glass surface. Microscope coverslips were modified with triethoxy(aminopropyl) silane and oligoglycine before incubation with EGFP-LPETGG-His 6 . The slides were washed with Sortase buffer containing 1% SDS and photographed using the FITC filter set on an Axiovert 200 microscope. (c) Glycine modified surface with EGFP-LPETGG-His 6 but no Sortase, (d) amino modified surface (i.e. without glycine) with EGFP-LPETGG-His 6 and Sortase, (e) Glycine modified surface with EGFP-LPETGG-His 6 and Sortase with same exposure settings as negative controls, (f) same as (e) with five-fold reduced exposure time.

Techniques Used: Ligation, Negative Control, Modification, Microscopy, Incubation

Fluorescence micrographs of labeled solid supports. (a) Diglycine GMA beads and (b) oligoglycine modified Affigel resin were separately labeled with EGFP and DsRed and then mixed. Fluorescence images were recorded as separate gray scale images (see Supplementary Figure S2 ) with FITC and Cy3 filter sets and then combined and false coloured.
Figure Legend Snippet: Fluorescence micrographs of labeled solid supports. (a) Diglycine GMA beads and (b) oligoglycine modified Affigel resin were separately labeled with EGFP and DsRed and then mixed. Fluorescence images were recorded as separate gray scale images (see Supplementary Figure S2 ) with FITC and Cy3 filter sets and then combined and false coloured.

Techniques Used: Fluorescence, Labeling, Modification

37) Product Images from "Self-cleavage of fusion protein in vivo using TEV protease to yield native protein"

Article Title: Self-cleavage of fusion protein in vivo using TEV protease to yield native protein

Journal: Protein Science : A Publication of the Protein Society

doi: 10.1110/ps.041129605

Cloning design and intracellular cleavage of otherwise identical MBP-TEVP-rsTEV-EGFP-His 6 fusion proteins with different amino acid residues in the P1′ position. ( A ) Introduction of an SnaBI site at the coding sequence of the TEVP recognition site. ( B ) Sticky-end PCR cloning strategy. One forward and two reverse PCR primers as well as PCR reactions were used in two separate tubes. An equal amount of the two PCR products were mixed, and then 5′ ends were phosphorylated with T4 polynucleotide kinase. After denaturing (95°C for 5 min) and renaturing (65°C for 5 min), ∼50% of the final products carry SnaBI (5′) and XhoI (3′) ends and are ready for ligation into the vector. The codon and anticodon of the amino acid residues in the P1′ position is indicated as “XXX” or “YYY,” respectively. ( C ) Schematic representation of the new MBP-TEVP-rsTEV-EGFP-His 6 fusion protein vector. The amino acid residue in the P1′ position is indicated as “Z.” ( D ) Samples of soluble protein lysates from IPTG-induced E. coli cells producing MBP-TEVP-rsTEV-EGFP-His 6 with different amino acid residues in the P1′ position (indicated in a single-letter code) were separated by SDS-PAGE and analyzed either by Coomassie blue staining or by Western blot using anti-His 6 antibody. The positions of MBP-TEVP-rsTEV and EGFP-His 6 protein bands are marked. The molecular weight standards are shown on the left .
Figure Legend Snippet: Cloning design and intracellular cleavage of otherwise identical MBP-TEVP-rsTEV-EGFP-His 6 fusion proteins with different amino acid residues in the P1′ position. ( A ) Introduction of an SnaBI site at the coding sequence of the TEVP recognition site. ( B ) Sticky-end PCR cloning strategy. One forward and two reverse PCR primers as well as PCR reactions were used in two separate tubes. An equal amount of the two PCR products were mixed, and then 5′ ends were phosphorylated with T4 polynucleotide kinase. After denaturing (95°C for 5 min) and renaturing (65°C for 5 min), ∼50% of the final products carry SnaBI (5′) and XhoI (3′) ends and are ready for ligation into the vector. The codon and anticodon of the amino acid residues in the P1′ position is indicated as “XXX” or “YYY,” respectively. ( C ) Schematic representation of the new MBP-TEVP-rsTEV-EGFP-His 6 fusion protein vector. The amino acid residue in the P1′ position is indicated as “Z.” ( D ) Samples of soluble protein lysates from IPTG-induced E. coli cells producing MBP-TEVP-rsTEV-EGFP-His 6 with different amino acid residues in the P1′ position (indicated in a single-letter code) were separated by SDS-PAGE and analyzed either by Coomassie blue staining or by Western blot using anti-His 6 antibody. The positions of MBP-TEVP-rsTEV and EGFP-His 6 protein bands are marked. The molecular weight standards are shown on the left .

Techniques Used: Clone Assay, Sequencing, Polymerase Chain Reaction, Ligation, Plasmid Preparation, SDS Page, Staining, Western Blot, Molecular Weight

38) Product Images from "Nuclear glutaredoxin 3 is critical for protection against oxidative stress-induced cell death"

Article Title: Nuclear glutaredoxin 3 is critical for protection against oxidative stress-induced cell death

Journal: Free radical biology & medicine

doi: 10.1016/j.freeradbiomed.2015.05.003

Nuclear-targeting Grx3 is sufficient to suppress the sensitivity of Grx3-KD cells to oxidative stress. (A) The subcellular localization of nuclear-targeted Grx3 fusion proteins in HeLa cells. HeLa cells were transfected with eGFP–NuGrx3 and the nuclei were stained with Sytox orange dye. The fluorescence images were taken by confocal microscopy 24 h after transfection. (B) Cell viability assay. Grx3-KD cells (shRNA 2 line) were transfected with eGFP as controls or eGFP–NuGrx3 and then seeded at 1×10 5 cells into each well (triplicates for each treatment) and grown for 24 h with normal growth medium. Cell viability assay was conducted after being treated with various concentrations of diamide as indicated for 14 h. Shown is one representative of two independent experiments with consistent results. Statistical analysis using a two-way ANOVA, n =3; * p
Figure Legend Snippet: Nuclear-targeting Grx3 is sufficient to suppress the sensitivity of Grx3-KD cells to oxidative stress. (A) The subcellular localization of nuclear-targeted Grx3 fusion proteins in HeLa cells. HeLa cells were transfected with eGFP–NuGrx3 and the nuclei were stained with Sytox orange dye. The fluorescence images were taken by confocal microscopy 24 h after transfection. (B) Cell viability assay. Grx3-KD cells (shRNA 2 line) were transfected with eGFP as controls or eGFP–NuGrx3 and then seeded at 1×10 5 cells into each well (triplicates for each treatment) and grown for 24 h with normal growth medium. Cell viability assay was conducted after being treated with various concentrations of diamide as indicated for 14 h. Shown is one representative of two independent experiments with consistent results. Statistical analysis using a two-way ANOVA, n =3; * p

Techniques Used: Transfection, Staining, Fluorescence, Confocal Microscopy, Viability Assay, shRNA

39) 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

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

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

40) Product Images from "Transgenic Mouse Expressing Optical MicroRNA Reporter for Monitoring MicroRNA-124 Action during Development"

Article Title: Transgenic Mouse Expressing Optical MicroRNA Reporter for Monitoring MicroRNA-124 Action during Development

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2016.00052

Bioluminescence images of candidate miR-124 reporter-expressing transgenic mice. (A) Whole body bioluminescence images of wt and transgenic mice harboring effluc-eGFP-miR-124_3 × PT acquired with D -luciferin injection. The acquisition time of line 67 (left) and line 18 (right) was 10 s and 600 s, respectively. (B) Ex vivo images of effluc reporter activity of fetuses of wt mouse and transgenic mice (line 67) at E16 period. (C) Ex vivo images of major organs excised from a wt and a transgenic mouse (line 67).
Figure Legend Snippet: Bioluminescence images of candidate miR-124 reporter-expressing transgenic mice. (A) Whole body bioluminescence images of wt and transgenic mice harboring effluc-eGFP-miR-124_3 × PT acquired with D -luciferin injection. The acquisition time of line 67 (left) and line 18 (right) was 10 s and 600 s, respectively. (B) Ex vivo images of effluc reporter activity of fetuses of wt mouse and transgenic mice (line 67) at E16 period. (C) Ex vivo images of major organs excised from a wt and a transgenic mouse (line 67).

Techniques Used: Expressing, Transgenic Assay, Mouse Assay, Injection, Ex Vivo, Activity Assay

MiR-124 action reporter vector and its validation. (A) Schematic diagram of miR-124 reporter transgene of lentiviral vector. The lentiviral construct consists of CMV promoter, effluc, IRES, and 3 × PT (triple perfect targets) including three repeat perfect target sequences complementary to mature miR-124. (B) Luminescence produced after 24 h of transient transfection of scramble or miR-124 showed working of effluc in the effluc-eGFP-miR-124_3 × PT transfected HeLa cells. Luciferase activity (photons/second) was quantified using Living image software. Data are represented by the means ± SEM ( n = 3).
Figure Legend Snippet: MiR-124 action reporter vector and its validation. (A) Schematic diagram of miR-124 reporter transgene of lentiviral vector. The lentiviral construct consists of CMV promoter, effluc, IRES, and 3 × PT (triple perfect targets) including three repeat perfect target sequences complementary to mature miR-124. (B) Luminescence produced after 24 h of transient transfection of scramble or miR-124 showed working of effluc in the effluc-eGFP-miR-124_3 × PT transfected HeLa cells. Luciferase activity (photons/second) was quantified using Living image software. Data are represented by the means ± SEM ( n = 3).

Techniques Used: Plasmid Preparation, Construct, Produced, Transfection, Luciferase, Activity Assay, Software

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Clone Assay:

Article Title: Cervical Cancer Cells with Positive Sox2 Expression Exhibit the Properties of Cancer Stem Cells
Article Snippet: .. The correct human Sox2 promoter, UTR/enhancer, EGFP, and vector were subsequently cloned using an In-Fusion PCR Cloning Kit, and the resulting vector was designated phSox2/EGFP (Takara Bio Inc, Dalian, China). .. Immunohistochemistry and Immunocytochemistry Immunohistochemistry was performed on 4-µm sections of paraffin-embedded tissues.

Article Title: Identifying Activated T Cells in Reconstituted RAG Deficient Mice Using Retrovirally Transduced Pax5 Deficient Pro-B Cells
Article Snippet: .. EGFP was cloned downstream of the retroviral LTR promoter followed by the CD40L promoter driving the expression of HcRed (Clontech) with the small t-intron and polyadenylation sequence from SV40 ( ). .. Consequently, cells transfected with this construct will constitutively express EGFP and will only express HcRed, a far-red fluorescent protein from Heteractis crispa , upon activation of CD40L promoter, e.g. upon antigen challenge.

Article Title: Map7/7D1 and Dvl form a feedback loop that facilitates microtubule remodeling and Wnt5a signaling
Article Snippet: .. To generate EGFP‐ or V5‐tagged proteins, the CDS was cloned into pEGFP‐N3 (Clontech) or pcDNA3.1‐V5His6 (Thermo Fisher Scientific), respectively. ..

Article Title: Regulation of ASIC channels by a stomatin/STOML3 complex located in a mobile vesicle pool in sensory neurons
Article Snippet: .. A red version of STOML3 was generated by replacing EGFP with mCherry red fluorescent protein and a Strep-tagged cyan version by cloning into pAmCyan1-N1 (Clontech). .. Mouse (1a, 1b, 4) and rat (2a, 2b, 3) full-length ASIC sequences were cloned into pEYFP-C1 (Clontech) and pFLAG-CMV-2 (Sigma).

Article Title: The LTB4–BLT1 axis regulates the polarized trafficking of chemoattractant GPCRs during neutrophil chemotaxis
Article Snippet: .. FPR1 and BLT1 were tagged with EGFP- or mCherry-expressing sequence at the C-terminus and cloned into the pMSCVneo vector (Clontech Laboratories). .. C5aR1–EFGP was amplified from Addgene plasmid #62612 (deposited by Heini Miettinen; ). pMSCV-based constructs were transfected using Lipofectamine 2000 (Invitrogen) into Phoenix 293T packaging cells to produce retroviruses, which were then used to infect and select PLB cells.

Multiple Displacement Amplification:

Article Title: Syk Interacts with and Phosphorylates Nucleolin To Stabilize Bcl-xL mRNA and Promote Cell Survival
Article Snippet: .. MDA-MB-231 cells expressing Syk-EGFP, Syk-EGFP(K396R), or EGFP were constructed using a Lenti-X Tet-On advanced inducible expression system (Clontech). .. To constitutively express the tetracycline-controlled transactivator rtTA in the Tet-On inducible system, cells were first infected with viral particles with the pLVX Tet-On advanced regulator.

Construct:

Article Title: Syk Interacts with and Phosphorylates Nucleolin To Stabilize Bcl-xL mRNA and Promote Cell Survival
Article Snippet: .. MDA-MB-231 cells expressing Syk-EGFP, Syk-EGFP(K396R), or EGFP were constructed using a Lenti-X Tet-On advanced inducible expression system (Clontech). .. To constitutively express the tetracycline-controlled transactivator rtTA in the Tet-On inducible system, cells were first infected with viral particles with the pLVX Tet-On advanced regulator.

Polymerase Chain Reaction:

Article Title: Cervical Cancer Cells with Positive Sox2 Expression Exhibit the Properties of Cancer Stem Cells
Article Snippet: .. The correct human Sox2 promoter, UTR/enhancer, EGFP, and vector were subsequently cloned using an In-Fusion PCR Cloning Kit, and the resulting vector was designated phSox2/EGFP (Takara Bio Inc, Dalian, China). .. Immunohistochemistry and Immunocytochemistry Immunohistochemistry was performed on 4-µm sections of paraffin-embedded tissues.

Generated:

Article Title: Regulation of ASIC channels by a stomatin/STOML3 complex located in a mobile vesicle pool in sensory neurons
Article Snippet: .. A red version of STOML3 was generated by replacing EGFP with mCherry red fluorescent protein and a Strep-tagged cyan version by cloning into pAmCyan1-N1 (Clontech). .. Mouse (1a, 1b, 4) and rat (2a, 2b, 3) full-length ASIC sequences were cloned into pEYFP-C1 (Clontech) and pFLAG-CMV-2 (Sigma).

Plasmid Preparation:

Article Title: Cervical Cancer Cells with Positive Sox2 Expression Exhibit the Properties of Cancer Stem Cells
Article Snippet: .. The correct human Sox2 promoter, UTR/enhancer, EGFP, and vector were subsequently cloned using an In-Fusion PCR Cloning Kit, and the resulting vector was designated phSox2/EGFP (Takara Bio Inc, Dalian, China). .. Immunohistochemistry and Immunocytochemistry Immunohistochemistry was performed on 4-µm sections of paraffin-embedded tissues.

Article Title: The LTB4–BLT1 axis regulates the polarized trafficking of chemoattractant GPCRs during neutrophil chemotaxis
Article Snippet: .. FPR1 and BLT1 were tagged with EGFP- or mCherry-expressing sequence at the C-terminus and cloned into the pMSCVneo vector (Clontech Laboratories). .. C5aR1–EFGP was amplified from Addgene plasmid #62612 (deposited by Heini Miettinen; ). pMSCV-based constructs were transfected using Lipofectamine 2000 (Invitrogen) into Phoenix 293T packaging cells to produce retroviruses, which were then used to infect and select PLB cells.

Article Title: Expression of ?5 integrin rescues fibronectin responsiveness in NT2N CNS neuronal cells
Article Snippet: .. The retroviral vector pMSCVpuro (Clontech), containing EGFP or α5 integrin cDNA inserts, was co-transfected along with a VSVG retroviral coat protein expression vector into GP2-293 packaging cells (Clontech). .. The resulting supernatants were used to transduce PT67 packaging cells (Clontech), and stably transduced, virus-producing PT67 cell populations were selected.

Expressing:

Article Title: Identifying Activated T Cells in Reconstituted RAG Deficient Mice Using Retrovirally Transduced Pax5 Deficient Pro-B Cells
Article Snippet: .. EGFP was cloned downstream of the retroviral LTR promoter followed by the CD40L promoter driving the expression of HcRed (Clontech) with the small t-intron and polyadenylation sequence from SV40 ( ). .. Consequently, cells transfected with this construct will constitutively express EGFP and will only express HcRed, a far-red fluorescent protein from Heteractis crispa , upon activation of CD40L promoter, e.g. upon antigen challenge.

Article Title: Syk Interacts with and Phosphorylates Nucleolin To Stabilize Bcl-xL mRNA and Promote Cell Survival
Article Snippet: .. MDA-MB-231 cells expressing Syk-EGFP, Syk-EGFP(K396R), or EGFP were constructed using a Lenti-X Tet-On advanced inducible expression system (Clontech). .. To constitutively express the tetracycline-controlled transactivator rtTA in the Tet-On inducible system, cells were first infected with viral particles with the pLVX Tet-On advanced regulator.

Article Title: Expression of ?5 integrin rescues fibronectin responsiveness in NT2N CNS neuronal cells
Article Snippet: .. The retroviral vector pMSCVpuro (Clontech), containing EGFP or α5 integrin cDNA inserts, was co-transfected along with a VSVG retroviral coat protein expression vector into GP2-293 packaging cells (Clontech). .. The resulting supernatants were used to transduce PT67 packaging cells (Clontech), and stably transduced, virus-producing PT67 cell populations were selected.

Sequencing:

Article Title: Identifying Activated T Cells in Reconstituted RAG Deficient Mice Using Retrovirally Transduced Pax5 Deficient Pro-B Cells
Article Snippet: .. EGFP was cloned downstream of the retroviral LTR promoter followed by the CD40L promoter driving the expression of HcRed (Clontech) with the small t-intron and polyadenylation sequence from SV40 ( ). .. Consequently, cells transfected with this construct will constitutively express EGFP and will only express HcRed, a far-red fluorescent protein from Heteractis crispa , upon activation of CD40L promoter, e.g. upon antigen challenge.

Article Title: The LTB4–BLT1 axis regulates the polarized trafficking of chemoattractant GPCRs during neutrophil chemotaxis
Article Snippet: .. FPR1 and BLT1 were tagged with EGFP- or mCherry-expressing sequence at the C-terminus and cloned into the pMSCVneo vector (Clontech Laboratories). .. C5aR1–EFGP was amplified from Addgene plasmid #62612 (deposited by Heini Miettinen; ). pMSCV-based constructs were transfected using Lipofectamine 2000 (Invitrogen) into Phoenix 293T packaging cells to produce retroviruses, which were then used to infect and select PLB cells.

Derivative Assay:

Article Title: PiggyBac transgenic strategies in the developing chicken spinal cord
Article Snippet: .. EGFP , destabilized GFP and DsRed vectors were derived from Clontech (pEGFP-N1, pd2EGFP, pDsRedN2 ). .. The PiggyBac and PBase expressing vectors were described previously ( ). pCYL50 , a PB vector containing multiple cloning sites, was used as the parental plasmid to construct all PB derivatives.

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  • 92
    TaKaRa hr5 ie1 egfp sv40 cassette
    Schematic representation of the B. mori W chromosome and donor plasmids. ( A ) Schematic representation of female-specific RAPD markers on the W chromosome (not to scale). The gray region represents the W chromosome. The black rectangles are the female-specific RAPD markers. A 50-bp fragment located in the Rikishi RAPD marker is the targeted site. The sequences in blue and underlined are the targets of the TALENs. A 17-bp sequence serves as a spacer between the two sites. ( B ) Schematic representation of the genomic target site and donor plasmid A. Donor A contains a cassette expressing DsRed2 under the control of the <t>HR5-IE1</t> promoter and the <t>SV40</t> polyadenylation site. Two homologous DNA fragments (L-homo and R-homo), each 1,000 bp in length at the 5′- and 3′-ends of the TALEN sites, were cloned separately into the left and right sides of the DsRed2 cassette. The two homologous fragments were both flanked by two of the 50-bp TALEN sites (blue). ( C ) Donor B contains two cassettes expressing <t>EGFP</t> under the control of an HR5-IE1 promoter and Cas9 driven by the embryo-specific nos promoter. Two homologous DNA fragments (L-homo and R-homo), each 1,000 bp in length at the 5′- and 3′-ends of the TALEN site, were cloned separately to the left of the Cas9 cassette and to the right of the EGFP cassette. All other descriptions are the same as in B .
    Hr5 Ie1 Egfp Sv40 Cassette, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hr5 ie1 egfp sv40 cassette/product/TaKaRa
    Average 92 stars, based on 2 article reviews
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    92
    TaKaRa egfp bax expression vector
    Enhanced green fluorescent protein <t>(EGFP)‐Bax</t> 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
    Egfp Bax Expression Vector, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/egfp bax expression vector/product/TaKaRa
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    Price from $9.99 to $1999.99
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    93
    TaKaRa bamhi h2b egfp
    Chromatin architecture demarcates the repair locus. ( A ) FLIM-FRET maps acquired in HeLa <t>H2B-2FP</t> cells 10 min before and 0, 30, and 60 min after microirradiation ( Upper ) and expanded images of the DSB ROI ( Lower ). ( B ) <t>eGFP-53BP1</t> intensity images acquired in HeLa cells 10 min before and 0, 30, and 60 min after microirradiation ( Upper ) and expanded images of the DSB ROI ( Lower ). ( C ) Correlation of compact chromatin foci localization along the horizontal axis as a function of time ( Top ; image from 0 min is shown), with 53BP1 localization ( Middle ; image from 0 min is shown), and mobility in terms of time delay ( Bottom ). eGFP-53BP1 localization and mobility are averaged along the horizontal axes (green plots). Red dashed box indicates laser microirradiation ROI. ( D ) Comparison of compact chromatin foci localization ( Top ), eGFP-53BP1 localization ( Middle ), and eGFP-53BP1 mobility ( Bottom ) in untreated HeLa cells (blue curve), KU-55933–treated HeLa cells (green curve), or RNF8 KO cells (red curve) 10 min before and 0, 30, and 60 min after microirradiation. Representative example of n = 3 shown. (Scale bars, 5 μm.)
    Bamhi H2b Egfp, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    TaKaRa egfp g3bp1 q325e mutant
    EMCV infection results in the cleavage of <t>G3BP1.</t> (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 <t>Q325E</t> 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.
    Egfp G3bp1 Q325e Mutant, 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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    Image Search Results


    Schematic representation of the B. mori W chromosome and donor plasmids. ( A ) Schematic representation of female-specific RAPD markers on the W chromosome (not to scale). The gray region represents the W chromosome. The black rectangles are the female-specific RAPD markers. A 50-bp fragment located in the Rikishi RAPD marker is the targeted site. The sequences in blue and underlined are the targets of the TALENs. A 17-bp sequence serves as a spacer between the two sites. ( B ) Schematic representation of the genomic target site and donor plasmid A. Donor A contains a cassette expressing DsRed2 under the control of the HR5-IE1 promoter and the SV40 polyadenylation site. Two homologous DNA fragments (L-homo and R-homo), each 1,000 bp in length at the 5′- and 3′-ends of the TALEN sites, were cloned separately into the left and right sides of the DsRed2 cassette. The two homologous fragments were both flanked by two of the 50-bp TALEN sites (blue). ( C ) Donor B contains two cassettes expressing EGFP under the control of an HR5-IE1 promoter and Cas9 driven by the embryo-specific nos promoter. Two homologous DNA fragments (L-homo and R-homo), each 1,000 bp in length at the 5′- and 3′-ends of the TALEN site, were cloned separately to the left of the Cas9 cassette and to the right of the EGFP cassette. All other descriptions are the same as in B .

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

    Article Title: Silkworm genetic sexing through W chromosome-linked, targeted gene integration

    doi: 10.1073/pnas.1810945115

    Figure Lengend Snippet: Schematic representation of the B. mori W chromosome and donor plasmids. ( A ) Schematic representation of female-specific RAPD markers on the W chromosome (not to scale). The gray region represents the W chromosome. The black rectangles are the female-specific RAPD markers. A 50-bp fragment located in the Rikishi RAPD marker is the targeted site. The sequences in blue and underlined are the targets of the TALENs. A 17-bp sequence serves as a spacer between the two sites. ( B ) Schematic representation of the genomic target site and donor plasmid A. Donor A contains a cassette expressing DsRed2 under the control of the HR5-IE1 promoter and the SV40 polyadenylation site. Two homologous DNA fragments (L-homo and R-homo), each 1,000 bp in length at the 5′- and 3′-ends of the TALEN sites, were cloned separately into the left and right sides of the DsRed2 cassette. The two homologous fragments were both flanked by two of the 50-bp TALEN sites (blue). ( C ) Donor B contains two cassettes expressing EGFP under the control of an HR5-IE1 promoter and Cas9 driven by the embryo-specific nos promoter. Two homologous DNA fragments (L-homo and R-homo), each 1,000 bp in length at the 5′- and 3′-ends of the TALEN site, were cloned separately to the left of the Cas9 cassette and to the right of the EGFP cassette. All other descriptions are the same as in B .

    Article Snippet: The HAs (1,000 bp) flanking TALEN sites were amplified using genomic DNA as the template and cloned into pGEMT_HR5-IE1-DsRed2-SV40 to generate donor A. Donor B (pGEMT_L-Homo_HR5-IE1-EGFP-SV40_Nos-Cas9-SV40_R-Homo): An HR5-IE1-EGFP-SV40 cassette was subcloned into the pGEMT-simple vector (Takara) to generate pGEMT_HR5-IE1-EGFP-SV40.

    Techniques: Marker, TALENs, Sequencing, Plasmid Preparation, Expressing, Clone Assay

    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

    Journal: Journal of Cellular and Molecular Medicine

    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

    doi: 10.1111/jcmm.14076

    Figure Lengend 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

    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.

    Techniques: 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

    Journal: Journal of Cellular and Molecular Medicine

    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

    doi: 10.1111/jcmm.14076

    Figure Lengend 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

    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.

    Techniques: 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

    Journal: Journal of Cellular and Molecular Medicine

    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

    doi: 10.1111/jcmm.14076

    Figure Lengend 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

    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.

    Techniques: Translocation Assay, High Throughput Screening Assay

    Chromatin architecture demarcates the repair locus. ( A ) FLIM-FRET maps acquired in HeLa H2B-2FP cells 10 min before and 0, 30, and 60 min after microirradiation ( Upper ) and expanded images of the DSB ROI ( Lower ). ( B ) eGFP-53BP1 intensity images acquired in HeLa cells 10 min before and 0, 30, and 60 min after microirradiation ( Upper ) and expanded images of the DSB ROI ( Lower ). ( C ) Correlation of compact chromatin foci localization along the horizontal axis as a function of time ( Top ; image from 0 min is shown), with 53BP1 localization ( Middle ; image from 0 min is shown), and mobility in terms of time delay ( Bottom ). eGFP-53BP1 localization and mobility are averaged along the horizontal axes (green plots). Red dashed box indicates laser microirradiation ROI. ( D ) Comparison of compact chromatin foci localization ( Top ), eGFP-53BP1 localization ( Middle ), and eGFP-53BP1 mobility ( Bottom ) in untreated HeLa cells (blue curve), KU-55933–treated HeLa cells (green curve), or RNF8 KO cells (red curve) 10 min before and 0, 30, and 60 min after microirradiation. Representative example of n = 3 shown. (Scale bars, 5 μm.)

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

    Article Title: Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response

    doi: 10.1073/pnas.1814965116

    Figure Lengend Snippet: Chromatin architecture demarcates the repair locus. ( A ) FLIM-FRET maps acquired in HeLa H2B-2FP cells 10 min before and 0, 30, and 60 min after microirradiation ( Upper ) and expanded images of the DSB ROI ( Lower ). ( B ) eGFP-53BP1 intensity images acquired in HeLa cells 10 min before and 0, 30, and 60 min after microirradiation ( Upper ) and expanded images of the DSB ROI ( Lower ). ( C ) Correlation of compact chromatin foci localization along the horizontal axis as a function of time ( Top ; image from 0 min is shown), with 53BP1 localization ( Middle ; image from 0 min is shown), and mobility in terms of time delay ( Bottom ). eGFP-53BP1 localization and mobility are averaged along the horizontal axes (green plots). Red dashed box indicates laser microirradiation ROI. ( D ) Comparison of compact chromatin foci localization ( Top ), eGFP-53BP1 localization ( Middle ), and eGFP-53BP1 mobility ( Bottom ) in untreated HeLa cells (blue curve), KU-55933–treated HeLa cells (green curve), or RNF8 KO cells (red curve) 10 min before and 0, 30, and 60 min after microirradiation. Representative example of n = 3 shown. (Scale bars, 5 μm.)

    Article Snippet: Plasmids for Cell Line and Vector Generation. pLXSN H2B-mCherry was a gift from Laure Crabbe, The Centre for Integrative Biology, Toulouse, France ( ). pWZL H2B-eGFP was created by PCR-amplifying H2B-eGFP from eGFP-N1 H2B-eGFP (a gift from Geoff Wahl, The Salk Institute for Biological Sciences, La Jolla, CA; Addgene plasmid no. 11680) ( ) using the BamHI H2B-eGFP forward and EcoRI H2B-eGFP reverse oligonucleotides (SI Appendix , Table S1 ), followed by in-fusion (Clontech) cloning into BamHI- and EcoRI-digested (New England Biolabs) pWZL vector (a gift from Scott Lowe, Memorial Sloan Kettering Cancer Center, New York; Addgene no. 18750). pLentiCRISPRv2 was a gift from Feng Zhang, Massachusetts Institute of Technology, Cambridge, MA (Addgene plasmid no. 52961) ( ). pLentiCRISPRv2-RNF8-2 was generated by cloning an sgRNA sequence targeting RNF8 exon 1 by annealing the sgRNF8-2 sense and antisense oligonucleotides (SI Appendix , Table S1 ) and in-fusion cloning into BsmBI-digested pLentiCRISPRv2. pcDNA5-FRT/TO-eGFP-53BP1 was a gift from Dan Durocher, The Lunenfeld-Tanenbaum Research Institute, Toronto (Addgene plasmid no. 60813) ( ).

    Techniques:

    Phasor approach to FLIM-FRET analysis of chromatin compaction. ( A ) HeLa H2B-2FP nucleus coexpressing H2B-eGFP and H2B-mCherry (H2B-mCh). ( B ) Graphical depiction of how increasing nucleosome proximity leads to increased FRET between fluorescent histones. ( C ) Graphical depiction of phasor transformation of HeLa H2B-2FP FLIM-FRET data. ( C , Left ) Fluorescence lifetime of H2B-eGFP reports on the degree of FRET interaction in each pixel. Each line represents the fluorescent lifetime from a different pixel. ( C , Right ) These data when phasor-transformed give rise to phasor coordinates ( s , g ). The donor phasor is right-shifted to shorter fluorescent lifetimes depending on the efficiency of FRET interaction. In HeLa H2B-2FP , decreasing lifetime and increasing FRET corresponds to more compact chromatin. ( D ) Untreated, TSA-treated, or Actinomycin D (Act D)-treated HeLa H2B-2FP nuclei, shown in the H2B-eGFP channel. ( E ) Combined phasor distribution of H2B-eGFP fluorescence lifetime from all conditions shown in D with the theoretical FRET trajectory superimposed to determine the range of FRET efficiencies in HeLa H2B-2FP . The linear combination of unquenched donor and background cellular autofluorescence (teal–bright green) (defined in SI Appendix , Fig. S3 ) follows a distinct trajectory from FRET (teal–red). ( F ) Fraction of pixels in a compact (red) vs. open (teal) chromatin state in control (Cntrl), TSA-, and Act D-treated cells. ( G ) Pseudocolored chromatin compaction maps of the cells in D according to the palette defined in the phasor plot data in E . (Scale bars, 5 μm.)

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

    Article Title: Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response

    doi: 10.1073/pnas.1814965116

    Figure Lengend Snippet: Phasor approach to FLIM-FRET analysis of chromatin compaction. ( A ) HeLa H2B-2FP nucleus coexpressing H2B-eGFP and H2B-mCherry (H2B-mCh). ( B ) Graphical depiction of how increasing nucleosome proximity leads to increased FRET between fluorescent histones. ( C ) Graphical depiction of phasor transformation of HeLa H2B-2FP FLIM-FRET data. ( C , Left ) Fluorescence lifetime of H2B-eGFP reports on the degree of FRET interaction in each pixel. Each line represents the fluorescent lifetime from a different pixel. ( C , Right ) These data when phasor-transformed give rise to phasor coordinates ( s , g ). The donor phasor is right-shifted to shorter fluorescent lifetimes depending on the efficiency of FRET interaction. In HeLa H2B-2FP , decreasing lifetime and increasing FRET corresponds to more compact chromatin. ( D ) Untreated, TSA-treated, or Actinomycin D (Act D)-treated HeLa H2B-2FP nuclei, shown in the H2B-eGFP channel. ( E ) Combined phasor distribution of H2B-eGFP fluorescence lifetime from all conditions shown in D with the theoretical FRET trajectory superimposed to determine the range of FRET efficiencies in HeLa H2B-2FP . The linear combination of unquenched donor and background cellular autofluorescence (teal–bright green) (defined in SI Appendix , Fig. S3 ) follows a distinct trajectory from FRET (teal–red). ( F ) Fraction of pixels in a compact (red) vs. open (teal) chromatin state in control (Cntrl), TSA-, and Act D-treated cells. ( G ) Pseudocolored chromatin compaction maps of the cells in D according to the palette defined in the phasor plot data in E . (Scale bars, 5 μm.)

    Article Snippet: Plasmids for Cell Line and Vector Generation. pLXSN H2B-mCherry was a gift from Laure Crabbe, The Centre for Integrative Biology, Toulouse, France ( ). pWZL H2B-eGFP was created by PCR-amplifying H2B-eGFP from eGFP-N1 H2B-eGFP (a gift from Geoff Wahl, The Salk Institute for Biological Sciences, La Jolla, CA; Addgene plasmid no. 11680) ( ) using the BamHI H2B-eGFP forward and EcoRI H2B-eGFP reverse oligonucleotides (SI Appendix , Table S1 ), followed by in-fusion (Clontech) cloning into BamHI- and EcoRI-digested (New England Biolabs) pWZL vector (a gift from Scott Lowe, Memorial Sloan Kettering Cancer Center, New York; Addgene no. 18750). pLentiCRISPRv2 was a gift from Feng Zhang, Massachusetts Institute of Technology, Cambridge, MA (Addgene plasmid no. 52961) ( ). pLentiCRISPRv2-RNF8-2 was generated by cloning an sgRNA sequence targeting RNF8 exon 1 by annealing the sgRNF8-2 sense and antisense oligonucleotides (SI Appendix , Table S1 ) and in-fusion cloning into BsmBI-digested pLentiCRISPRv2. pcDNA5-FRT/TO-eGFP-53BP1 was a gift from Dan Durocher, The Lunenfeld-Tanenbaum Research Institute, Toronto (Addgene plasmid no. 60813) ( ).

    Techniques: Transformation Assay, Fluorescence, Activated Clotting Time Assay

    FLIM-FRET analysis of chromatin compaction reveals chromatin architectural changes during the DDR. ( A and B ) Time series of H2B-eGFP fluorescence intensity images ( A ) and lifetime maps ( B ) acquired in a HeLa H2B-2FP 10 min before and at hourly intervals after NIR irradiation. The white square indicates the NIR laser-treated locus. ( C ) Digital enlargement of the DNA damage site selected in B and the corresponding time series of lifetime maps within this ROI. ( D ) Masks selected for analysis of the number of pixels in a compacted (high-FRET) vs. noncompacted (low-FRET) state at the DNA damage site vs. outside this ROI. ( E and F ) Fraction of pixels within ( E ) and outside ( F ) of the NIR-irradiated ROI that are in a compacted state during the DDR (red curve) vs. an unperturbed cell (black curve) ( n = 10 cells, mean ± SEM). (Scale bars, 5 μm.)

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

    Article Title: Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response

    doi: 10.1073/pnas.1814965116

    Figure Lengend Snippet: FLIM-FRET analysis of chromatin compaction reveals chromatin architectural changes during the DDR. ( A and B ) Time series of H2B-eGFP fluorescence intensity images ( A ) and lifetime maps ( B ) acquired in a HeLa H2B-2FP 10 min before and at hourly intervals after NIR irradiation. The white square indicates the NIR laser-treated locus. ( C ) Digital enlargement of the DNA damage site selected in B and the corresponding time series of lifetime maps within this ROI. ( D ) Masks selected for analysis of the number of pixels in a compacted (high-FRET) vs. noncompacted (low-FRET) state at the DNA damage site vs. outside this ROI. ( E and F ) Fraction of pixels within ( E ) and outside ( F ) of the NIR-irradiated ROI that are in a compacted state during the DDR (red curve) vs. an unperturbed cell (black curve) ( n = 10 cells, mean ± SEM). (Scale bars, 5 μm.)

    Article Snippet: Plasmids for Cell Line and Vector Generation. pLXSN H2B-mCherry was a gift from Laure Crabbe, The Centre for Integrative Biology, Toulouse, France ( ). pWZL H2B-eGFP was created by PCR-amplifying H2B-eGFP from eGFP-N1 H2B-eGFP (a gift from Geoff Wahl, The Salk Institute for Biological Sciences, La Jolla, CA; Addgene plasmid no. 11680) ( ) using the BamHI H2B-eGFP forward and EcoRI H2B-eGFP reverse oligonucleotides (SI Appendix , Table S1 ), followed by in-fusion (Clontech) cloning into BamHI- and EcoRI-digested (New England Biolabs) pWZL vector (a gift from Scott Lowe, Memorial Sloan Kettering Cancer Center, New York; Addgene no. 18750). pLentiCRISPRv2 was a gift from Feng Zhang, Massachusetts Institute of Technology, Cambridge, MA (Addgene plasmid no. 52961) ( ). pLentiCRISPRv2-RNF8-2 was generated by cloning an sgRNA sequence targeting RNF8 exon 1 by annealing the sgRNF8-2 sense and antisense oligonucleotides (SI Appendix , Table S1 ) and in-fusion cloning into BsmBI-digested pLentiCRISPRv2. pcDNA5-FRT/TO-eGFP-53BP1 was a gift from Dan Durocher, The Lunenfeld-Tanenbaum Research Institute, Toronto (Addgene plasmid no. 60813) ( ).

    Techniques: Fluorescence, Irradiation

    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.

    Journal: Journal of Virology

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

    doi: 10.1128/JVI.03248-12

    Figure Lengend 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.

    Article Snippet: To generate HeLa cells stably expressing the EGFP-G3BP1 wild type (wt) and the EGFP-G3BP1 Q325E mutant, pEGFP-C1-G3BP1 and pEGFP-C1-G3BP1 Q325E mutant expression constructs were linearized by using the restriction enzyme ApaLI (TaKaRa, Japan).

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