Structured Review

Millipore anti flag
<t>MLKL</t> directly binds to Listeria and inhibits their replication. (A) HT-29 cells transiently expressing <t>FLAG-tagged</t> MLKL were infected with Listeria (MOI of 30) and cultured for 6 h. The cells were fixed and stained with anti-FLAG antibody and anti- Listeria antibody, and analyzed by confocal microscope. Arrowheads show the colocalization of MLKL with Listeria . Bar, 10 µm. (B) HeLa cell lysates containing either VENUS or NBB 140 -2xFV-VENUS were treated with vehicle or DIM, and incubated with Listeria for 1 h. Listeria -bound NBB 140 -2xFV-VENUS was detected by Western blotting. To determine the oligomerization state of NBB 140 -2xFV in the presence and absence of DIM treatment, cell lysates were treated with a cross-linker disuccinimidyl suberate (+DSS). (C) HeLa cell lysates containing either VENUS or NBB 140 -2xFV-VENUS were treated with vehicle (EtOH) or DIM, and incubated with the indicated bacteria for 2 h. Bacterial CFU relative to vehicle-treated, VENUS lysate sample is shown. (mean ± SEM; n = 6; **, P
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Images

1) Product Images from "Necroptosis mediators RIPK3 and MLKL suppress intracellular Listeria replication independently of host cell killing"

Article Title: Necroptosis mediators RIPK3 and MLKL suppress intracellular Listeria replication independently of host cell killing

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201810014

MLKL directly binds to Listeria and inhibits their replication. (A) HT-29 cells transiently expressing FLAG-tagged MLKL were infected with Listeria (MOI of 30) and cultured for 6 h. The cells were fixed and stained with anti-FLAG antibody and anti- Listeria antibody, and analyzed by confocal microscope. Arrowheads show the colocalization of MLKL with Listeria . Bar, 10 µm. (B) HeLa cell lysates containing either VENUS or NBB 140 -2xFV-VENUS were treated with vehicle or DIM, and incubated with Listeria for 1 h. Listeria -bound NBB 140 -2xFV-VENUS was detected by Western blotting. To determine the oligomerization state of NBB 140 -2xFV in the presence and absence of DIM treatment, cell lysates were treated with a cross-linker disuccinimidyl suberate (+DSS). (C) HeLa cell lysates containing either VENUS or NBB 140 -2xFV-VENUS were treated with vehicle (EtOH) or DIM, and incubated with the indicated bacteria for 2 h. Bacterial CFU relative to vehicle-treated, VENUS lysate sample is shown. (mean ± SEM; n = 6; **, P
Figure Legend Snippet: MLKL directly binds to Listeria and inhibits their replication. (A) HT-29 cells transiently expressing FLAG-tagged MLKL were infected with Listeria (MOI of 30) and cultured for 6 h. The cells were fixed and stained with anti-FLAG antibody and anti- Listeria antibody, and analyzed by confocal microscope. Arrowheads show the colocalization of MLKL with Listeria . Bar, 10 µm. (B) HeLa cell lysates containing either VENUS or NBB 140 -2xFV-VENUS were treated with vehicle or DIM, and incubated with Listeria for 1 h. Listeria -bound NBB 140 -2xFV-VENUS was detected by Western blotting. To determine the oligomerization state of NBB 140 -2xFV in the presence and absence of DIM treatment, cell lysates were treated with a cross-linker disuccinimidyl suberate (+DSS). (C) HeLa cell lysates containing either VENUS or NBB 140 -2xFV-VENUS were treated with vehicle (EtOH) or DIM, and incubated with the indicated bacteria for 2 h. Bacterial CFU relative to vehicle-treated, VENUS lysate sample is shown. (mean ± SEM; n = 6; **, P

Techniques Used: Expressing, Infection, Cell Culture, Staining, Microscopy, Incubation, Western Blot

Listeria invasion–induced phosphorylation of MLKL does not lead to necroptotic cell death. (A) FLAG-tagged RIPK3 stably expressing (RIPK3+) HeLa cells were either treated with 50 ng/ml TNF, 100 nM SMAC mimetic, 20 µM Q-VD-OPh (TSQ) for the indicated time periods, or infected with Listeria (MOI of 10 and 100) for 24 h. Phosphorylation (top panel) and oligomerization (bottom panel) of MLKL were analyzed by Western blotting. *, Nonspecific bands. (B and C) Control and RIPK3+ HeLa cells were either treated with TSQ for the indicated time periods, or infected with Listeria (MOI of 10 and 100) and cultured for 24 h. Phosphorylation level of MLKL (B, Western blotting), bright-field images of the cells (B, right panels), and cell viability measured by crystal violet staining (C) are shown. Bar, 200 µm. (D) Control and FLAG-RIPK3 stably expressing (RIPK3 + ) HeLa cells were either treated with TSQ or infected with Listeria (MOI of 100) and cultured for the indicated time periods. Floating dead cells were washed out after the culture. Membrane permeability of the remaining adherent cells was measured by incorporation of ethidium homodimer III (mean ± SEM; n = 3; ***, P
Figure Legend Snippet: Listeria invasion–induced phosphorylation of MLKL does not lead to necroptotic cell death. (A) FLAG-tagged RIPK3 stably expressing (RIPK3+) HeLa cells were either treated with 50 ng/ml TNF, 100 nM SMAC mimetic, 20 µM Q-VD-OPh (TSQ) for the indicated time periods, or infected with Listeria (MOI of 10 and 100) for 24 h. Phosphorylation (top panel) and oligomerization (bottom panel) of MLKL were analyzed by Western blotting. *, Nonspecific bands. (B and C) Control and RIPK3+ HeLa cells were either treated with TSQ for the indicated time periods, or infected with Listeria (MOI of 10 and 100) and cultured for 24 h. Phosphorylation level of MLKL (B, Western blotting), bright-field images of the cells (B, right panels), and cell viability measured by crystal violet staining (C) are shown. Bar, 200 µm. (D) Control and FLAG-RIPK3 stably expressing (RIPK3 + ) HeLa cells were either treated with TSQ or infected with Listeria (MOI of 100) and cultured for the indicated time periods. Floating dead cells were washed out after the culture. Membrane permeability of the remaining adherent cells was measured by incorporation of ethidium homodimer III (mean ± SEM; n = 3; ***, P

Techniques Used: Stable Transfection, Expressing, Infection, Western Blot, Cell Culture, Staining, Permeability

RIPK3-MLKL pathway suppresses intracellular replication of Listeria. (A and B) Control, FLAG-tagged RIPK3 stably expressing (RIPK3+) HeLa cells (A), and HT-29 cells (B) were infected with Listeria (MOI of 10) and cultured for the indicated periods. Phosphorylation levels of MLKL (pMLKL) and RIPK3 (pRIPK3) were analyzed by Western blotting. Gentamicin was added to the culture to eliminate extracellular Listeria . (C and D) Control and RIPK3+ HeLa cells were infected with Listeria (MOI of 10) and cultured for the indicated periods. The cells were stained with anti- Listeria antibody, phalloidin, and DAPI at the indicated time point (C). Bar, 20 µm. Fold replication of Listeria (relative to the number of bacteria at 2 hpi) was measured (D). (E) Control and RIPK3+ HeLa cells were treated with either vehicle (DMSO) or 1 µM GSK’872 for 30 min. The cells were then infected with Listeria (MOI of 10) and cultured for 24 h with either DMSO or 1 µM GSK’872. Listeria CFU relative to vehicle-treated control is shown. (F and G) Control, RIPK3+ HeLa (F), and HT-29 cells (G) were treated with either vehicle (DMSO) or 1 µM NSA for 30 min. The cells were then infected with Listeria (MOI of 10) and cultured for 24 h with either DMSO or 1 µM NSA. Listeria CFU relative to vehicle-treated control is shown (mean ± SEM; n = 3; *, P
Figure Legend Snippet: RIPK3-MLKL pathway suppresses intracellular replication of Listeria. (A and B) Control, FLAG-tagged RIPK3 stably expressing (RIPK3+) HeLa cells (A), and HT-29 cells (B) were infected with Listeria (MOI of 10) and cultured for the indicated periods. Phosphorylation levels of MLKL (pMLKL) and RIPK3 (pRIPK3) were analyzed by Western blotting. Gentamicin was added to the culture to eliminate extracellular Listeria . (C and D) Control and RIPK3+ HeLa cells were infected with Listeria (MOI of 10) and cultured for the indicated periods. The cells were stained with anti- Listeria antibody, phalloidin, and DAPI at the indicated time point (C). Bar, 20 µm. Fold replication of Listeria (relative to the number of bacteria at 2 hpi) was measured (D). (E) Control and RIPK3+ HeLa cells were treated with either vehicle (DMSO) or 1 µM GSK’872 for 30 min. The cells were then infected with Listeria (MOI of 10) and cultured for 24 h with either DMSO or 1 µM GSK’872. Listeria CFU relative to vehicle-treated control is shown. (F and G) Control, RIPK3+ HeLa (F), and HT-29 cells (G) were treated with either vehicle (DMSO) or 1 µM NSA for 30 min. The cells were then infected with Listeria (MOI of 10) and cultured for 24 h with either DMSO or 1 µM NSA. Listeria CFU relative to vehicle-treated control is shown (mean ± SEM; n = 3; *, P

Techniques Used: Stable Transfection, Expressing, Infection, Cell Culture, Western Blot, Staining

2) Product Images from "SUV420H1 enhances the phosphorylation and transcription of ERK1 in cancer cells"

Article Title: SUV420H1 enhances the phosphorylation and transcription of ERK1 in cancer cells

Journal: Oncotarget

doi:

Effects of SUV420H1-dependent methylation on ERK1 activity ( A ) HeLa cells were co-transfected with HA-SUV420H1 and FLAG-ERK1-WT, or expression vectors containing the deletion variants (FLAG-ERK1-K302A, FLAG-ERK1-K361A). Cell lysates were immunoprecipitated with anti-FLAG M2 agarose beads. Samples were fractionated by SDS-PAGE and immunoblotted with anti-FLAG and anti-phospho ERK1 (Thr 202/Tyr 204) antibodies. Graphical representation of p-ERK1 levels normalized by FLAG-ERK1. ( B ) 293T cells were transfected with FLAG-ERK1-WT and HA-Mock or HA-SUV420H1. Cells were lysed with CelLytic ™ M 48 hours after transfection, followed by immunoprecipitation using anti-FLAG M2 agarose. Samples were immunoblotted with anti-FLAG and anti-phospho ERK1 (Thr 202/Tyr 204) antibodies.
Figure Legend Snippet: Effects of SUV420H1-dependent methylation on ERK1 activity ( A ) HeLa cells were co-transfected with HA-SUV420H1 and FLAG-ERK1-WT, or expression vectors containing the deletion variants (FLAG-ERK1-K302A, FLAG-ERK1-K361A). Cell lysates were immunoprecipitated with anti-FLAG M2 agarose beads. Samples were fractionated by SDS-PAGE and immunoblotted with anti-FLAG and anti-phospho ERK1 (Thr 202/Tyr 204) antibodies. Graphical representation of p-ERK1 levels normalized by FLAG-ERK1. ( B ) 293T cells were transfected with FLAG-ERK1-WT and HA-Mock or HA-SUV420H1. Cells were lysed with CelLytic ™ M 48 hours after transfection, followed by immunoprecipitation using anti-FLAG M2 agarose. Samples were immunoblotted with anti-FLAG and anti-phospho ERK1 (Thr 202/Tyr 204) antibodies.

Techniques Used: Methylation, Activity Assay, Transfection, Expressing, Immunoprecipitation, SDS Page

Confirmation of lysine 302 and lysine 361 methylation by specific antibodies in vivo ( A ) Determination of the titer and specificity of the anti-tri-methylated K302 ERK1 (Sigma-Aldrich) antibody analyzed by ELISA. ( B ) The FLAG-ERK1-WT or FLAG-ERK1-K302A vector was co-transfected with the HA-Mock or HA-SUV420H1 vector into HeLa cells. Whole cell lysates were immunoprecipitated with anti-FLAG M2 agarose beads. Immunoprecipitants were immunoblotted with anti-ERK1-K302me3 and anti-FLAG (Sigma-Aldrich) antibodies. Graphical representation of ERK1-K302me3 levels normalized by FLAG-ERK1. ( C ) Determination of the titer and specificity of the anti-tri-methylated K361 ERK1 (Sigma-Aldrich) antibody analyzed by ELISA. ( D ) The FLAG-ERK1-WT or FLAG-ERK1-K361A vector was co-transfected with the HA-Mock or HA-SUV420H1 vector into HeLa cells. Whole cell lysates were immunoprecipitated with anti-FLAG M2 agarose beads. Immunoprecipitants were immunoblotted with anti-ERK1-K361me3 and anti-FLAG (Sigma-Aldrich) antibodies. Graphical representation of ERK1-K361me3 levels normalized by FLAG-ERK1.
Figure Legend Snippet: Confirmation of lysine 302 and lysine 361 methylation by specific antibodies in vivo ( A ) Determination of the titer and specificity of the anti-tri-methylated K302 ERK1 (Sigma-Aldrich) antibody analyzed by ELISA. ( B ) The FLAG-ERK1-WT or FLAG-ERK1-K302A vector was co-transfected with the HA-Mock or HA-SUV420H1 vector into HeLa cells. Whole cell lysates were immunoprecipitated with anti-FLAG M2 agarose beads. Immunoprecipitants were immunoblotted with anti-ERK1-K302me3 and anti-FLAG (Sigma-Aldrich) antibodies. Graphical representation of ERK1-K302me3 levels normalized by FLAG-ERK1. ( C ) Determination of the titer and specificity of the anti-tri-methylated K361 ERK1 (Sigma-Aldrich) antibody analyzed by ELISA. ( D ) The FLAG-ERK1-WT or FLAG-ERK1-K361A vector was co-transfected with the HA-Mock or HA-SUV420H1 vector into HeLa cells. Whole cell lysates were immunoprecipitated with anti-FLAG M2 agarose beads. Immunoprecipitants were immunoblotted with anti-ERK1-K361me3 and anti-FLAG (Sigma-Aldrich) antibodies. Graphical representation of ERK1-K361me3 levels normalized by FLAG-ERK1.

Techniques Used: Methylation, In Vivo, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Transfection, Immunoprecipitation

3) Product Images from "The TEA domain family transcription factor TEAD4 represses murine adipogenesis by recruiting the cofactors VGLL4 and CtBP2 into a transcriptional complex"

Article Title: The TEA domain family transcription factor TEAD4 represses murine adipogenesis by recruiting the cofactors VGLL4 and CtBP2 into a transcriptional complex

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.003608

CtBP2 formed a complex with TEAD4-VGLL4. A , co-IP assay of the interaction between CtBP2 and VGLL4. The indicated plasmids were transfected into 293T cells and analyzed by co-IP. IB , immunoblot; MW , molecular weight. B , the endogenous interaction between CtBP2 and VGLL4. 293T cells lysates were incubated with CtBP2 or IgG antibodies, followed by immunoprecipitation and Western blot analysis with the indicated antibodies. The arrowhead indicates the IgG heavy chain. C and D , co-IP assay of the interaction between TEAD4 and CtBP2 ( C ) and TEAD4 and VGLL4 ( D ). The indicated plasmids were transfected into 293T cells and analyzed by co-IP. E , an MBP pulldown assay showed that the TEAD4 and TEAD4 C termini were pulled down by MBP-CtBP2. MBP or MBP-CtBP2 bound to MBP beads were incubated with IVTT 35 S-labeled TEAD4/TEAD4-N/TEAD4-C for 3 h. Immobilized complexes were then washed and subjected to SDS-PAGE. The input was 10% of the total amount of IVTT 35 S-TEAD4/TEAD4-N/TEAD4-C. CBB , Coomassie Brilliant Blue staining. F , an MBP pulldown assay showed that VGLL4 and VGLL4 N termini were pulled down by MBP-CtBP2. MBP or MBP-CtBP2 bound to MBP beads were incubated with IVTT 35 S-labeled VGLL4/VGLL4-N/VGLL4-C for 3 h. Immobilized complexes were then washed and subjected to SDS-PAGE. The input was 10% of the total amount of IVTT 35 S-VGLL4/VGLL4-N/VGLL4-C. G , 293T cells expressing the indicated proteins were collected for two-step immunoprecipitation and analyzed by Western blotting. Cell lysates were first co-immunoprecipitated with M2-FLAG beads and then eluted for a second immunoprecipitation with Myc antibody. The arrowheads indicate the IgG heavy chains.
Figure Legend Snippet: CtBP2 formed a complex with TEAD4-VGLL4. A , co-IP assay of the interaction between CtBP2 and VGLL4. The indicated plasmids were transfected into 293T cells and analyzed by co-IP. IB , immunoblot; MW , molecular weight. B , the endogenous interaction between CtBP2 and VGLL4. 293T cells lysates were incubated with CtBP2 or IgG antibodies, followed by immunoprecipitation and Western blot analysis with the indicated antibodies. The arrowhead indicates the IgG heavy chain. C and D , co-IP assay of the interaction between TEAD4 and CtBP2 ( C ) and TEAD4 and VGLL4 ( D ). The indicated plasmids were transfected into 293T cells and analyzed by co-IP. E , an MBP pulldown assay showed that the TEAD4 and TEAD4 C termini were pulled down by MBP-CtBP2. MBP or MBP-CtBP2 bound to MBP beads were incubated with IVTT 35 S-labeled TEAD4/TEAD4-N/TEAD4-C for 3 h. Immobilized complexes were then washed and subjected to SDS-PAGE. The input was 10% of the total amount of IVTT 35 S-TEAD4/TEAD4-N/TEAD4-C. CBB , Coomassie Brilliant Blue staining. F , an MBP pulldown assay showed that VGLL4 and VGLL4 N termini were pulled down by MBP-CtBP2. MBP or MBP-CtBP2 bound to MBP beads were incubated with IVTT 35 S-labeled VGLL4/VGLL4-N/VGLL4-C for 3 h. Immobilized complexes were then washed and subjected to SDS-PAGE. The input was 10% of the total amount of IVTT 35 S-VGLL4/VGLL4-N/VGLL4-C. G , 293T cells expressing the indicated proteins were collected for two-step immunoprecipitation and analyzed by Western blotting. Cell lysates were first co-immunoprecipitated with M2-FLAG beads and then eluted for a second immunoprecipitation with Myc antibody. The arrowheads indicate the IgG heavy chains.

Techniques Used: Co-Immunoprecipitation Assay, Transfection, Molecular Weight, Incubation, Immunoprecipitation, Western Blot, Labeling, SDS Page, Staining, Expressing

4) Product Images from "A New Cell-penetrating Peptide That Blocks the Autoinhibitory XIP Domain of NCX1 and Enhances Antiporter Activity"

Article Title: A New Cell-penetrating Peptide That Blocks the Autoinhibitory XIP Domain of NCX1 and Enhances Antiporter Activity

Journal: Molecular Therapy

doi: 10.1038/mt.2014.231

Interaction between EGFP-P1 and NCX1. ( a ) Top, representative scheme of P1 Flag structure. Bottom, immunocitochemical assay of the time- and concentration-dependent penetration of P1 Flag (red) in BHK-NCX1 cells. Hoecst dye (blue) was used to mark nuclei.
Figure Legend Snippet: Interaction between EGFP-P1 and NCX1. ( a ) Top, representative scheme of P1 Flag structure. Bottom, immunocitochemical assay of the time- and concentration-dependent penetration of P1 Flag (red) in BHK-NCX1 cells. Hoecst dye (blue) was used to mark nuclei.

Techniques Used: Concentration Assay

5) Product Images from "Polycystin-1 regulates bone development through an interaction with the transcriptional coactivator TAZ"

Article Title: Polycystin-1 regulates bone development through an interaction with the transcriptional coactivator TAZ

Journal: Human Molecular Genetics

doi: 10.1093/hmg/ddy322

PC1-CTT binds to TAZ without disrupting the TAZ-14-3-3 interaction. (A) HEK293 cells were co-transfected with FLAG-TAZ or FLAG-YAP and HA-PC1-CTT. Cell lysates were subjected to immunoprecipitation using anti-FLAG sepharose, then blotted with the indicated antibodies. (B) HEK293 cells were co-transfected with FLAG-TAZ and HA-PC1-CTT. Cell lysates were subjected to immunoprecipitation using anti-HA sepharose, then blotted with the indicated antibodies. (C) A GST-tagged construct containing the C-terminal 91 amino acids of the PC1-CTT (p91) was produced in BL21 bacteria and purified on glutathione-sepharose 4B beads. The GST-p91 coated glutathione beads were then exposed to lysates from HEK293 cells expressing FLAG-TAZ and the resulting complexes were blotted with the indicated antibodies. (D) HEK293 cells were co-transfected with FLAG-TAZ(WT) or FLAG-TAZ(S89A) and HA-PC1-CTT. Cell lysates were subjected to immunoprecipitation using anti-FLAG sepharose, then blotted with the indicated antibodies.
Figure Legend Snippet: PC1-CTT binds to TAZ without disrupting the TAZ-14-3-3 interaction. (A) HEK293 cells were co-transfected with FLAG-TAZ or FLAG-YAP and HA-PC1-CTT. Cell lysates were subjected to immunoprecipitation using anti-FLAG sepharose, then blotted with the indicated antibodies. (B) HEK293 cells were co-transfected with FLAG-TAZ and HA-PC1-CTT. Cell lysates were subjected to immunoprecipitation using anti-HA sepharose, then blotted with the indicated antibodies. (C) A GST-tagged construct containing the C-terminal 91 amino acids of the PC1-CTT (p91) was produced in BL21 bacteria and purified on glutathione-sepharose 4B beads. The GST-p91 coated glutathione beads were then exposed to lysates from HEK293 cells expressing FLAG-TAZ and the resulting complexes were blotted with the indicated antibodies. (D) HEK293 cells were co-transfected with FLAG-TAZ(WT) or FLAG-TAZ(S89A) and HA-PC1-CTT. Cell lysates were subjected to immunoprecipitation using anti-FLAG sepharose, then blotted with the indicated antibodies.

Techniques Used: Transfection, Immunoprecipitation, Construct, Produced, Purification, Expressing

6) Product Images from "Modulation of Smooth Muscle Gene Expression by Association of Histone Acetyltransferases and Deacetylases with Myocardin"

Article Title: Modulation of Smooth Muscle Gene Expression by Association of Histone Acetyltransferases and Deacetylases with Myocardin

Journal:

doi: 10.1128/MCB.25.1.364-376.2005

Simultaneous interaction of myocardin with p300 and HDAC5. COS cells were transiently transfected with expression vectors encoding hemagglutinin (HA)-tagged p300, FLAG-tagged myocardin, and Myc-tagged HDAC5. FLAG-tagged myocardin was immunoprecipitated
Figure Legend Snippet: Simultaneous interaction of myocardin with p300 and HDAC5. COS cells were transiently transfected with expression vectors encoding hemagglutinin (HA)-tagged p300, FLAG-tagged myocardin, and Myc-tagged HDAC5. FLAG-tagged myocardin was immunoprecipitated

Techniques Used: Transfection, Expressing, Immunoprecipitation

Acetylation of histones associated with smooth muscle gene promoters in response to myocardin and modulation of myocardin myogenic activity by p300 and HDAC5. (A) 10T1/2 cells were transiently transfected with a FLAG-tagged myocardin expression vector
Figure Legend Snippet: Acetylation of histones associated with smooth muscle gene promoters in response to myocardin and modulation of myocardin myogenic activity by p300 and HDAC5. (A) 10T1/2 cells were transiently transfected with a FLAG-tagged myocardin expression vector

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

7) Product Images from "Cell-cycle dependent localization of MELK and its new partner RACK1 in epithelial versus mesenchyme-like cells in Xenopus embryo"

Article Title: Cell-cycle dependent localization of MELK and its new partner RACK1 in epithelial versus mesenchyme-like cells in Xenopus embryo

Journal: Biology Open

doi: 10.1242/bio.20136080

RACK1 regulates localization of iMELK. (A) Gastrula embryos expressing FLAG-RACK1 FL, FLAG-RACK1 WD1–4 and FLAG-RACK1 WD5–7 were fixed and processed for indirect immunofluorescence with anti-FLAG (a,d,g) and anti-xMELK antibodies (b,e,h). Pictures were merged (merge, c,f,i) together with pictures of DNA (blue) at the same confocal planes to visualize co-localization of xMELK (red) with FLAG-RACK1 constructs (green). Embryos expressing FLAG-RACK1 WD5–7 were incubated with a rabbit polyclonal anti-FLAG (j) and a mouse monoclonal anti-tubulin (k) antibody. Pictures were merged together with pictures of DNA (blue) to visualize FLAG-RACK1 WD5–7 and microtubules. White dashed arrows in panels c, f and i indicate the plane used for orthogonal projections of confocal planes shown on the right. Asterisks indicate cytokinetic cells. Arrowheads point on xMELK concentrated at the tight junctions. Scale bars: 20 µm (a–i), 10 µm (j–l). Intensity of the xMELK fluorescent signals at the cell–cell contacts in embryos expressing FLAG-RACK1 FL, FLAG-RACK1 WD1–4 and FLAG-RACK1 WD5–7 were quantified in interphase and mitotic cells for each 0.5 µm confocal plane. (B) Embryos were coinjected with FLAG-RACK1 FL, FLAG-RACK1 WD1–4 and FLAG-RACK1 WD5–7 mRNAs with GFP-xMELK KR or GFP-gpi mRNAs. White dashed lines mark the plane used for orthogonal projections of confocal planes shown in the center. Arrows points to the apical junctional complex. Black and white arrows points to GFP-xMELK and GFP-gpi, respectively. Scale bars: 20 µm. The intensity of the GFP-xMELK KR and GFP-gpi fluorescent signals at the cell–cell contacts in embryos expressing FLAG-RACK1 FL, FLAG-RACK1 WD1–4 and FLAG-RACK1 WD5–7 was quantified in interphase and mitotic cells for each 0.5 µm confocal plane. Statistical analysis was performed. *GFP-xMELK KR+ FLAG-RACK1 WD5–7 is significantly different from GFP-xMELK KR+ FLAG-RACK1 FL at p
Figure Legend Snippet: RACK1 regulates localization of iMELK. (A) Gastrula embryos expressing FLAG-RACK1 FL, FLAG-RACK1 WD1–4 and FLAG-RACK1 WD5–7 were fixed and processed for indirect immunofluorescence with anti-FLAG (a,d,g) and anti-xMELK antibodies (b,e,h). Pictures were merged (merge, c,f,i) together with pictures of DNA (blue) at the same confocal planes to visualize co-localization of xMELK (red) with FLAG-RACK1 constructs (green). Embryos expressing FLAG-RACK1 WD5–7 were incubated with a rabbit polyclonal anti-FLAG (j) and a mouse monoclonal anti-tubulin (k) antibody. Pictures were merged together with pictures of DNA (blue) to visualize FLAG-RACK1 WD5–7 and microtubules. White dashed arrows in panels c, f and i indicate the plane used for orthogonal projections of confocal planes shown on the right. Asterisks indicate cytokinetic cells. Arrowheads point on xMELK concentrated at the tight junctions. Scale bars: 20 µm (a–i), 10 µm (j–l). Intensity of the xMELK fluorescent signals at the cell–cell contacts in embryos expressing FLAG-RACK1 FL, FLAG-RACK1 WD1–4 and FLAG-RACK1 WD5–7 were quantified in interphase and mitotic cells for each 0.5 µm confocal plane. (B) Embryos were coinjected with FLAG-RACK1 FL, FLAG-RACK1 WD1–4 and FLAG-RACK1 WD5–7 mRNAs with GFP-xMELK KR or GFP-gpi mRNAs. White dashed lines mark the plane used for orthogonal projections of confocal planes shown in the center. Arrows points to the apical junctional complex. Black and white arrows points to GFP-xMELK and GFP-gpi, respectively. Scale bars: 20 µm. The intensity of the GFP-xMELK KR and GFP-gpi fluorescent signals at the cell–cell contacts in embryos expressing FLAG-RACK1 FL, FLAG-RACK1 WD1–4 and FLAG-RACK1 WD5–7 was quantified in interphase and mitotic cells for each 0.5 µm confocal plane. Statistical analysis was performed. *GFP-xMELK KR+ FLAG-RACK1 WD5–7 is significantly different from GFP-xMELK KR+ FLAG-RACK1 FL at p

Techniques Used: Expressing, Immunofluorescence, Construct, Incubation

8) Product Images from "The Hemagglutinin of Bat-Associated Influenza Viruses Is Activated by TMPRSS2 for pH-Dependent Entry into Bat but Not Human Cells"

Article Title: The Hemagglutinin of Bat-Associated Influenza Viruses Is Activated by TMPRSS2 for pH-Dependent Entry into Bat but Not Human Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0152134

Human proteases that activate FLUAV-HA for cell entry also activate batFLUAV-HAL. (A) HEK-293T cells were transfected with plasmids encoding HA or HAL proteins and either trypsin treated or cotransfected with plasmids encoding type II transmembrane serine proteases. Transfection of empty vector served as negative control. Cleavage of HA/HAL proteins was analyzed by SDS-PAGE and Western blotting, employing antibodies against FLUAV-HA (α-FLUAV) and the FLAG epitope (α-FLAG). Detection of ß-actin served as loading control. Signals corresponding to uncleaved precursor proteins are marked by black circles, while products of proteolytic cleavage are indicated by white circles. The results were confirmed in a separate experiment. To assess proteolytic activation of HA/HAL proteins, vesicular stomatitis virus-based pseudotypes (VSVpp) were produced in cells transfected to express the indicated type II transmembrane serine proteases (B) or different amounts of TMPRSS2 (C). Pseudotypes were either directly used for transduction of EpoNi/22.1 cells (black bars) or previously treated with trypsin (white bars). At 24 h post inoculation, transduction efficiency was measured by quantification of the activity of VSVpp-encoded luciferase in cell lysates. For normalization, transduction by HA- or HAL-bearing pseudotypes that were produced in the absence of type II transmembrane serine protease expression (empty vector) and not treated with trypsin was set as 1. The result of a single representative experiment carried out with quadruplicate samples is presented. Similar results were obtained in three independent experiments carried out with separate pseudotype preparations. Error bars indicate standard deviations. A two-tailed, unpaired student’s t-test was used to test statistical significance (* = p
Figure Legend Snippet: Human proteases that activate FLUAV-HA for cell entry also activate batFLUAV-HAL. (A) HEK-293T cells were transfected with plasmids encoding HA or HAL proteins and either trypsin treated or cotransfected with plasmids encoding type II transmembrane serine proteases. Transfection of empty vector served as negative control. Cleavage of HA/HAL proteins was analyzed by SDS-PAGE and Western blotting, employing antibodies against FLUAV-HA (α-FLUAV) and the FLAG epitope (α-FLAG). Detection of ß-actin served as loading control. Signals corresponding to uncleaved precursor proteins are marked by black circles, while products of proteolytic cleavage are indicated by white circles. The results were confirmed in a separate experiment. To assess proteolytic activation of HA/HAL proteins, vesicular stomatitis virus-based pseudotypes (VSVpp) were produced in cells transfected to express the indicated type II transmembrane serine proteases (B) or different amounts of TMPRSS2 (C). Pseudotypes were either directly used for transduction of EpoNi/22.1 cells (black bars) or previously treated with trypsin (white bars). At 24 h post inoculation, transduction efficiency was measured by quantification of the activity of VSVpp-encoded luciferase in cell lysates. For normalization, transduction by HA- or HAL-bearing pseudotypes that were produced in the absence of type II transmembrane serine protease expression (empty vector) and not treated with trypsin was set as 1. The result of a single representative experiment carried out with quadruplicate samples is presented. Similar results were obtained in three independent experiments carried out with separate pseudotype preparations. Error bars indicate standard deviations. A two-tailed, unpaired student’s t-test was used to test statistical significance (* = p

Techniques Used: Transfection, Plasmid Preparation, Negative Control, SDS Page, Western Blot, FLAG-tag, Activation Assay, Produced, Transduction, Activity Assay, Luciferase, Expressing, Two Tailed Test

9) Product Images from "Phosphatidylinositol glycan anchor biosynthesis, class X containing complex promotes cancer cell proliferation through suppression of EHD2 and ZIC1, putative tumor suppressors"

Article Title: Phosphatidylinositol glycan anchor biosynthesis, class X containing complex promotes cancer cell proliferation through suppression of EHD2 and ZIC1, putative tumor suppressors

Journal: International Journal of Oncology

doi: 10.3892/ijo.2016.3607

PIGX functions as a core in the complex with RCN1 and RCN2. (A) Western blot analysis of overexpressed PIGX in two different reducing conditions (+β-Me and +DTT). Two different sized bands were observed in mild reducing condition with DTT. β-Me, β-mercaptoethanol; DTT, dithiothreitol. (B) Co-immunoprecipitation analysis of PIGX multimer formation. FLAG- or HA-tagged PIGX was co-overexpressed in 293T cells and immunoprecipitated using FLAG tag. IP, immunoprecipitation; IB, immunoblotting. (C and D) Co-immunoprecipitation analysis of the interaction between RCN1 and RCN2 with PIGX knockdown. Knockdown effect was validated by qRT-PCR. siNC, siNegative control. (E and F) Co-immunoprecipitation analysis between PIGX and RCN1 or RCN2 with knockdown of the other RCN protein. FLAG-tagged PIGX was overexpressed in 293T cells and interaction with endogenous RCN1 or RCN2 were analyzed.
Figure Legend Snippet: PIGX functions as a core in the complex with RCN1 and RCN2. (A) Western blot analysis of overexpressed PIGX in two different reducing conditions (+β-Me and +DTT). Two different sized bands were observed in mild reducing condition with DTT. β-Me, β-mercaptoethanol; DTT, dithiothreitol. (B) Co-immunoprecipitation analysis of PIGX multimer formation. FLAG- or HA-tagged PIGX was co-overexpressed in 293T cells and immunoprecipitated using FLAG tag. IP, immunoprecipitation; IB, immunoblotting. (C and D) Co-immunoprecipitation analysis of the interaction between RCN1 and RCN2 with PIGX knockdown. Knockdown effect was validated by qRT-PCR. siNC, siNegative control. (E and F) Co-immunoprecipitation analysis between PIGX and RCN1 or RCN2 with knockdown of the other RCN protein. FLAG-tagged PIGX was overexpressed in 293T cells and interaction with endogenous RCN1 or RCN2 were analyzed.

Techniques Used: Western Blot, Immunoprecipitation, FLAG-tag, Quantitative RT-PCR

PIGX and RCN proteins interact with each other in ER. (A) Immunocytochemical analysis of HeLa cells transfected with PIGX expression vector. Signal of exogenous PIGX was merged with that of ER marker, KDEL. Scale bar, 20 μm. (B and C) Immunocytochemical analysis of HeLa cells for RCN1 (B) or RCN2 (C). Endogenous RCN1 or RCN2 was detected and merged with ER marker. Scale bar, 20 μm. (D and E) Co-immunoprecipitation analysis. GST-FLAG tagged PIGX was co-overexpressed with HA-tagged RCN1 or RCN2 and pulled down using GST-tag. (F and G) Cell growth of T-47D with knockdown of RCN1 (F) and RCN2 (G) was examined by MTT assay. The knockdown effect was validated by qRT-PCR. Results are the mean ± SD of three independent experiments and P-values were calculated with Student’s t-test ( * P
Figure Legend Snippet: PIGX and RCN proteins interact with each other in ER. (A) Immunocytochemical analysis of HeLa cells transfected with PIGX expression vector. Signal of exogenous PIGX was merged with that of ER marker, KDEL. Scale bar, 20 μm. (B and C) Immunocytochemical analysis of HeLa cells for RCN1 (B) or RCN2 (C). Endogenous RCN1 or RCN2 was detected and merged with ER marker. Scale bar, 20 μm. (D and E) Co-immunoprecipitation analysis. GST-FLAG tagged PIGX was co-overexpressed with HA-tagged RCN1 or RCN2 and pulled down using GST-tag. (F and G) Cell growth of T-47D with knockdown of RCN1 (F) and RCN2 (G) was examined by MTT assay. The knockdown effect was validated by qRT-PCR. Results are the mean ± SD of three independent experiments and P-values were calculated with Student’s t-test ( * P

Techniques Used: Transfection, Expressing, Plasmid Preparation, Marker, Immunoprecipitation, MTT Assay, Quantitative RT-PCR

10) Product Images from "FAM21 directs SNX27–retromer cargoes to the plasma membrane by preventing transport to the Golgi apparatus"

Article Title: FAM21 directs SNX27–retromer cargoes to the plasma membrane by preventing transport to the Golgi apparatus

Journal: Nature Communications

doi: 10.1038/ncomms10939

SNX27 interacts directly with FAM21 among the WASH complex components. ( a ) HEK293T cells were transfected with 3 × FLAG-tagged-WASH complex proteins. Lysates were incubated with GST or GST-SNX27 proteins and precipitated with Glutathione Sepharose 4B beads. Precipitates were then immunoblotted as shown. ( b ) Schematic representation of FAM21 constructs used. LFa motifs and α-helices are indicated. ( c , d ) HA-SNX27 was co-transfected with the indicated Myc-tagged constructs in HEK293T cells, and lysates were immunoprecipitated with anti-haemagglutinin (HA) antibodies and immunoblotted. Asterisks (*) denote IgG heavy chains. ( e ) GST or GST-SNX27 fusion proteins were incubated with indicated His-tagged FAM21 proteins and precipitated using Glutathione Sepharose 4B beads. Precipitates were immunoblotted as indicated. ( f ) Biotinylated peptides corresponding to FAM21 residues 40–79 and 592–600 were incubated with CBP-SNX27 and precipitated using streptavidin-conjugated beads. Precipitates were immunoblotted for CBP.
Figure Legend Snippet: SNX27 interacts directly with FAM21 among the WASH complex components. ( a ) HEK293T cells were transfected with 3 × FLAG-tagged-WASH complex proteins. Lysates were incubated with GST or GST-SNX27 proteins and precipitated with Glutathione Sepharose 4B beads. Precipitates were then immunoblotted as shown. ( b ) Schematic representation of FAM21 constructs used. LFa motifs and α-helices are indicated. ( c , d ) HA-SNX27 was co-transfected with the indicated Myc-tagged constructs in HEK293T cells, and lysates were immunoprecipitated with anti-haemagglutinin (HA) antibodies and immunoblotted. Asterisks (*) denote IgG heavy chains. ( e ) GST or GST-SNX27 fusion proteins were incubated with indicated His-tagged FAM21 proteins and precipitated using Glutathione Sepharose 4B beads. Precipitates were immunoblotted as indicated. ( f ) Biotinylated peptides corresponding to FAM21 residues 40–79 and 592–600 were incubated with CBP-SNX27 and precipitated using streptavidin-conjugated beads. Precipitates were immunoblotted for CBP.

Techniques Used: Transfection, Incubation, Construct, Immunoprecipitation

11) Product Images from "Intrinsic dynamics study identifies two amino acids of TIMP-1 critical for its LRP-1-mediated endocytosis in neurons"

Article Title: Intrinsic dynamics study identifies two amino acids of TIMP-1 critical for its LRP-1-mediated endocytosis in neurons

Journal: Scientific Reports

doi: 10.1038/s41598-017-05039-z

Production of TIMP-1 mutants. ( a ) Schematic representation of T1-WT, T1-F12A and T1-K47A proteins. ( b ) A representative western blot with the indicated antibodies of cell extracts and conditioned medium (cond. med) of stably transfected CHO cells with p3X-FLAG-CMV-14 expressing T1-WT, T1-F12A or T1-K47A. The relative protein level based on three independent experiments is indicated under each band. The gels were run under the same experimental conditions and were shown as cropped gels/blots (Cropped gels/blots are shown in Supplementary Figure S1 ).
Figure Legend Snippet: Production of TIMP-1 mutants. ( a ) Schematic representation of T1-WT, T1-F12A and T1-K47A proteins. ( b ) A representative western blot with the indicated antibodies of cell extracts and conditioned medium (cond. med) of stably transfected CHO cells with p3X-FLAG-CMV-14 expressing T1-WT, T1-F12A or T1-K47A. The relative protein level based on three independent experiments is indicated under each band. The gels were run under the same experimental conditions and were shown as cropped gels/blots (Cropped gels/blots are shown in Supplementary Figure S1 ).

Techniques Used: Western Blot, Stable Transfection, Transfection, Expressing

12) Product Images from "A CRISPR Activation Screen Identifies Genes That Protect against Zika Virus Infection"

Article Title: A CRISPR Activation Screen Identifies Genes That Protect against Zika Virus Infection

Journal: Journal of Virology

doi: 10.1128/JVI.00211-19

Immunofluorescence microscopy of cells expressing IFI6-FLAG. (A) A schematic representation of IFI6 structural domain organization. (B) Huh7 cells were transfected with IFI6-FLAG. After 24 h, the cells were stained with IFI6-specific and FLAG-specific antibodies. Scale bar = 10 μm. (C) Huh7 cells were transfected with IFI6-FLAG and after 24 h, the cells were stained with MitoTracker, fixed, and costained with anti-FLAG and anti-GRP94 antibodies. Bottom panel is 3.5-fold enlargement of the area labeled with a white square. The cells were visualized using a confocal microscope. The contour of the IFI6-expressing cell is outlined in white. Scale bar = 10 μm. (D) Graph indicates the Pearson correlation coefficients of IFI6 and MitoTracker or GRP94 localization in 16 cells. ****, P
Figure Legend Snippet: Immunofluorescence microscopy of cells expressing IFI6-FLAG. (A) A schematic representation of IFI6 structural domain organization. (B) Huh7 cells were transfected with IFI6-FLAG. After 24 h, the cells were stained with IFI6-specific and FLAG-specific antibodies. Scale bar = 10 μm. (C) Huh7 cells were transfected with IFI6-FLAG and after 24 h, the cells were stained with MitoTracker, fixed, and costained with anti-FLAG and anti-GRP94 antibodies. Bottom panel is 3.5-fold enlargement of the area labeled with a white square. The cells were visualized using a confocal microscope. The contour of the IFI6-expressing cell is outlined in white. Scale bar = 10 μm. (D) Graph indicates the Pearson correlation coefficients of IFI6 and MitoTracker or GRP94 localization in 16 cells. ****, P

Techniques Used: Immunofluorescence, Microscopy, Expressing, Transfection, Staining, Labeling

13) Product Images from "GSDMB promotes non-canonical pyroptosis by enhancing caspase-4 activity"

Article Title: GSDMB promotes non-canonical pyroptosis by enhancing caspase-4 activity

Journal: Journal of Molecular Cell Biology

doi: 10.1093/jmcb/mjy056

Full-length GSDMB promotes the enzyme activity of caspase-4 through the GSDMB N-terminus (1–83 aa) binding with the CARD domain of caspase-4. ( A ) Schematic of the GSDMB protein and the truncation fragments: fragment 1 (1–83 aa), fragment 2 (84–167 aa), fragment 3 (168–251 aa), fragment 4 (252–335 aa), and fragment 5 (336–417 aa). ( B ) 293T cells were transfected with the caspase-4-Myc expression plasmid and plasmids expressing the indicated FLAG-tagged GSDMB truncation form. Immunoprecipitation was performed using a monoclonal FLAG antibody, and samples were analyzed with a monoclonal Myc antibody via western blotting. ( C ) Graphic representation of the caspase-4 protein and the functional domains: CARD domain (1–80 aa), subunit 1 (81–270 aa), and subunit 2 (291–380 aa). ( D ) 293T cells were transfected with FLAG-GSDMB expression plasmid and plasmids expressing the indicated caspase-4-Myc truncation form. Immunoprecipitation was performed using a monoclonal Myc antibody, and the samples were analyzed with a monoclonal FLAG antibody via western blotting. ( E ) Graphic representation of GSDMB protein and the caspase cleavage site. ( F ) Assays of GSDMB cleavage by members of the human caspase family overexpressed in cells. 293T cells were co-transfected with plasmids encoding 3×FLAG-mouse GSDMB and the indicated pcs2 (+)-human caspase constructs. Total cell lysates were analyzed via anti-FLAG and anti-GAPDH immunoblotting. ( G ) The enzyme activity of caspase-4 in 293T cells. 293T cells were co-transfected with plasmids encoding pcs2 (+)-caspase-4 and either pcs2 (+) vector, pcs2 (+)-GSDMB N-terminus, pcs2 (+)-GSDMB C-terminus, or pcs2 (+)-full-length GSDMB. ( H and I ) 293T cells were transfected with full-length GSDMB (1–417 aa), GSDMB N-terminus (1–91 aa), GSDMB C-terminus (92–417 aa), and GSDMD N-terminus (1–275 aa) plasmids. ( H ) LDH released from 293T cells after 16 h of transfection. ( I ) The cell morphology of 293T cells after 16 h of transfection. The graphs show mean ± SD of triplicate wells and represent three independent experiments. * P
Figure Legend Snippet: Full-length GSDMB promotes the enzyme activity of caspase-4 through the GSDMB N-terminus (1–83 aa) binding with the CARD domain of caspase-4. ( A ) Schematic of the GSDMB protein and the truncation fragments: fragment 1 (1–83 aa), fragment 2 (84–167 aa), fragment 3 (168–251 aa), fragment 4 (252–335 aa), and fragment 5 (336–417 aa). ( B ) 293T cells were transfected with the caspase-4-Myc expression plasmid and plasmids expressing the indicated FLAG-tagged GSDMB truncation form. Immunoprecipitation was performed using a monoclonal FLAG antibody, and samples were analyzed with a monoclonal Myc antibody via western blotting. ( C ) Graphic representation of the caspase-4 protein and the functional domains: CARD domain (1–80 aa), subunit 1 (81–270 aa), and subunit 2 (291–380 aa). ( D ) 293T cells were transfected with FLAG-GSDMB expression plasmid and plasmids expressing the indicated caspase-4-Myc truncation form. Immunoprecipitation was performed using a monoclonal Myc antibody, and the samples were analyzed with a monoclonal FLAG antibody via western blotting. ( E ) Graphic representation of GSDMB protein and the caspase cleavage site. ( F ) Assays of GSDMB cleavage by members of the human caspase family overexpressed in cells. 293T cells were co-transfected with plasmids encoding 3×FLAG-mouse GSDMB and the indicated pcs2 (+)-human caspase constructs. Total cell lysates were analyzed via anti-FLAG and anti-GAPDH immunoblotting. ( G ) The enzyme activity of caspase-4 in 293T cells. 293T cells were co-transfected with plasmids encoding pcs2 (+)-caspase-4 and either pcs2 (+) vector, pcs2 (+)-GSDMB N-terminus, pcs2 (+)-GSDMB C-terminus, or pcs2 (+)-full-length GSDMB. ( H and I ) 293T cells were transfected with full-length GSDMB (1–417 aa), GSDMB N-terminus (1–91 aa), GSDMB C-terminus (92–417 aa), and GSDMD N-terminus (1–275 aa) plasmids. ( H ) LDH released from 293T cells after 16 h of transfection. ( I ) The cell morphology of 293T cells after 16 h of transfection. The graphs show mean ± SD of triplicate wells and represent three independent experiments. * P

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

GSDMB enhances the enzyme activity of caspase-4 by binding to caspase-4. ( A , C , E ) Western blot analyses show the cleavage of GSDMD in 293T cells. ( B , D , F ) Graphs show GSDMD N-terminus levels normalized to GAPDH expression using the ImageJ software program. ( A and B ) 293T cells were co-transfected with plasmids encoding 3×FLAG-full-length GSDMD and either pcs2 (+) vector, pcs2 (+)-caspase-4, or pcs2 (+)-caspase-4 plus 3×FLAG-full-length GSDMB. ( C and D ) 293T cells were co-transfected with plasmids encoding 3×FLAG-full-length GSDMD and either pcs2 (+) vector, pcs2 (+)-caspase-11, or pcs2 (+)-caspase-11 plus 3×FLAG-full-length GSDMB. ( E and F ) 293T cells were co-transfected with plasmids encoding 3×FLAG-full-length GSDMD and either pcs2 (+) vector, pcs2 (+)-caspase-1, or pcs2 (+)-caspase-1 plus 3×FLAG-full-length GSDMB. ( G ) GSDMB did not interact with GSDMD. Co-IP analysis was performed in GSDMB-GFP- and FLAG-GSDMD-transfected 293T cells. ( H and I ) GSDMB interacted with caspase-4. Co-IP analysis was performed in caspase-4-Myc- and FLAG-GSDMB-transfected 293T cells. ( J ) The enzyme activity of caspase-4 in 293T cells. 293T cells were co-transfected with plasmids encoding pcs2 (+)-caspase-4 and either pcs2 (+) vector or pcs2 (+)-GSDMB. Graphs show mean ± SD of triplicate wells and represent three independent experiments. * P
Figure Legend Snippet: GSDMB enhances the enzyme activity of caspase-4 by binding to caspase-4. ( A , C , E ) Western blot analyses show the cleavage of GSDMD in 293T cells. ( B , D , F ) Graphs show GSDMD N-terminus levels normalized to GAPDH expression using the ImageJ software program. ( A and B ) 293T cells were co-transfected with plasmids encoding 3×FLAG-full-length GSDMD and either pcs2 (+) vector, pcs2 (+)-caspase-4, or pcs2 (+)-caspase-4 plus 3×FLAG-full-length GSDMB. ( C and D ) 293T cells were co-transfected with plasmids encoding 3×FLAG-full-length GSDMD and either pcs2 (+) vector, pcs2 (+)-caspase-11, or pcs2 (+)-caspase-11 plus 3×FLAG-full-length GSDMB. ( E and F ) 293T cells were co-transfected with plasmids encoding 3×FLAG-full-length GSDMD and either pcs2 (+) vector, pcs2 (+)-caspase-1, or pcs2 (+)-caspase-1 plus 3×FLAG-full-length GSDMB. ( G ) GSDMB did not interact with GSDMD. Co-IP analysis was performed in GSDMB-GFP- and FLAG-GSDMD-transfected 293T cells. ( H and I ) GSDMB interacted with caspase-4. Co-IP analysis was performed in caspase-4-Myc- and FLAG-GSDMB-transfected 293T cells. ( J ) The enzyme activity of caspase-4 in 293T cells. 293T cells were co-transfected with plasmids encoding pcs2 (+)-caspase-4 and either pcs2 (+) vector or pcs2 (+)-GSDMB. Graphs show mean ± SD of triplicate wells and represent three independent experiments. * P

Techniques Used: Activity Assay, Binding Assay, Western Blot, Expressing, Software, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay

14) Product Images from "A New Cell-penetrating Peptide That Blocks the Autoinhibitory XIP Domain of NCX1 and Enhances Antiporter Activity"

Article Title: A New Cell-penetrating Peptide That Blocks the Autoinhibitory XIP Domain of NCX1 and Enhances Antiporter Activity

Journal: Molecular Therapy

doi: 10.1038/mt.2014.231

Interaction between EGFP-P1 and NCX1. ( a ) Top, representative scheme of P1 Flag structure. Bottom, immunocitochemical assay of the time- and concentration-dependent penetration of P1 Flag (red) in BHK-NCX1 cells. Hoecst dye (blue) was used to mark nuclei.
Figure Legend Snippet: Interaction between EGFP-P1 and NCX1. ( a ) Top, representative scheme of P1 Flag structure. Bottom, immunocitochemical assay of the time- and concentration-dependent penetration of P1 Flag (red) in BHK-NCX1 cells. Hoecst dye (blue) was used to mark nuclei.

Techniques Used: Concentration Assay

15) Product Images from "Characterization of Leber Congenital Amaurosis-associated NMNAT1 Mutants *"

Article Title: Characterization of Leber Congenital Amaurosis-associated NMNAT1 Mutants *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.637850

Subcellular localization of wild type and LCA-associated NMNAT1 mutants. Subcellular localization of wild type or mutant NMNAT1 with antibodies to ( a ) FLAG or ( b ) HA epitope tags was analyzed by immunohistochemistry. All NMNAT1 mutant proteins were localized
Figure Legend Snippet: Subcellular localization of wild type and LCA-associated NMNAT1 mutants. Subcellular localization of wild type or mutant NMNAT1 with antibodies to ( a ) FLAG or ( b ) HA epitope tags was analyzed by immunohistochemistry. All NMNAT1 mutant proteins were localized

Techniques Used: Mutagenesis, Immunohistochemistry

16) Product Images from "Metnase Mediates Loading of Exonuclease 1 onto Single Strand Overhang DNA for End Resection at Stalled Replication Forks *"

Article Title: Metnase Mediates Loading of Exonuclease 1 onto Single Strand Overhang DNA for End Resection at Stalled Replication Forks *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.745646

Metnase mediates Exo1 loading onto single strand overhang DNA. A , ss-overhang is required for binding of WT Metnase ( Met ) to duplex DNA. FLAG-tagged WT Metnase (1.0 μg) was incubated with 25 pmol of the 3′-biotinylated partial ( top ) or full ( bottom ) duplex DNA to assay protein-DNA binding by streptavidin-agarose beads. “ B ” indicates biotin label. The protein-DNA interaction was analyzed by Western blotting ( WB ) using an anti-FLAG antibody. L.C. represents the loading control of WT Metnase (0.1 μg). B , WT Metnase is required for Exo1 binding to a partial duplex DNA with 3′-ss-overhang in streptavidin-agarose pulldown assay. Where indicated, 1 μg of WT Metnase and two concentrations of Exo1 (0.5 and 1 μg) were used. C , a diagram of Metnase domains. Two DNA binding motifs (HTH and NUMOD1 motifs) and the catalytic motif reside within the transposase domain. D , silver staining of purified WT Metnase and the mutants following 8% SDS-PAGE. E , two Arg residues in the HTH motif are essential for Metnase-DNA interaction. Where indicated, 25 pmol of the 3′-biotinylated partial duplex DNA was used. F , Metnase mutants lacking DNA binding activity failed to mediate loading of WT Exo1 onto ss-overhang DNA. Where indicated, 1 μg of either WT or mutant Metnase and 0.5 and 1 μg of Exo1 were used. G , a mutation at a key DNA binding or DNA cleavage site of Metnase has no effect on the Metnase-Exo1 interaction. HEK293 cells stably expressing V5-Exo1 were transfected with WT or the mutant FLAG-Metnase and immunoprecipitated ( IP ) with FLAG ( left panel ) or V5 antibody ( right panel ), and Western blots were probed with V5, FLAG, and β-actin antibodies.
Figure Legend Snippet: Metnase mediates Exo1 loading onto single strand overhang DNA. A , ss-overhang is required for binding of WT Metnase ( Met ) to duplex DNA. FLAG-tagged WT Metnase (1.0 μg) was incubated with 25 pmol of the 3′-biotinylated partial ( top ) or full ( bottom ) duplex DNA to assay protein-DNA binding by streptavidin-agarose beads. “ B ” indicates biotin label. The protein-DNA interaction was analyzed by Western blotting ( WB ) using an anti-FLAG antibody. L.C. represents the loading control of WT Metnase (0.1 μg). B , WT Metnase is required for Exo1 binding to a partial duplex DNA with 3′-ss-overhang in streptavidin-agarose pulldown assay. Where indicated, 1 μg of WT Metnase and two concentrations of Exo1 (0.5 and 1 μg) were used. C , a diagram of Metnase domains. Two DNA binding motifs (HTH and NUMOD1 motifs) and the catalytic motif reside within the transposase domain. D , silver staining of purified WT Metnase and the mutants following 8% SDS-PAGE. E , two Arg residues in the HTH motif are essential for Metnase-DNA interaction. Where indicated, 25 pmol of the 3′-biotinylated partial duplex DNA was used. F , Metnase mutants lacking DNA binding activity failed to mediate loading of WT Exo1 onto ss-overhang DNA. Where indicated, 1 μg of either WT or mutant Metnase and 0.5 and 1 μg of Exo1 were used. G , a mutation at a key DNA binding or DNA cleavage site of Metnase has no effect on the Metnase-Exo1 interaction. HEK293 cells stably expressing V5-Exo1 were transfected with WT or the mutant FLAG-Metnase and immunoprecipitated ( IP ) with FLAG ( left panel ) or V5 antibody ( right panel ), and Western blots were probed with V5, FLAG, and β-actin antibodies.

Techniques Used: Binding Assay, Incubation, Western Blot, Silver Staining, Purification, SDS Page, Activity Assay, Mutagenesis, Stable Transfection, Expressing, Transfection, Immunoprecipitation

17) Product Images from "TRIM25 binds RNA to modulate cellular anti-viral defense"

Article Title: TRIM25 binds RNA to modulate cellular anti-viral defense

Journal: Journal of molecular biology

doi: 10.1016/j.jmb.2018.10.003

RNA binding is important for TRIM25’s ability to inhibit the replication of representative members of three different RNA virus families. ( a ) TRIM25 -KO HEK 293T cells were transiently transfected with either empty vector, FLAG-tagged TRIM25 (WT), or the 7KA mutant (7KA). Twenty-four hours later, cells were infected with DENV (MOI 4) for 24 h, and then stained for the viral prM protein. The percentage of prM-positive cells were determined by flow cytometry. Error bars represent the standard deviation of three biological replicates. ( b ) HEK 293T cells, transfected as in (A), were infected with VSV-eGFP (MOI 0.01). Seventeen hours later, the percentage of eGFP-positive cells were determined by flow cytometry. Error bars represent the standard deviation of three biological replicates. ( c ) TRIM25 -KO HEK 293T cells were transfected as in (a). Twenty-four hours later, cells were infected with IAV (MOI 0.5) for 72 h, and then immunoblotted with anti-NS1 to determine expression levels of the viral NS1 protein. For all viral assays, whole cell lysates (WCLs) were further immunoblotted with anti-FLAG antibody to confirm expression of TRIM25 WT and 7KA proteins. Actin was used as loading control. ( d ) IAV replication in TRIM25 -KO HEK 293T cells that were transfected as in (a), and infected with IAV (MOI of 0.1) for 24 h. Error bars represent the standard deviation of three biological replicates. *P
Figure Legend Snippet: RNA binding is important for TRIM25’s ability to inhibit the replication of representative members of three different RNA virus families. ( a ) TRIM25 -KO HEK 293T cells were transiently transfected with either empty vector, FLAG-tagged TRIM25 (WT), or the 7KA mutant (7KA). Twenty-four hours later, cells were infected with DENV (MOI 4) for 24 h, and then stained for the viral prM protein. The percentage of prM-positive cells were determined by flow cytometry. Error bars represent the standard deviation of three biological replicates. ( b ) HEK 293T cells, transfected as in (A), were infected with VSV-eGFP (MOI 0.01). Seventeen hours later, the percentage of eGFP-positive cells were determined by flow cytometry. Error bars represent the standard deviation of three biological replicates. ( c ) TRIM25 -KO HEK 293T cells were transfected as in (a). Twenty-four hours later, cells were infected with IAV (MOI 0.5) for 72 h, and then immunoblotted with anti-NS1 to determine expression levels of the viral NS1 protein. For all viral assays, whole cell lysates (WCLs) were further immunoblotted with anti-FLAG antibody to confirm expression of TRIM25 WT and 7KA proteins. Actin was used as loading control. ( d ) IAV replication in TRIM25 -KO HEK 293T cells that were transfected as in (a), and infected with IAV (MOI of 0.1) for 24 h. Error bars represent the standard deviation of three biological replicates. *P

Techniques Used: RNA Binding Assay, Transfection, Plasmid Preparation, Mutagenesis, Infection, Staining, Flow Cytometry, Cytometry, Standard Deviation, Expressing

RNA-binding activity of TRIM25 is required for GST-2CARD ubiquitination and induction of interferon. ( a ) TRIM25 ]). Twenty-four hours later, IFN-β promoter activity was measured by a luciferase assay, and values were normalized to β-galactosidase levels. Whole cell lysates (WCL) were analyzed by immunoblotting with anti-FLAG to determine ectopic TRIM25 expression, with actin as loading control. ( b ) HEK 293T cells were transfected with GST or GST-2CARD together with empty vector or the indicated FLAG-tagged TRIM25 constructs (WT or 7KA mutant). Forty-eight hours later, WCL were subjected to pulldown with anti-GST followed by immunoblotting with anti-Ub and anti-GST antibodies. WCL were further immunoblotted with anti-FLAG to determine expression levels of the TRIM25 WT and 7KA proteins. Actin was used as a loading control. ( c ) HEK 293T cells were transfected as in (B). Forty-eight hours later, WCL were subjected to pulldown with anti-FLAG followed by immunoblotting with anti-GST and anti-FLAG antibodies. WCLs were further immunoblotted with anti-GST to determine expression levels of GST-2CARD.
Figure Legend Snippet: RNA-binding activity of TRIM25 is required for GST-2CARD ubiquitination and induction of interferon. ( a ) TRIM25 ]). Twenty-four hours later, IFN-β promoter activity was measured by a luciferase assay, and values were normalized to β-galactosidase levels. Whole cell lysates (WCL) were analyzed by immunoblotting with anti-FLAG to determine ectopic TRIM25 expression, with actin as loading control. ( b ) HEK 293T cells were transfected with GST or GST-2CARD together with empty vector or the indicated FLAG-tagged TRIM25 constructs (WT or 7KA mutant). Forty-eight hours later, WCL were subjected to pulldown with anti-GST followed by immunoblotting with anti-Ub and anti-GST antibodies. WCL were further immunoblotted with anti-FLAG to determine expression levels of the TRIM25 WT and 7KA proteins. Actin was used as a loading control. ( c ) HEK 293T cells were transfected as in (B). Forty-eight hours later, WCL were subjected to pulldown with anti-FLAG followed by immunoblotting with anti-GST and anti-FLAG antibodies. WCLs were further immunoblotted with anti-GST to determine expression levels of GST-2CARD.

Techniques Used: RNA Binding Assay, Activity Assay, Luciferase, Expressing, Transfection, Plasmid Preparation, Construct, Mutagenesis

18) Product Images from "CPI motif interaction is necessary for capping protein function in cells"

Article Title: CPI motif interaction is necessary for capping protein function in cells

Journal: Nature Communications

doi: 10.1038/ncomms9415

CPI-motif binding-site mutations impair CPI–CP interaction. ( a ) Interaction of purified CP and CARMIL-CBR, assayed by SPR (surface plasmon resonance). The SPR chip contains GST fused to CBR of human CARMIL1. CP at 20 nM was flowed over the chip. Black: wild-type CP, green: Y79A, red: R15A, blue: R15A/Y79A. One representative experiment, among four, is shown. ( b ) SPR traces as in a , with concentrations of CP as follows: wild-type 20 nM, R15A 200 nM, Y79A 200 nM, R15A/Y79A 2,000 nM. One representative experiment, among four, is shown. ( c ) Impaired association of CP mutants with CPI-motif proteins in cells. HT1080 cells expressed CP mutants tested in a and b . Cells were co-transfected with YFP-CPα1 and either GFP-CPβ2, GFP-CPβ2 R15A, GFP-CPβ2 Y79A or GFP-CPβ2 R15A/Y79A. CP was immunoprecipitated from whole-cell lysates with anti-GFP, and precipitates were probed with antibodies to endogenous CD2AP and Fam21C. For CARMIL1 and CARMIL2, endogenous levels were low, so cells were co-transfected with FLAG-tagged expression constructs, and IPs were probed with anti-FLAG.
Figure Legend Snippet: CPI-motif binding-site mutations impair CPI–CP interaction. ( a ) Interaction of purified CP and CARMIL-CBR, assayed by SPR (surface plasmon resonance). The SPR chip contains GST fused to CBR of human CARMIL1. CP at 20 nM was flowed over the chip. Black: wild-type CP, green: Y79A, red: R15A, blue: R15A/Y79A. One representative experiment, among four, is shown. ( b ) SPR traces as in a , with concentrations of CP as follows: wild-type 20 nM, R15A 200 nM, Y79A 200 nM, R15A/Y79A 2,000 nM. One representative experiment, among four, is shown. ( c ) Impaired association of CP mutants with CPI-motif proteins in cells. HT1080 cells expressed CP mutants tested in a and b . Cells were co-transfected with YFP-CPα1 and either GFP-CPβ2, GFP-CPβ2 R15A, GFP-CPβ2 Y79A or GFP-CPβ2 R15A/Y79A. CP was immunoprecipitated from whole-cell lysates with anti-GFP, and precipitates were probed with antibodies to endogenous CD2AP and Fam21C. For CARMIL1 and CARMIL2, endogenous levels were low, so cells were co-transfected with FLAG-tagged expression constructs, and IPs were probed with anti-FLAG.

Techniques Used: Binding Assay, Purification, SPR Assay, Chromatin Immunoprecipitation, Transfection, Immunoprecipitation, Expressing, Construct

19) Product Images from "Characterization of Np95 expression in mouse brain from embryo to adult: A novel marker for proliferating neural stem/precursor cells"

Article Title: Characterization of Np95 expression in mouse brain from embryo to adult: A novel marker for proliferating neural stem/precursor cells

Journal: Neurogenesis

doi: 10.4161/23262133.2014.976026

Np95 is expressed abundantly in the brain at midgestation, when a large number of NS/PCs exist. ( A ) Confirmation of antibody reactivity to mouse Np95. HEK293T cells were transfected with FLAG-tagged Np95 expression plasmid and immunoblotted with anti-FLAG, -Np95 and -UHRF1 antibodies. ( B ) Western blot analysis of mouse whole brain at various stages of development, from E11 to postnatal day 7 (P7) and in 8-week-old adults, using anti-DNMT1, -Np95 and -actin antibodies. Np95 and DNMT1 bands were strongest in E11 brains. ( C ) Representative immunofluorescence images for Np95, Ki67 and Sox2 in E14 mouse forebrain sections. Scale bar: 250 μm. ( D ) Confocal immunofluorescence images for Np95, Ki67 and Sox2 in E14 mouse forebrain sections. Scale bars: 10 μm. ( E–G ) Representative immunofluorescence images of E14 mouse forebrain sections. Immunostaining of Np95 (red), Nestin (cyan) and Ki67 ( E ), DCX (F) or MAP2ab ( G ) (green). The insets in Merge images show Hoechst staining. Np95 expression was observed only in Ki67+ or Nestin+ proliferating NS/PCs. Scale bars: 100 μm. VZ: ventricular zone; SVZ: subventricular zone; IZ: intermediate zone; CP: cortical plate.
Figure Legend Snippet: Np95 is expressed abundantly in the brain at midgestation, when a large number of NS/PCs exist. ( A ) Confirmation of antibody reactivity to mouse Np95. HEK293T cells were transfected with FLAG-tagged Np95 expression plasmid and immunoblotted with anti-FLAG, -Np95 and -UHRF1 antibodies. ( B ) Western blot analysis of mouse whole brain at various stages of development, from E11 to postnatal day 7 (P7) and in 8-week-old adults, using anti-DNMT1, -Np95 and -actin antibodies. Np95 and DNMT1 bands were strongest in E11 brains. ( C ) Representative immunofluorescence images for Np95, Ki67 and Sox2 in E14 mouse forebrain sections. Scale bar: 250 μm. ( D ) Confocal immunofluorescence images for Np95, Ki67 and Sox2 in E14 mouse forebrain sections. Scale bars: 10 μm. ( E–G ) Representative immunofluorescence images of E14 mouse forebrain sections. Immunostaining of Np95 (red), Nestin (cyan) and Ki67 ( E ), DCX (F) or MAP2ab ( G ) (green). The insets in Merge images show Hoechst staining. Np95 expression was observed only in Ki67+ or Nestin+ proliferating NS/PCs. Scale bars: 100 μm. VZ: ventricular zone; SVZ: subventricular zone; IZ: intermediate zone; CP: cortical plate.

Techniques Used: Transfection, Expressing, Plasmid Preparation, Western Blot, Immunofluorescence, Immunostaining, Staining

20) Product Images from "PRMT1 promotes mitosis of cancer cells through arginine methylation of INCENP"

Article Title: PRMT1 promotes mitosis of cancer cells through arginine methylation of INCENP

Journal: Oncotarget

doi:

In vivo methylation of INCENP by PRMT1 A. Determination of the titer and specificity of the anti-mono-methylated R887 INCENP antibody analyzed by ELISA. B. Validation of the anti-mono-methylated R887 INCENP antibody. Recombinant GST-tagged INCENP protein and S-adenosyl-L-methionine (SAM) were incubated in the presence of BSA or recombinant PRMT1. Samples were immunoblotted with the anti-mono-methylated R887 INCENP antibody, and amounts of loading proteins were evaluated by staining with MemCode TM Reversible Protein Stain. C. 293T cells were co-transfected with a FLAG-INCENP-WT (amino acids 821-918) vector or a FLAG-INCENP-R887A (amino acids 821-918) vector and an HA-PRMT1 vector. The samples were immunoblotted with anti-mono-methylated R887 INCENP, anti-FLAG and anti-AURKB antibodies after immunoprecipitating with anti-FLAG M2 agarose (Sigma-Aldrich). D. HeLa cells were transfected with FLAG-PRMT1-WT and immunocytochemistry was performed with anti-FLAG (Sigma-Aldrich, M2, dilution: 1:100, green) and anti-R887meINCENP (dilution: 1:100, green) antibodies. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Transfected HeLa cells exhibited higher INCENP and R887meINCENP integrated densities.
Figure Legend Snippet: In vivo methylation of INCENP by PRMT1 A. Determination of the titer and specificity of the anti-mono-methylated R887 INCENP antibody analyzed by ELISA. B. Validation of the anti-mono-methylated R887 INCENP antibody. Recombinant GST-tagged INCENP protein and S-adenosyl-L-methionine (SAM) were incubated in the presence of BSA or recombinant PRMT1. Samples were immunoblotted with the anti-mono-methylated R887 INCENP antibody, and amounts of loading proteins were evaluated by staining with MemCode TM Reversible Protein Stain. C. 293T cells were co-transfected with a FLAG-INCENP-WT (amino acids 821-918) vector or a FLAG-INCENP-R887A (amino acids 821-918) vector and an HA-PRMT1 vector. The samples were immunoblotted with anti-mono-methylated R887 INCENP, anti-FLAG and anti-AURKB antibodies after immunoprecipitating with anti-FLAG M2 agarose (Sigma-Aldrich). D. HeLa cells were transfected with FLAG-PRMT1-WT and immunocytochemistry was performed with anti-FLAG (Sigma-Aldrich, M2, dilution: 1:100, green) and anti-R887meINCENP (dilution: 1:100, green) antibodies. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Transfected HeLa cells exhibited higher INCENP and R887meINCENP integrated densities.

Techniques Used: In Vivo, Methylation, Enzyme-linked Immunosorbent Assay, Recombinant, Incubation, Staining, Transfection, Plasmid Preparation, Immunocytochemistry

21) Product Images from "PRMT1 promotes mitosis of cancer cells through arginine methylation of INCENP"

Article Title: PRMT1 promotes mitosis of cancer cells through arginine methylation of INCENP

Journal: Oncotarget

doi:

In vivo methylation of INCENP by PRMT1 A. Determination of the titer and specificity of the anti-mono-methylated R887 INCENP antibody analyzed by ELISA. B. Validation of the anti-mono-methylated R887 INCENP antibody. Recombinant GST-tagged INCENP protein and S-adenosyl-L-methionine (SAM) were incubated in the presence of BSA or recombinant PRMT1. Samples were immunoblotted with the anti-mono-methylated R887 INCENP antibody, and amounts of loading proteins were evaluated by staining with MemCode TM Reversible Protein Stain. C. 293T cells were co-transfected with a FLAG-INCENP-WT (amino acids 821-918) vector or a FLAG-INCENP-R887A (amino acids 821-918) vector and an HA-PRMT1 vector. The samples were immunoblotted with anti-mono-methylated R887 INCENP, anti-FLAG and anti-AURKB antibodies after immunoprecipitating with anti-FLAG M2 agarose (Sigma-Aldrich). D. HeLa cells were transfected with FLAG-PRMT1-WT and immunocytochemistry was performed with anti-FLAG (Sigma-Aldrich, M2, dilution: 1:100, green) and anti-R887meINCENP (dilution: 1:100, green) antibodies. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Transfected HeLa cells exhibited higher INCENP and R887meINCENP integrated densities.
Figure Legend Snippet: In vivo methylation of INCENP by PRMT1 A. Determination of the titer and specificity of the anti-mono-methylated R887 INCENP antibody analyzed by ELISA. B. Validation of the anti-mono-methylated R887 INCENP antibody. Recombinant GST-tagged INCENP protein and S-adenosyl-L-methionine (SAM) were incubated in the presence of BSA or recombinant PRMT1. Samples were immunoblotted with the anti-mono-methylated R887 INCENP antibody, and amounts of loading proteins were evaluated by staining with MemCode TM Reversible Protein Stain. C. 293T cells were co-transfected with a FLAG-INCENP-WT (amino acids 821-918) vector or a FLAG-INCENP-R887A (amino acids 821-918) vector and an HA-PRMT1 vector. The samples were immunoblotted with anti-mono-methylated R887 INCENP, anti-FLAG and anti-AURKB antibodies after immunoprecipitating with anti-FLAG M2 agarose (Sigma-Aldrich). D. HeLa cells were transfected with FLAG-PRMT1-WT and immunocytochemistry was performed with anti-FLAG (Sigma-Aldrich, M2, dilution: 1:100, green) and anti-R887meINCENP (dilution: 1:100, green) antibodies. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Transfected HeLa cells exhibited higher INCENP and R887meINCENP integrated densities.

Techniques Used: In Vivo, Methylation, Enzyme-linked Immunosorbent Assay, Recombinant, Incubation, Staining, Transfection, Plasmid Preparation, Immunocytochemistry

22) Product Images from "Pathogenic exon-trapping by SVA retrotransposon and rescue in Fukuyama muscular dystrophy"

Article Title: Pathogenic exon-trapping by SVA retrotransposon and rescue in Fukuyama muscular dystrophy

Journal: Nature

doi: 10.1038/nature10456

Abnormal fukutin protein in FCMD a-c, Immunoprecipitation analysis of fukutin protein in human lymphoblasts ( a ), both skeletal muscle and brain tissues from Hp/Hp mice ( b ), and brain tissue from FCMD patients ( c ). Closed arrow, abnormal fukutin. N, normal sample; F, FCMD patient sample. Hn, Hn/Hn mice; Hp, Hp/Hp mice; PI, preimmune serum. D, Duchenne muscular dystrophy (DMD) patient. d, The subcellular localization of fukutin. Top, normal fukutin; middle, mis-spliced fukutin; bottom, truncated fukutin. Stained with anti-FLAG (Left, to detect fukutin), anti-GM130 (middle, Golgi marker, top) and anti-KDEL (ER marker, middle and bottom), and merge (right, with DAPI stain). Scale bar, 10 μm.
Figure Legend Snippet: Abnormal fukutin protein in FCMD a-c, Immunoprecipitation analysis of fukutin protein in human lymphoblasts ( a ), both skeletal muscle and brain tissues from Hp/Hp mice ( b ), and brain tissue from FCMD patients ( c ). Closed arrow, abnormal fukutin. N, normal sample; F, FCMD patient sample. Hn, Hn/Hn mice; Hp, Hp/Hp mice; PI, preimmune serum. D, Duchenne muscular dystrophy (DMD) patient. d, The subcellular localization of fukutin. Top, normal fukutin; middle, mis-spliced fukutin; bottom, truncated fukutin. Stained with anti-FLAG (Left, to detect fukutin), anti-GM130 (middle, Golgi marker, top) and anti-KDEL (ER marker, middle and bottom), and merge (right, with DAPI stain). Scale bar, 10 μm.

Techniques Used: Immunoprecipitation, Mouse Assay, Staining, Marker

Related Articles

Clone Assay:

Article Title: Sipl1 and Rbck1 Are Novel Eya1-Binding Proteins with a Role in Craniofacial Development ▿
Article Snippet: Yeast expression constructs for Eya1, Eya2, and Eya3 were obtained by PCR amplification of the respective cDNA fragments and subsequent cloning into pGBT9 for interaction assays or pGBKT7 for expression analysis. .. Mouse monoclonal anti-Flag M2 (Sigma), anti-HA 6E2 and anti-c-Myc 9B11 (Cell Signaling), and anti-β-actin (ab8224; Abcam) antibodies were purchased from the indicated manufacturers.

Amplification:

Article Title: Sipl1 and Rbck1 Are Novel Eya1-Binding Proteins with a Role in Craniofacial Development ▿
Article Snippet: Yeast expression constructs for Eya1, Eya2, and Eya3 were obtained by PCR amplification of the respective cDNA fragments and subsequent cloning into pGBT9 for interaction assays or pGBKT7 for expression analysis. .. Mouse monoclonal anti-Flag M2 (Sigma), anti-HA 6E2 and anti-c-Myc 9B11 (Cell Signaling), and anti-β-actin (ab8224; Abcam) antibodies were purchased from the indicated manufacturers.

Whole Genome Amplification:

Article Title: Lipopolysaccharide (LPS)-binding protein stimulates CD14-dependent Toll-like receptor 4 internalization and LPS-induced TBK1–IKKϵ–IRF3 axis activation
Article Snippet: CF®488A-conjugated WGA was from Biotium Inc. (Fremont, CA). .. Other Abs were purchased from the following companies: mouse anti-FLAG M2 mAb, Sigma; rabbit anti-GFP Ab, anti-EEA1 antibody, mouse anti-HA (TANA2) mAb, MBL, Nagoya, Japan; mouse anti-human LBP mAb (biG412), Biometec GmbH, Greifswald, Germany; mouse anti-IκBα (L35A4), anti-phospho-IκBα (Ser-32/36) (5A5), rabbit anti-phospho-TBK1 (Ser-172) (D52C2), anti-phospho-IKKϵ (Ser-172) (D1B7), anti-IRF3 (D83B9), anti-phospho-IRF3 (Ser-396) (4D4G), anti-IRAK1 (D51G7), anti-TLR4 (D8L5W) mAb, anti-phospho-p44/42 MAPK (Erk1/2) (Thr-202/Tyr-204), anti-phospho-p38 MAPK (Thr-180/Tyr-182), anti-phospho-SAPK/JNK (Thr-183/Tyr-185), anti-TRAF3 Ab, and HRP-conjugated goat anti-rabbit IgG Ab, Cell Signaling Technology, Danvers, MA; mouse anti-RIP mAb (38/RIP), BD Biosciences, San Jose, CA; HRP-conjugated goat anti-mouse IgG Ab, Jackson ImmunoResearch Laboratories; mouse anti-human/mouse TLR2 (T2.5) mAb, allophycocyanin- and phycoerythrin (PE)-conjugated goat anti-mouse IgG, anti-rat IgG, PE- and HRP-conjugated streptavidin (Stv), BioLegend, San Diego, CA; and Alexa 546-conjugated F(ab′)2 goat anti-mouse IgG (H+L), Invitrogen; rabbit anti-NAK/TBK1 (EP611Y), anti-TRAF6 (EP591Y) mAb, Alexa 488-conjugated preadsorbed goat anti-rabbit IgG H & L, Abcam, Cambridge, UK.

Blocking Assay:

Article Title: The Autism-Related Protein PX-RICS Mediates GABAergic Synaptic Plasticity in Hippocampal Neurons and Emotional Learning in Mice
Article Snippet: After being fixed with 2% paraformaldehyde in PBS for 15 min at room temperature, the neurons were incubated with unconjugated anti–rabbit IgG (50 μg/ml; Sigma-Aldrich) for 1 h at room temperature to block the unlabeled primary antibody remaining on the neuronal surface, rinsed in PBS and then permeabilized with 0.2% Triton X-100 in PBS for 5 min. .. Alternatively, in , the neurons were double-stained with anti–γ2 and mouse mAb against FLAG (M2; 1:500; Sigma-Aldrich), and then with Alexa Fluor 647–labeled anti–rabbit IgG and Alexa Fluor 594–labeled anti–mouse IgG.

Incubation:

Article Title: Identification of Cysteine Ubiquitylation Sites on the Sec23A Protein of the COPII Complex Required for Vesicle Formation from the ER
Article Snippet: Equal amounts of cell lysates were incubated with mouse anti-HA monoclonal antibody (F-7; Santa Cruz Biotechnology) overnight at 4°C, followed by incubation with protein A-Sepharose beads (GE Healthcare) for 1 hour at 4°C. .. The immune complexes were analysed by SDS-PAGE (10%) and revealed by mouse anti-FLAG monoclonal antibody (M2, Sigma-Aldrich), mouse anti-HA antibody and mouse monoclonal anti-ʏ tubulin (Santa Cruz Biotechnology).

Article Title: The Autism-Related Protein PX-RICS Mediates GABAergic Synaptic Plasticity in Hippocampal Neurons and Emotional Learning in Mice
Article Snippet: The neurons were again incubated with anti–γ2 for 60 min at room temperature, followed by staining with Alexa Fluor 594– or Alexa Fluor 647–conjugated anti–rabbit IgG (1:500; Thermo Fisher Scientific) for 60 min at room temperature. .. Alternatively, in , the neurons were double-stained with anti–γ2 and mouse mAb against FLAG (M2; 1:500; Sigma-Aldrich), and then with Alexa Fluor 647–labeled anti–rabbit IgG and Alexa Fluor 594–labeled anti–mouse IgG.

Article Title: Interferon regulatory factor 3 controls interleukin-17 expression in CD8 T lymphocytes
Article Snippet: .. Whole-cell lysates were incubated overnight at 4 °C with rabbit polyclonal anti-Rorγt (H190; Santa Cruz Biotechnology), polyclonal rabbit anti-IRF3 (ZM3; Invitrogen), rabbit polyclonal anti-T7 (MBL), or monoclonal mouse anti-FLAG (M2; Sigma) covalently linked to protein G magnetic-activated beads (Dynabeads Protein G, Invitrogen). ..

Article Title: The Delta intracellular domain mediates TGF-?/Activin signaling through binding to Smads and has an important bi-directional function in the Notch-Delta signaling pathway
Article Snippet: .. Forty-eight hours after transfection, cells were disrupted and aliquots of 20 μg of cell lysates were incubated with 1 μg of rabbit anti-Dll1IC antibody or 1 μg of mouse monoclonal anti-FLAG M2 antibody (Sigma), followed by immunoprecipitation and western blotting as described ( ). .. Establishment of Dll1IC expressing P19 cell lines and neuronal induction P19 cells were cultured in alpha-modified Eagle's essential medium (Sigma) containing 10% FCS and 2 mM l -glutamine.

Luciferase:

Article Title: Sipl1 and Rbck1 Are Novel Eya1-Binding Proteins with a Role in Craniofacial Development ▿
Article Snippet: Yeast expression constructs for mouse Eya4 were kindly provided by Richard J. H. Smith, and the plasmids pGL3-MEF3/TATA, pGL3-TATA, and pCR3-Six4 used for luciferase reporter assays were provided by Pascal Maire. .. Mouse monoclonal anti-Flag M2 (Sigma), anti-HA 6E2 and anti-c-Myc 9B11 (Cell Signaling), and anti-β-actin (ab8224; Abcam) antibodies were purchased from the indicated manufacturers.

Activity Assay:

Article Title: An Aplysia Type 4 Phosphodiesterase Homolog Localizes at the Presynaptic Terminals of Aplysia Neuron and Regulates Synaptic Facilitation
Article Snippet: The principle of this method is that 3 H-labeled cAMP is converted to 3 H-labeled 5′-AMP by phosphodiesterase activity. .. We confirmed that the overexpressed apPDE protein mainly existed in the soluble part by Western blotting using monoclonal mouse anti-FLAG (Sigma) (data not shown).

Expressing:

Article Title: Potent HIV-1-Specific CD8 T Cell Responses Induced in Mice after Priming with a Multiepitopic DNA-TMEP and Boosting with the HIV Vaccine MVA-B
Article Snippet: .. At 6 h post-infection, cells were harvested, washed with PBS and lysed in Laemmli buffer with β-mercaptoethanol; cell extracts were fractionated by 8% SDS-PAGE and analyzed by Western blot using mouse monoclonal anti-FLAG M2 antibody (1:1000; Sigma-Aldrich) to evaluate TMEP-B expression. .. Anti-mouse-horseradish peroxidase (1:2000; SIGMA-ALDRICH) was used as conjugated secondary antibody.

Article Title: Identification of Cysteine Ubiquitylation Sites on the Sec23A Protein of the COPII Complex Required for Vesicle Formation from the ER
Article Snippet: 2.3 Co-Immunoprecipitation 10 cm dish-cultured HuH7 or Hek 293 T cells were co-transfected with either the p3xFLAG-Sec23a or the p3XFLAG empty vector plus the same DNA amount of the pcDNA3/HA-Ubiquitin expression vector [ ]. .. The immune complexes were analysed by SDS-PAGE (10%) and revealed by mouse anti-FLAG monoclonal antibody (M2, Sigma-Aldrich), mouse anti-HA antibody and mouse monoclonal anti-ʏ tubulin (Santa Cruz Biotechnology).

Article Title: Sipl1 and Rbck1 Are Novel Eya1-Binding Proteins with a Role in Craniofacial Development ▿
Article Snippet: Paragraph title: Expression constructs and antibodies. ... Mouse monoclonal anti-Flag M2 (Sigma), anti-HA 6E2 and anti-c-Myc 9B11 (Cell Signaling), and anti-β-actin (ab8224; Abcam) antibodies were purchased from the indicated manufacturers.

Modification:

Article Title: An Aplysia Type 4 Phosphodiesterase Homolog Localizes at the Presynaptic Terminals of Aplysia Neuron and Regulates Synaptic Facilitation
Article Snippet: We assayed PDE activity by a modified procedure of Thompson and Appleman ( ) and Bauer and Schwabe ( ). .. We confirmed that the overexpressed apPDE protein mainly existed in the soluble part by Western blotting using monoclonal mouse anti-FLAG (Sigma) (data not shown).

Western Blot:

Article Title: Potent HIV-1-Specific CD8 T Cell Responses Induced in Mice after Priming with a Multiepitopic DNA-TMEP and Boosting with the HIV Vaccine MVA-B
Article Snippet: .. At 6 h post-infection, cells were harvested, washed with PBS and lysed in Laemmli buffer with β-mercaptoethanol; cell extracts were fractionated by 8% SDS-PAGE and analyzed by Western blot using mouse monoclonal anti-FLAG M2 antibody (1:1000; Sigma-Aldrich) to evaluate TMEP-B expression. .. Anti-mouse-horseradish peroxidase (1:2000; SIGMA-ALDRICH) was used as conjugated secondary antibody.

Article Title: An Aplysia Type 4 Phosphodiesterase Homolog Localizes at the Presynaptic Terminals of Aplysia Neuron and Regulates Synaptic Facilitation
Article Snippet: .. We confirmed that the overexpressed apPDE protein mainly existed in the soluble part by Western blotting using monoclonal mouse anti-FLAG (Sigma) (data not shown). ..

Article Title: Interferon regulatory factor 3 controls interleukin-17 expression in CD8 T lymphocytes
Article Snippet: Paragraph title: Western Blotting and Co-IP Experiments. ... Whole-cell lysates were incubated overnight at 4 °C with rabbit polyclonal anti-Rorγt (H190; Santa Cruz Biotechnology), polyclonal rabbit anti-IRF3 (ZM3; Invitrogen), rabbit polyclonal anti-T7 (MBL), or monoclonal mouse anti-FLAG (M2; Sigma) covalently linked to protein G magnetic-activated beads (Dynabeads Protein G, Invitrogen).

Article Title: Termination factor Rho: From the control of pervasive transcription to cell fate determination in Bacillus subtilis
Article Snippet: Paragraph title: Western blotting ... The SPA-tagged Rho, KinA and KinB proteins were visualized using the primary mouse ANTI-FLAG M2 monoclonal antibodies (Sigma-Aldrich; dilution 1:5,000) and the secondary goat peroxidase-coupled anti-mouse IgG antibodies (Sigma-Aldrich; dilution 1:20,000).

Article Title: Pseudomonas aeruginosa injects NDK into host cells through a type III secretion system
Article Snippet: Paragraph title: Western blotting. ... Proteins were transferred onto PVDF membranes and subjected to immunoblotting using an anti-FLAG antibody (mouse M2 monoclonal Ab; Sigma) for NDK and anti-β-actin (Santa Cruz) for actin, with a 1000-fold dilution.

Article Title: The Delta intracellular domain mediates TGF-?/Activin signaling through binding to Smads and has an important bi-directional function in the Notch-Delta signaling pathway
Article Snippet: .. Forty-eight hours after transfection, cells were disrupted and aliquots of 20 μg of cell lysates were incubated with 1 μg of rabbit anti-Dll1IC antibody or 1 μg of mouse monoclonal anti-FLAG M2 antibody (Sigma), followed by immunoprecipitation and western blotting as described ( ). .. Establishment of Dll1IC expressing P19 cell lines and neuronal induction P19 cells were cultured in alpha-modified Eagle's essential medium (Sigma) containing 10% FCS and 2 mM l -glutamine.

Transfection:

Article Title: Potent HIV-1-Specific CD8 T Cell Responses Induced in Mice after Priming with a Multiepitopic DNA-TMEP and Boosting with the HIV Vaccine MVA-B
Article Snippet: Paragraph title: 2.5. Transfection Assay and Expression of TMEP-B Protein by Western Blot Analysis ... At 6 h post-infection, cells were harvested, washed with PBS and lysed in Laemmli buffer with β-mercaptoethanol; cell extracts were fractionated by 8% SDS-PAGE and analyzed by Western blot using mouse monoclonal anti-FLAG M2 antibody (1:1000; Sigma-Aldrich) to evaluate TMEP-B expression.

Article Title: Interferon regulatory factor 3 controls interleukin-17 expression in CD8 T lymphocytes
Article Snippet: Then, 48 h after transfection, cells were lysed in lysis buffer [0.5% Nonidet P-40, 50 mM Tris (pH 7.4), 200 mM NaCl, 10% (vol/vol) glycerol 1 mM DTT, and protease and phosphatase inhibitors). .. Whole-cell lysates were incubated overnight at 4 °C with rabbit polyclonal anti-Rorγt (H190; Santa Cruz Biotechnology), polyclonal rabbit anti-IRF3 (ZM3; Invitrogen), rabbit polyclonal anti-T7 (MBL), or monoclonal mouse anti-FLAG (M2; Sigma) covalently linked to protein G magnetic-activated beads (Dynabeads Protein G, Invitrogen).

Article Title: Role of CCAAT/Enhancer-Binding Protein Alpha (C/EBP?) in Activation of the Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Lytic-Cycle Replication-Associated Protein (RAP) Promoter in Cooperation with the KSHV Replication and Transcription Activator (RTA) and RAP
Article Snippet: The IFA was performed at 40 h after transfection of BCBL-1 cells. .. Secondary donkey- or goat-derived fluorescein isothiocyanate- or rhodamine-conjugated anti-rabbit or anti-mouse immunoglobulin G (Jackson Pharmaceuticals, West Grove, Pa.) was used to detect the primary antibodies, which included rabbit antipeptide antiserum against KSHV RAP ( ) and mouse anti-Flag monoclonal antibody (MAb) (Sigma).

Article Title: The Delta intracellular domain mediates TGF-?/Activin signaling through binding to Smads and has an important bi-directional function in the Notch-Delta signaling pathway
Article Snippet: .. Forty-eight hours after transfection, cells were disrupted and aliquots of 20 μg of cell lysates were incubated with 1 μg of rabbit anti-Dll1IC antibody or 1 μg of mouse monoclonal anti-FLAG M2 antibody (Sigma), followed by immunoprecipitation and western blotting as described ( ). .. Establishment of Dll1IC expressing P19 cell lines and neuronal induction P19 cells were cultured in alpha-modified Eagle's essential medium (Sigma) containing 10% FCS and 2 mM l -glutamine.

Protease Inhibitor:

Article Title: Identification of Cysteine Ubiquitylation Sites on the Sec23A Protein of the COPII Complex Required for Vesicle Formation from the ER
Article Snippet: At 48 hours, the cells were subjected to drug treatments, as indicated, before to be harvested in the lysis buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA pH 8.0, 1% Triton X-100) supplemented by protease inhibitor cocktail (Roche). .. The immune complexes were analysed by SDS-PAGE (10%) and revealed by mouse anti-FLAG monoclonal antibody (M2, Sigma-Aldrich), mouse anti-HA antibody and mouse monoclonal anti-ʏ tubulin (Santa Cruz Biotechnology).

Infection:

Article Title: Potent HIV-1-Specific CD8 T Cell Responses Induced in Mice after Priming with a Multiepitopic DNA-TMEP and Boosting with the HIV Vaccine MVA-B
Article Snippet: Transfection Assay and Expression of TMEP-B Protein by Western Blot Analysis To determine the correct expression of TMEP-B protein from DNA-TMEP vector, 293T cells (1 × 106 ) were mock-infected or infected with 5 pfu/cell of WR or VT7 viruses and transfected 1 h later with 5 μg of DNA-TMEP or pMax-GFP (Lonza, Basel, Switzerland) using Lipofectamine 2000 (Invitrogen) according to manufacturer’s recommendations. .. At 6 h post-infection, cells were harvested, washed with PBS and lysed in Laemmli buffer with β-mercaptoethanol; cell extracts were fractionated by 8% SDS-PAGE and analyzed by Western blot using mouse monoclonal anti-FLAG M2 antibody (1:1000; Sigma-Aldrich) to evaluate TMEP-B expression.

Generated:

Article Title: Sipl1 and Rbck1 Are Novel Eya1-Binding Proteins with a Role in Craniofacial Development ▿
Article Snippet: Mammalian expression constructs for mouse Flag-Sipl1 and Flag-Rbck1 as well as bacterial expression constructs for glutathione S -transferase (GST)-Sipl1 and GST-Rbck1 were generated by cloning of the full-length cDNAs into pcDNA3.1-Flag and pGEX-KG , respectively. .. Mouse monoclonal anti-Flag M2 (Sigma), anti-HA 6E2 and anti-c-Myc 9B11 (Cell Signaling), and anti-β-actin (ab8224; Abcam) antibodies were purchased from the indicated manufacturers.

other:

Article Title: ErbB-2 signals through Plexin-B1 to promote breast cancer metastasis
Article Snippet: The following antibodies were used: rabbit polyclonal anti–cleaved caspase-3 (Cell Signaling Technology), rabbit polyclonal anti-CD31 (Abcam), mouse monoclonal anti–ErbB-2 (clone E2-4001, Invitrogen), rabbit polyclonal anti–phospho–ErbB-2(Y1248) (Cell Signaling Technology), rabbit polyclonal anti–phospho-ErbB-2(Y1248) (Sigma-Aldrich), rat monoclonal anti–Mac-3 (clone M3/84, BD Biosciences — Pharmingen), goat polyclonal anti–Plexin-B1 (R & D Systems), mouse monoclonal anti–Plexin-B1 (clone 439512, R & D Systems), rabbit monoclonal anti-RhoA (clone 67B9, Cell Signaling Technology), rabbit polyclonal anti-RhoB (Cell Signaling Technology), rabbit monoclonal anti-RhoC (clone D40E4, Cell Signaling Technology), mouse monoclonal anti–α-tubulin (Sigma-Aldrich), goat polyclonal anti-VSV (Thermo), mouse monoclonal anti-phosphotyrosine (clone 4G10, Upstate, Millipore), mouse monoclonal anti-FLAG (clone M2, Sigma-Aldrich), rabbit polyclonal anti-Myc (Sigma-Aldrich), mouse monoclonal anti-HA (clone HA-7, Sigma-Aldrich), and trastuzumab (Genentech).

Polymerase Chain Reaction:

Article Title: Sipl1 and Rbck1 Are Novel Eya1-Binding Proteins with a Role in Craniofacial Development ▿
Article Snippet: Yeast expression constructs for Eya1, Eya2, and Eya3 were obtained by PCR amplification of the respective cDNA fragments and subsequent cloning into pGBT9 for interaction assays or pGBKT7 for expression analysis. .. Mouse monoclonal anti-Flag M2 (Sigma), anti-HA 6E2 and anti-c-Myc 9B11 (Cell Signaling), and anti-β-actin (ab8224; Abcam) antibodies were purchased from the indicated manufacturers.

Injection:

Article Title: Pseudomonas aeruginosa injects NDK into host cells through a type III secretion system
Article Snippet: Secretion and injection samples were loaded and separated on 4–20 % gradient SDS-PAGE gels (Bio-Rad). .. Proteins were transferred onto PVDF membranes and subjected to immunoblotting using an anti-FLAG antibody (mouse M2 monoclonal Ab; Sigma) for NDK and anti-β-actin (Santa Cruz) for actin, with a 1000-fold dilution.

Recombinant:

Article Title: Lipopolysaccharide (LPS)-binding protein stimulates CD14-dependent Toll-like receptor 4 internalization and LPS-induced TBK1–IKKϵ–IRF3 axis activation
Article Snippet: Other Abs were purchased from the following companies: mouse anti-FLAG M2 mAb, Sigma; rabbit anti-GFP Ab, anti-EEA1 antibody, mouse anti-HA (TANA2) mAb, MBL, Nagoya, Japan; mouse anti-human LBP mAb (biG412), Biometec GmbH, Greifswald, Germany; mouse anti-IκBα (L35A4), anti-phospho-IκBα (Ser-32/36) (5A5), rabbit anti-phospho-TBK1 (Ser-172) (D52C2), anti-phospho-IKKϵ (Ser-172) (D1B7), anti-IRF3 (D83B9), anti-phospho-IRF3 (Ser-396) (4D4G), anti-IRAK1 (D51G7), anti-TLR4 (D8L5W) mAb, anti-phospho-p44/42 MAPK (Erk1/2) (Thr-202/Tyr-204), anti-phospho-p38 MAPK (Thr-180/Tyr-182), anti-phospho-SAPK/JNK (Thr-183/Tyr-185), anti-TRAF3 Ab, and HRP-conjugated goat anti-rabbit IgG Ab, Cell Signaling Technology, Danvers, MA; mouse anti-RIP mAb (38/RIP), BD Biosciences, San Jose, CA; HRP-conjugated goat anti-mouse IgG Ab, Jackson ImmunoResearch Laboratories; mouse anti-human/mouse TLR2 (T2.5) mAb, allophycocyanin- and phycoerythrin (PE)-conjugated goat anti-mouse IgG, anti-rat IgG, PE- and HRP-conjugated streptavidin (Stv), BioLegend, San Diego, CA; and Alexa 546-conjugated F(ab′)2 goat anti-mouse IgG (H+L), Invitrogen; rabbit anti-NAK/TBK1 (EP611Y), anti-TRAF6 (EP591Y) mAb, Alexa 488-conjugated preadsorbed goat anti-rabbit IgG H & L, Abcam, Cambridge, UK. .. Recombinant sCD14, LBP, and LBP-N tagged with FLAG sequences at the C terminus were prepared as previously described ( , ).

Immunofluorescence:

Article Title: Role of CCAAT/Enhancer-Binding Protein Alpha (C/EBP?) in Activation of the Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Lytic-Cycle Replication-Associated Protein (RAP) Promoter in Cooperation with the KSHV Replication and Transcription Activator (RTA) and RAP
Article Snippet: Paragraph title: Indirect IFA. ... Secondary donkey- or goat-derived fluorescein isothiocyanate- or rhodamine-conjugated anti-rabbit or anti-mouse immunoglobulin G (Jackson Pharmaceuticals, West Grove, Pa.) was used to detect the primary antibodies, which included rabbit antipeptide antiserum against KSHV RAP ( ) and mouse anti-Flag monoclonal antibody (MAb) (Sigma).

Microscopy:

Article Title: The Autism-Related Protein PX-RICS Mediates GABAergic Synaptic Plasticity in Hippocampal Neurons and Emotional Learning in Mice
Article Snippet: Alternatively, in , the neurons were double-stained with anti–γ2 and mouse mAb against FLAG (M2; 1:500; Sigma-Aldrich), and then with Alexa Fluor 647–labeled anti–rabbit IgG and Alexa Fluor 594–labeled anti–mouse IgG. .. The cell images were obtained with an LSM510META laser scanning confocal microscope (ZEISS).

Purification:

Article Title: An Aplysia Type 4 Phosphodiesterase Homolog Localizes at the Presynaptic Terminals of Aplysia Neuron and Regulates Synaptic Facilitation
Article Snippet: Degraded 3 H-labeled cAMP is purified with QAE-Sephadex (Sigma, St. Louis, MO) columns. .. We confirmed that the overexpressed apPDE protein mainly existed in the soluble part by Western blotting using monoclonal mouse anti-FLAG (Sigma) (data not shown).

Labeling:

Article Title: The Autism-Related Protein PX-RICS Mediates GABAergic Synaptic Plasticity in Hippocampal Neurons and Emotional Learning in Mice
Article Snippet: Paragraph title: 2.7. Labeling of Surface and Internal γ2 Subunits ... Alternatively, in , the neurons were double-stained with anti–γ2 and mouse mAb against FLAG (M2; 1:500; Sigma-Aldrich), and then with Alexa Fluor 647–labeled anti–rabbit IgG and Alexa Fluor 594–labeled anti–mouse IgG.

Lysis:

Article Title: Identification of Cysteine Ubiquitylation Sites on the Sec23A Protein of the COPII Complex Required for Vesicle Formation from the ER
Article Snippet: At 48 hours, the cells were subjected to drug treatments, as indicated, before to be harvested in the lysis buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA pH 8.0, 1% Triton X-100) supplemented by protease inhibitor cocktail (Roche). .. The immune complexes were analysed by SDS-PAGE (10%) and revealed by mouse anti-FLAG monoclonal antibody (M2, Sigma-Aldrich), mouse anti-HA antibody and mouse monoclonal anti-ʏ tubulin (Santa Cruz Biotechnology).

Article Title: Interferon regulatory factor 3 controls interleukin-17 expression in CD8 T lymphocytes
Article Snippet: For co-IP of endogenous IRF-3 and Rorγt, CD8 T cells were differentiated with IL-6 and TGF-β for 3 d and were lysed in the lysis buffer described above. .. Whole-cell lysates were incubated overnight at 4 °C with rabbit polyclonal anti-Rorγt (H190; Santa Cruz Biotechnology), polyclonal rabbit anti-IRF3 (ZM3; Invitrogen), rabbit polyclonal anti-T7 (MBL), or monoclonal mouse anti-FLAG (M2; Sigma) covalently linked to protein G magnetic-activated beads (Dynabeads Protein G, Invitrogen).

SDS Page:

Article Title: Potent HIV-1-Specific CD8 T Cell Responses Induced in Mice after Priming with a Multiepitopic DNA-TMEP and Boosting with the HIV Vaccine MVA-B
Article Snippet: .. At 6 h post-infection, cells were harvested, washed with PBS and lysed in Laemmli buffer with β-mercaptoethanol; cell extracts were fractionated by 8% SDS-PAGE and analyzed by Western blot using mouse monoclonal anti-FLAG M2 antibody (1:1000; Sigma-Aldrich) to evaluate TMEP-B expression. .. Anti-mouse-horseradish peroxidase (1:2000; SIGMA-ALDRICH) was used as conjugated secondary antibody.

Article Title: Identification of Cysteine Ubiquitylation Sites on the Sec23A Protein of the COPII Complex Required for Vesicle Formation from the ER
Article Snippet: .. The immune complexes were analysed by SDS-PAGE (10%) and revealed by mouse anti-FLAG monoclonal antibody (M2, Sigma-Aldrich), mouse anti-HA antibody and mouse monoclonal anti-ʏ tubulin (Santa Cruz Biotechnology). .. HRP-conjugated goat anti-mouse antibody (Sigma-Aldrich) was used as the secondary antibody.

Article Title: Pseudomonas aeruginosa injects NDK into host cells through a type III secretion system
Article Snippet: Secretion and injection samples were loaded and separated on 4–20 % gradient SDS-PAGE gels (Bio-Rad). .. Proteins were transferred onto PVDF membranes and subjected to immunoblotting using an anti-FLAG antibody (mouse M2 monoclonal Ab; Sigma) for NDK and anti-β-actin (Santa Cruz) for actin, with a 1000-fold dilution.

Plasmid Preparation:

Article Title: Potent HIV-1-Specific CD8 T Cell Responses Induced in Mice after Priming with a Multiepitopic DNA-TMEP and Boosting with the HIV Vaccine MVA-B
Article Snippet: Transfection Assay and Expression of TMEP-B Protein by Western Blot Analysis To determine the correct expression of TMEP-B protein from DNA-TMEP vector, 293T cells (1 × 106 ) were mock-infected or infected with 5 pfu/cell of WR or VT7 viruses and transfected 1 h later with 5 μg of DNA-TMEP or pMax-GFP (Lonza, Basel, Switzerland) using Lipofectamine 2000 (Invitrogen) according to manufacturer’s recommendations. .. At 6 h post-infection, cells were harvested, washed with PBS and lysed in Laemmli buffer with β-mercaptoethanol; cell extracts were fractionated by 8% SDS-PAGE and analyzed by Western blot using mouse monoclonal anti-FLAG M2 antibody (1:1000; Sigma-Aldrich) to evaluate TMEP-B expression.

Article Title: Identification of Cysteine Ubiquitylation Sites on the Sec23A Protein of the COPII Complex Required for Vesicle Formation from the ER
Article Snippet: 2.3 Co-Immunoprecipitation 10 cm dish-cultured HuH7 or Hek 293 T cells were co-transfected with either the p3xFLAG-Sec23a or the p3XFLAG empty vector plus the same DNA amount of the pcDNA3/HA-Ubiquitin expression vector [ ]. .. The immune complexes were analysed by SDS-PAGE (10%) and revealed by mouse anti-FLAG monoclonal antibody (M2, Sigma-Aldrich), mouse anti-HA antibody and mouse monoclonal anti-ʏ tubulin (Santa Cruz Biotechnology).

Article Title: Sipl1 and Rbck1 Are Novel Eya1-Binding Proteins with a Role in Craniofacial Development ▿
Article Snippet: Similarly, Sipl1 and its deletion fragments were introduced into the yeast expression vector pGADT7. .. Mouse monoclonal anti-Flag M2 (Sigma), anti-HA 6E2 and anti-c-Myc 9B11 (Cell Signaling), and anti-β-actin (ab8224; Abcam) antibodies were purchased from the indicated manufacturers.

Article Title: Role of CCAAT/Enhancer-Binding Protein Alpha (C/EBP?) in Activation of the Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Lytic-Cycle Replication-Associated Protein (RAP) Promoter in Cooperation with the KSHV Replication and Transcription Activator (RTA) and RAP
Article Snippet: Secondary donkey- or goat-derived fluorescein isothiocyanate- or rhodamine-conjugated anti-rabbit or anti-mouse immunoglobulin G (Jackson Pharmaceuticals, West Grove, Pa.) was used to detect the primary antibodies, which included rabbit antipeptide antiserum against KSHV RAP ( ) and mouse anti-Flag monoclonal antibody (MAb) (Sigma). .. Mounting solution with 4′,6′-diamidino-2-phenylindole (DAPI) (Vector Shield) was used to visualize cellular DNA.

Software:

Article Title: The Autism-Related Protein PX-RICS Mediates GABAergic Synaptic Plasticity in Hippocampal Neurons and Emotional Learning in Mice
Article Snippet: Alternatively, in , the neurons were double-stained with anti–γ2 and mouse mAb against FLAG (M2; 1:500; Sigma-Aldrich), and then with Alexa Fluor 647–labeled anti–rabbit IgG and Alexa Fluor 594–labeled anti–mouse IgG. .. The surface-expressed γ2 levels were quantitatively evaluated by analyzing fluorescent images with ImageJ software.

Co-Immunoprecipitation Assay:

Article Title: Interferon regulatory factor 3 controls interleukin-17 expression in CD8 T lymphocytes
Article Snippet: Paragraph title: Western Blotting and Co-IP Experiments. ... Whole-cell lysates were incubated overnight at 4 °C with rabbit polyclonal anti-Rorγt (H190; Santa Cruz Biotechnology), polyclonal rabbit anti-IRF3 (ZM3; Invitrogen), rabbit polyclonal anti-T7 (MBL), or monoclonal mouse anti-FLAG (M2; Sigma) covalently linked to protein G magnetic-activated beads (Dynabeads Protein G, Invitrogen).

Immunoprecipitation:

Article Title: The Delta intracellular domain mediates TGF-?/Activin signaling through binding to Smads and has an important bi-directional function in the Notch-Delta signaling pathway
Article Snippet: .. Forty-eight hours after transfection, cells were disrupted and aliquots of 20 μg of cell lysates were incubated with 1 μg of rabbit anti-Dll1IC antibody or 1 μg of mouse monoclonal anti-FLAG M2 antibody (Sigma), followed by immunoprecipitation and western blotting as described ( ). .. Establishment of Dll1IC expressing P19 cell lines and neuronal induction P19 cells were cultured in alpha-modified Eagle's essential medium (Sigma) containing 10% FCS and 2 mM l -glutamine.

Construct:

Article Title: Sipl1 and Rbck1 Are Novel Eya1-Binding Proteins with a Role in Craniofacial Development ▿
Article Snippet: Paragraph title: Expression constructs and antibodies. ... Mouse monoclonal anti-Flag M2 (Sigma), anti-HA 6E2 and anti-c-Myc 9B11 (Cell Signaling), and anti-β-actin (ab8224; Abcam) antibodies were purchased from the indicated manufacturers.

Staining:

Article Title: The Autism-Related Protein PX-RICS Mediates GABAergic Synaptic Plasticity in Hippocampal Neurons and Emotional Learning in Mice
Article Snippet: The neurons were again incubated with anti–γ2 for 60 min at room temperature, followed by staining with Alexa Fluor 594– or Alexa Fluor 647–conjugated anti–rabbit IgG (1:500; Thermo Fisher Scientific) for 60 min at room temperature. .. Alternatively, in , the neurons were double-stained with anti–γ2 and mouse mAb against FLAG (M2; 1:500; Sigma-Aldrich), and then with Alexa Fluor 647–labeled anti–rabbit IgG and Alexa Fluor 594–labeled anti–mouse IgG.

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  • 90
    Millipore anti flag tag rabbit antibody
    <t>Deaf1-MeCP2</t> interaction enhances their recruitment to the HTR1A promoter Deaf1 site. ( A ) MeCP2 increases Deaf1 transactivation of the human HTR1A C(-1019) site independent of promoter methylation. At left, yeast were transduced with a combination of Deaf1-GAL4AD or vector (pACT2), MSssI or vector (pA7WA), and MeCP2 or vector (pRS423) and the β-Gal gene fused to three copies of the human 5-HT1A 26 bp Deaf1 site with the C (1019-C) in the p8op vector. The level of β-galactosidase activity is shown as a measure of transactivation. At right, yeast were transduced with a combination of MeCP2-GAL4AD or vector (pACT2), MSssI or vector, and either the C or G allele of the HTR1A Deaf1 element (1019-C or 1019-G). Data shown as mean ± S.E., n = 3. ( B ) His-Deaf1 and <t>Flag-MeCP2</t> co-precipitate in co-transfected human SKN-SH neuroblastoma cells. His-Deaf1, FLAG-MeCP2 or FLAG-vector were co-transfected in HEK-293 cells and cell extracts (Input) analyzed by pull-down (PD) followed by Western blot for Deaf1 or FLAG (MeCP2). Left, FLAG-MeCP2 but not FLAG was enriched upon of His-Deaf1; right, His-Deaf1 was enriched upon pull-down of FLAG (red boxes). ( C ) Deaf1 recruits MeCP2 to the Deaf1 sites on the mouse HTR1A promoter. Deaf1 −/− and +/+ MEF extracts were analyzed by ChIP using pull-down with pre-immune rabbit IgG (Ig), n = 8; Deaf1 antibody (D1), n = 8; MeCP2 antibody (Me), n = 4; or histone H1 antibody (H1), n = 4. PCRs of precipitated DNA were run on 2% agarose gel. Above, HTR1A promoter ChIP. The mean total luminosity within a standard band template was quantified and normalized to the appropriate pre-immune IgG and plotted as –fold IgG ± SE, *p
    Anti Flag Tag Rabbit Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti flag tag rabbit antibody/product/Millipore
    Average 90 stars, based on 2 article reviews
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    anti flag tag rabbit antibody - by Bioz Stars, 2020-04
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    92
    Millipore anti flag igg solution buffer
    The membrane integration of TIC40 depends on SCY2/SECE2. Arabidopsis chloroplast lysate ( scy2 sece2 double mutant complemented with <t>SCY2-HA/FLAG-SECE2</t> ) was mixed with anti-FLAG, anti-HA, or unrelated anti-ALB3 <t>IgG</t> for 1 h before being
    Anti Flag Igg Solution Buffer, supplied by Millipore, 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/anti flag igg solution buffer/product/Millipore
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti flag igg solution buffer - by Bioz Stars, 2020-04
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    99
    Millipore monoclonal anti flag m2 antibody
    V H H3-sIgA subunits physically associate with one another. N. benthamiana leaf tissue co-infiltrated with all V H H3-sIgA subunits was collected at 6 dpi, and recombinant proteins were immunoprecipitated with an <t>anti-c-Myc</t> antibody. (A,B) Both cell extracts and immunoprecipitates were resolved by SDS-PAGE under reducing conditions. (C,D) SDS-PAGE performed under non-reducing conditions. (A,C) Immunoblots were detected with <t>anti-FLAG</t> antibodies. (B,D) Immunoblots were detected with anti-HA antibodies. Arrow points to a faint but nonetheless present monomeric JC band. TSP from p19-infiltrated N. benthamiana leaves was used as negative control.
    Monoclonal Anti Flag M2 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2407 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti flag m2 antibody/product/Millipore
    Average 99 stars, based on 2407 article reviews
    Price from $9.99 to $1999.99
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    Image Search Results


    Deaf1-MeCP2 interaction enhances their recruitment to the HTR1A promoter Deaf1 site. ( A ) MeCP2 increases Deaf1 transactivation of the human HTR1A C(-1019) site independent of promoter methylation. At left, yeast were transduced with a combination of Deaf1-GAL4AD or vector (pACT2), MSssI or vector (pA7WA), and MeCP2 or vector (pRS423) and the β-Gal gene fused to three copies of the human 5-HT1A 26 bp Deaf1 site with the C (1019-C) in the p8op vector. The level of β-galactosidase activity is shown as a measure of transactivation. At right, yeast were transduced with a combination of MeCP2-GAL4AD or vector (pACT2), MSssI or vector, and either the C or G allele of the HTR1A Deaf1 element (1019-C or 1019-G). Data shown as mean ± S.E., n = 3. ( B ) His-Deaf1 and Flag-MeCP2 co-precipitate in co-transfected human SKN-SH neuroblastoma cells. His-Deaf1, FLAG-MeCP2 or FLAG-vector were co-transfected in HEK-293 cells and cell extracts (Input) analyzed by pull-down (PD) followed by Western blot for Deaf1 or FLAG (MeCP2). Left, FLAG-MeCP2 but not FLAG was enriched upon of His-Deaf1; right, His-Deaf1 was enriched upon pull-down of FLAG (red boxes). ( C ) Deaf1 recruits MeCP2 to the Deaf1 sites on the mouse HTR1A promoter. Deaf1 −/− and +/+ MEF extracts were analyzed by ChIP using pull-down with pre-immune rabbit IgG (Ig), n = 8; Deaf1 antibody (D1), n = 8; MeCP2 antibody (Me), n = 4; or histone H1 antibody (H1), n = 4. PCRs of precipitated DNA were run on 2% agarose gel. Above, HTR1A promoter ChIP. The mean total luminosity within a standard band template was quantified and normalized to the appropriate pre-immune IgG and plotted as –fold IgG ± SE, *p

    Journal: Scientific Reports

    Article Title: Loss of MeCP2 in adult 5-HT neurons induces 5-HT1A autoreceptors, with opposite sex-dependent anxiety and depression phenotypes

    doi: 10.1038/s41598-018-24167-8

    Figure Lengend Snippet: Deaf1-MeCP2 interaction enhances their recruitment to the HTR1A promoter Deaf1 site. ( A ) MeCP2 increases Deaf1 transactivation of the human HTR1A C(-1019) site independent of promoter methylation. At left, yeast were transduced with a combination of Deaf1-GAL4AD or vector (pACT2), MSssI or vector (pA7WA), and MeCP2 or vector (pRS423) and the β-Gal gene fused to three copies of the human 5-HT1A 26 bp Deaf1 site with the C (1019-C) in the p8op vector. The level of β-galactosidase activity is shown as a measure of transactivation. At right, yeast were transduced with a combination of MeCP2-GAL4AD or vector (pACT2), MSssI or vector, and either the C or G allele of the HTR1A Deaf1 element (1019-C or 1019-G). Data shown as mean ± S.E., n = 3. ( B ) His-Deaf1 and Flag-MeCP2 co-precipitate in co-transfected human SKN-SH neuroblastoma cells. His-Deaf1, FLAG-MeCP2 or FLAG-vector were co-transfected in HEK-293 cells and cell extracts (Input) analyzed by pull-down (PD) followed by Western blot for Deaf1 or FLAG (MeCP2). Left, FLAG-MeCP2 but not FLAG was enriched upon of His-Deaf1; right, His-Deaf1 was enriched upon pull-down of FLAG (red boxes). ( C ) Deaf1 recruits MeCP2 to the Deaf1 sites on the mouse HTR1A promoter. Deaf1 −/− and +/+ MEF extracts were analyzed by ChIP using pull-down with pre-immune rabbit IgG (Ig), n = 8; Deaf1 antibody (D1), n = 8; MeCP2 antibody (Me), n = 4; or histone H1 antibody (H1), n = 4. PCRs of precipitated DNA were run on 2% agarose gel. Above, HTR1A promoter ChIP. The mean total luminosity within a standard band template was quantified and normalized to the appropriate pre-immune IgG and plotted as –fold IgG ± SE, *p

    Article Snippet: Entire sample supernatants were loaded on Western blot, stained with either Anti-FLAG tag rabbit antibody (1:2000; Millipore); anti-Deaf1 rabbit polyclonal antibody against homemade recombinant full-length human Deaf1 (1:500 v/v); anti-MeCP2 chicken antibody (Millipore CAT.# ABE171; 1:1000 v/v); or anti-β-actin mouse antibody (Sigma), followed up by the appropriate HRP-conjugated (Sigma; 1:4000) secondary antibody, and HRP substrate (Millipore).

    Techniques: Methylation, Transduction, Plasmid Preparation, Activity Assay, Transfection, Western Blot, Chromatin Immunoprecipitation, Agarose Gel Electrophoresis

    The membrane integration of TIC40 depends on SCY2/SECE2. Arabidopsis chloroplast lysate ( scy2 sece2 double mutant complemented with SCY2-HA/FLAG-SECE2 ) was mixed with anti-FLAG, anti-HA, or unrelated anti-ALB3 IgG for 1 h before being

    Journal: Plant Physiology

    Article Title: Identification of Putative Substrates of SEC2, a Chloroplast Inner Envelope Translocase 1Identification of Putative Substrates of SEC2, a Chloroplast Inner Envelope Translocase 1 [OPEN]

    doi: 10.1104/pp.17.00012

    Figure Lengend Snippet: The membrane integration of TIC40 depends on SCY2/SECE2. Arabidopsis chloroplast lysate ( scy2 sece2 double mutant complemented with SCY2-HA/FLAG-SECE2 ) was mixed with anti-FLAG, anti-HA, or unrelated anti-ALB3 IgG for 1 h before being

    Article Snippet: Before use, 100 μL of anti-FLAG IgG solution buffer was exchanged by three rounds of concentration to ∼10 μL with Microcon Centrifugal Filter Device YM-30 (EMD Millipore) followed by the addition of 100 μL of 20 m m HEPES-KOH, pH 7.5.

    Techniques: Mutagenesis

    V H H3-sIgA subunits physically associate with one another. N. benthamiana leaf tissue co-infiltrated with all V H H3-sIgA subunits was collected at 6 dpi, and recombinant proteins were immunoprecipitated with an anti-c-Myc antibody. (A,B) Both cell extracts and immunoprecipitates were resolved by SDS-PAGE under reducing conditions. (C,D) SDS-PAGE performed under non-reducing conditions. (A,C) Immunoblots were detected with anti-FLAG antibodies. (B,D) Immunoblots were detected with anti-HA antibodies. Arrow points to a faint but nonetheless present monomeric JC band. TSP from p19-infiltrated N. benthamiana leaves was used as negative control.

    Journal: Frontiers in Plant Science

    Article Title: Plant-Produced Chimeric VHH-sIgA Against Enterohemorrhagic E. coli Intimin Shows Cross-Serotype Inhibition of Bacterial Adhesion to Epithelial Cells

    doi: 10.3389/fpls.2019.00270

    Figure Lengend Snippet: V H H3-sIgA subunits physically associate with one another. N. benthamiana leaf tissue co-infiltrated with all V H H3-sIgA subunits was collected at 6 dpi, and recombinant proteins were immunoprecipitated with an anti-c-Myc antibody. (A,B) Both cell extracts and immunoprecipitates were resolved by SDS-PAGE under reducing conditions. (C,D) SDS-PAGE performed under non-reducing conditions. (A,C) Immunoblots were detected with anti-FLAG antibodies. (B,D) Immunoblots were detected with anti-HA antibodies. Arrow points to a faint but nonetheless present monomeric JC band. TSP from p19-infiltrated N. benthamiana leaves was used as negative control.

    Article Snippet: The recombinant proteins were detected with one of the following primary antibodies: mouse anti-c-Myc monoclonal antibody (GenScript, Cat. No. A00864), mouse anti-HA monoclonal antibody (Millipore Sigma, Cat. No. H3663), mouse anti-FLAG monoclonal antibody (Millipore Sigma, Cat. No. F3165), and HRP-conjugated goat anti-mouse IgG secondary antibody (Bio-Rad, Cat. No. 170-6516).

    Techniques: Recombinant, Immunoprecipitation, SDS Page, Western Blot, Negative Control

    Vacuum infiltration and purification of V H H9-sIgA. N. benthamiana leaves were vacuum infiltrated with a mixture of V H H9-Fc/SC/JC and p19. Tissue was collected at 12 dpi. Cell extracts were prepared under native conditions and separated with SDS-PAGE under non-reducing conditions. (A) Secretory IgA was purified with peptide M Agarose. Western blots were detected with anti-c-Myc antibody. Arrows indicate the expected size of fully assembled sIgA (No. 1), tetrameric (No. 2, ∼176 kDa), trimeric (No. 3, ∼132 kDa), dimeric (No. 4, ∼88 kDa), and monomeric (No. 5, ∼44 kDa) V H H9-Fc. (B) Secretory IgA was purified with anti-FLAG agarose. Western blots were detected with anti-Flag antibody. Arrows indicate the expected size of fully assembled sIgA (No. 1, ∼66 kDa), SC/trimeric V H H9-Fc/JC (No. 2, ∼206 kDa), SC/dimeric V H H9-Fc (No. 3, ∼160), SC/monomeric V H H9-Fc (No. 4, ∼110 kDa), and monomeric SC (No. 5). 10 μl of cell extract was loaded as a snapshot of the antibody produced in vivo .

    Journal: Frontiers in Plant Science

    Article Title: Plant-Produced Chimeric VHH-sIgA Against Enterohemorrhagic E. coli Intimin Shows Cross-Serotype Inhibition of Bacterial Adhesion to Epithelial Cells

    doi: 10.3389/fpls.2019.00270

    Figure Lengend Snippet: Vacuum infiltration and purification of V H H9-sIgA. N. benthamiana leaves were vacuum infiltrated with a mixture of V H H9-Fc/SC/JC and p19. Tissue was collected at 12 dpi. Cell extracts were prepared under native conditions and separated with SDS-PAGE under non-reducing conditions. (A) Secretory IgA was purified with peptide M Agarose. Western blots were detected with anti-c-Myc antibody. Arrows indicate the expected size of fully assembled sIgA (No. 1), tetrameric (No. 2, ∼176 kDa), trimeric (No. 3, ∼132 kDa), dimeric (No. 4, ∼88 kDa), and monomeric (No. 5, ∼44 kDa) V H H9-Fc. (B) Secretory IgA was purified with anti-FLAG agarose. Western blots were detected with anti-Flag antibody. Arrows indicate the expected size of fully assembled sIgA (No. 1, ∼66 kDa), SC/trimeric V H H9-Fc/JC (No. 2, ∼206 kDa), SC/dimeric V H H9-Fc (No. 3, ∼160), SC/monomeric V H H9-Fc (No. 4, ∼110 kDa), and monomeric SC (No. 5). 10 μl of cell extract was loaded as a snapshot of the antibody produced in vivo .

    Article Snippet: The recombinant proteins were detected with one of the following primary antibodies: mouse anti-c-Myc monoclonal antibody (GenScript, Cat. No. A00864), mouse anti-HA monoclonal antibody (Millipore Sigma, Cat. No. H3663), mouse anti-FLAG monoclonal antibody (Millipore Sigma, Cat. No. F3165), and HRP-conjugated goat anti-mouse IgG secondary antibody (Bio-Rad, Cat. No. 170-6516).

    Techniques: Purification, SDS Page, Western Blot, Produced, In Vivo

    Binding of plant-produced V H H9-sIgA to EHEC O157:H7 intimin. (A) SPR binding of V H H9-sIgA purified using peptide M. Either plant-produced V H H9-sIgA (top) or E. coli -produced V H H9 monomer (bottom) was immobilized on CM5 Series S sensor chips via amine coupling and MBP-Int277 was flowed over the resulting surfaces at concentrations ranging from 0.3 to 5 nM. The experiment was conducted in duplicate. Black lines show data and red lines show fits. (B) ELISA binding of plant-produced V H H9-sIgA purified using either peptide M (left) or anti-FLAG antibody (right) and detected using either anti-bovine IgA antibody (top) or anti-FLAG antibody (bottom). Results are representative of two independent experiments.

    Journal: Frontiers in Plant Science

    Article Title: Plant-Produced Chimeric VHH-sIgA Against Enterohemorrhagic E. coli Intimin Shows Cross-Serotype Inhibition of Bacterial Adhesion to Epithelial Cells

    doi: 10.3389/fpls.2019.00270

    Figure Lengend Snippet: Binding of plant-produced V H H9-sIgA to EHEC O157:H7 intimin. (A) SPR binding of V H H9-sIgA purified using peptide M. Either plant-produced V H H9-sIgA (top) or E. coli -produced V H H9 monomer (bottom) was immobilized on CM5 Series S sensor chips via amine coupling and MBP-Int277 was flowed over the resulting surfaces at concentrations ranging from 0.3 to 5 nM. The experiment was conducted in duplicate. Black lines show data and red lines show fits. (B) ELISA binding of plant-produced V H H9-sIgA purified using either peptide M (left) or anti-FLAG antibody (right) and detected using either anti-bovine IgA antibody (top) or anti-FLAG antibody (bottom). Results are representative of two independent experiments.

    Article Snippet: The recombinant proteins were detected with one of the following primary antibodies: mouse anti-c-Myc monoclonal antibody (GenScript, Cat. No. A00864), mouse anti-HA monoclonal antibody (Millipore Sigma, Cat. No. H3663), mouse anti-FLAG monoclonal antibody (Millipore Sigma, Cat. No. F3165), and HRP-conjugated goat anti-mouse IgG secondary antibody (Bio-Rad, Cat. No. 170-6516).

    Techniques: Binding Assay, Produced, SPR Assay, Purification, Enzyme-linked Immunosorbent Assay

    Design and production of individual subunits required for chimeric secretory IgA assembly. (A) Schematic of all produced subunits fully assembled into a chimeric antibody intended for secretory IgA functionality. It notably differs from the structure of native secretory IgA by the replacement of the Fab region with a camelid-derived variable heavy chain fragment (V H H). (B) Schematic representation of constructs used for Agrobacterium -mediated transient expression in N. benthamiana leaves. CaMV 35S, cauliflower mosaic virus 35S promoter; CPMV 5′UTR, 5′-untranslated region of Cowpea mosaic virus; PR1b, tobacco pathogenesis-related protein 1b signal peptide; V H Hx-Fc, fusion of a camelid-derived V H H to a bovine Fc where x is either 1, 3, 9, or 10, corresponding to the isolated V H Hs; SC, bovine secretory component; JC, bovine JC; c-Myc, FLAG, HA, detection tags; KDEL, endoplasmic reticulum retrieval tetra-peptide; CPMV 3′UTR, 3′-untranslated region of Cowpea mosaic virus; nos, nopaline synthase terminator sequence; the cassettes were cloned into pEAQ-DEST-1 plant expression vectors. Schematic not drawn to scale. Bold outlines indicate translated regions. (C) Monovalent affinities and kinetics of the interaction between V H Hs and MBP-Int277 by SPR (pH 7.4, 25°C). (D) Predicted protein size and number of glycosylation sites for each subunit. (E–G) Western blots of crude extract from leaves of N. benthamiana harvested at 6 dpi expressing V H H1, 3, 9, and 10-Fc along with p19, a suppressor of gene silencing (E) , SC (F) , and JC (G) . 10 μg of TSP was loaded in each lane.

    Journal: Frontiers in Plant Science

    Article Title: Plant-Produced Chimeric VHH-sIgA Against Enterohemorrhagic E. coli Intimin Shows Cross-Serotype Inhibition of Bacterial Adhesion to Epithelial Cells

    doi: 10.3389/fpls.2019.00270

    Figure Lengend Snippet: Design and production of individual subunits required for chimeric secretory IgA assembly. (A) Schematic of all produced subunits fully assembled into a chimeric antibody intended for secretory IgA functionality. It notably differs from the structure of native secretory IgA by the replacement of the Fab region with a camelid-derived variable heavy chain fragment (V H H). (B) Schematic representation of constructs used for Agrobacterium -mediated transient expression in N. benthamiana leaves. CaMV 35S, cauliflower mosaic virus 35S promoter; CPMV 5′UTR, 5′-untranslated region of Cowpea mosaic virus; PR1b, tobacco pathogenesis-related protein 1b signal peptide; V H Hx-Fc, fusion of a camelid-derived V H H to a bovine Fc where x is either 1, 3, 9, or 10, corresponding to the isolated V H Hs; SC, bovine secretory component; JC, bovine JC; c-Myc, FLAG, HA, detection tags; KDEL, endoplasmic reticulum retrieval tetra-peptide; CPMV 3′UTR, 3′-untranslated region of Cowpea mosaic virus; nos, nopaline synthase terminator sequence; the cassettes were cloned into pEAQ-DEST-1 plant expression vectors. Schematic not drawn to scale. Bold outlines indicate translated regions. (C) Monovalent affinities and kinetics of the interaction between V H Hs and MBP-Int277 by SPR (pH 7.4, 25°C). (D) Predicted protein size and number of glycosylation sites for each subunit. (E–G) Western blots of crude extract from leaves of N. benthamiana harvested at 6 dpi expressing V H H1, 3, 9, and 10-Fc along with p19, a suppressor of gene silencing (E) , SC (F) , and JC (G) . 10 μg of TSP was loaded in each lane.

    Article Snippet: The recombinant proteins were detected with one of the following primary antibodies: mouse anti-c-Myc monoclonal antibody (GenScript, Cat. No. A00864), mouse anti-HA monoclonal antibody (Millipore Sigma, Cat. No. H3663), mouse anti-FLAG monoclonal antibody (Millipore Sigma, Cat. No. F3165), and HRP-conjugated goat anti-mouse IgG secondary antibody (Bio-Rad, Cat. No. 170-6516).

    Techniques: Produced, Derivative Assay, Construct, Expressing, Isolation, Sequencing, Clone Assay, SPR Assay, Western Blot