anti rabbit  (Millipore)

 
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    Name:
    ANTI TYROSINASE C TERMINAL antibody
    Description:

    Catalog Number:
    SAB1306598
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    None
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    Structured Review

    Millipore anti rabbit
    ANTI TYROSINASE C TERMINAL antibody

    https://www.bioz.com/result/anti rabbit/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti rabbit - by Bioz Stars, 2021-07
    99/100 stars

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    other:

    Article Title: Endothelial dysfunction and compromised eNOS/Akt signaling in the thoracic aorta during the progression of Marfan syndrome
    Article Snippet: Ketamine hydrochloride and xylazine hydrochloride (Research Biochemicals International, Natick, MA, USA); phenylephrine, ACh, KCl, SNP, L -NAME, pluronic acid, HEPES, chemicals for preparing Krebs solution, anti-rabbit and anti-mouse IgG peroxidase-conjugated secondary antibodies (Sigma, Oakville, Canada); polyvinyldifluoride membranes (BioRad, Hercules, USA); anti-eNOS antibody (BD BioSciences, Mississauga, Canada), anti-phospho-eNOSSer1177 , Akt, phospho-AktThr308 and phospho-AktSer473 antibodies (Cell Signaling, Beverly, Massachusetts).

    Incubation:

    Article Title: Antibodies to a Surface-Exposed, N-terminal Domain of Aggregation Substance Are Not Protective in the Rabbit Model of Enterococcus faecalis Infective Endocarditis
    Article Snippet: .. Both were used at a 1:1,000 dilution and incubated for 1 h. Cells were washed three times with PBS and resuspended in 100 μl of PBSM containing either anti-rabbit or anti-mouse fluorescein isothiocyanate conjugate (each, 1:1,000 dilution) (Sigma) and incubated for 30 min. .. Cells were washed three times with PBS and resuspended in fluorescence-activated cell sorter buffer.

    Article Title: Comprehensive Characterization of the Pyroglutamate Amyloid-β Induced Motor Neurodegenerative Phenotype of TBA2.1 Mice
    Article Snippet: After a further washing step, sections were incubated with the primary antibody over night at 4°C in a humid chamber (6E10:1:1000, Bio Legend, San Diego, CA, USA; GFAP: 1:1000, DAKO, Agilent Technologies, Santa Clara, CA, USA; NeuN: 1:1000 Merck Millipore, Darmstadt, Germany, CD11b: 1:2500 Abcam, Cambridge, UK) in Tris buffered saline with 1% Triton X100 (TBST) with 1% bovine serum albumin (BSA). .. Afterwards, sections were incubated with a biotinylated secondary anti-mouse or anti-rabbit antibody (both 1:1000 in TBST+1% BSA, Sigma-Aldrich, Germany). .. Staining was visualized by use of 3, 3’-Diaminobenzidine (DAB) enhanced with saturated nickel ammonium sulphate solution (homozygous mice, and ), or by use of secondary antibodies labeled with different fluorescence dyes (Goat anti-mouse IgG (H + L), Alexa Fluor 568, Goat anti-rabbit IgG (H + L), Alexa Fluor 488, Invitrogen, USA).

    Article Title: Cytokine-stimulated release of decay-accelerating factor (DAF; CD55) from HT-29 human intestinal epithelial cells
    Article Snippet: .. After further incubation and washing, bound rabbit antibody was detected with HRP-labelled goat F(ab′)2 anti-rabbit IgG and 2,2′-amino-bis-3-ethylbenzo-thiazoline-6-sulphonic acid (Sigma) as substrate. .. Optical densities (OD) at 415 nm were measured on an automated ELISA plate reader.

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  • 94
    Millipore pax6
    Hes5 and <t>Pax6</t> expressions in injured and non-injured lungs
    Pax6, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pax6/product/Millipore
    Average 94 stars, based on 1 article reviews
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    99
    Millipore rabbit anti myc pab
    PLSCR1 inhibits the nuclear import of NP. (A, B) Empty retrovirus-transduced control A549 cells (A) or PLSCR1-overexpressing A549 cells (B) were infected with WSN virus at an MOI of 5. At 4, 6, 8, 10, and 12 h p.i., the infected cells were fixed and stained with mouse anti-NP <t>mAb</t> and rabbit anti-PLSCR1 <t>pAb,</t> followed by incubation with Alexa Fluor 488 donkey anti-rabbit IgG (H+L) (green) and Alexa Fluor 633 goat anti-mouse IgG (H+L) (red). The nuclei were stained with DAPI. (C) Quantitative analysis of NP localization in virus-infected cells. On the basis of the confocal microscopy in panels A and B, the localization of NP (indicative of vRNP) after nuclear import was categorized into four types, clear nuclear localization, simultaneous localization at the edge of the nucleus and the cytoplasm, predominant cytoplasmic localization, and close to the cytoplasmic membrane. The results shown are calculated from one hundred cells viewed under a confocal microscope with a 40X objective lens. (D). PLSCR1 inhibits the nuclear import of NP in transfected A549 cells. A549 cells were transfected with pCAGGS-WSNNP alone or were cotransfected with pCAGGS-WSNNP and pCAGGS-PLSCR1 and were assessed by confocal microscopy. NP was detected with a mouse anti-NP mAb and visualized with Alexa Fluor 633 (red). PLSCR1 was detected with a rabbit anti-PLSCR1 pAb and visualized with Alexa Fluor 488 (green). Yellow in the merged image indicates the colocalization of NP and PLSCR1. (E) PLSCR1-overexpressing or empty retrovirus-transduced control A549 cells were infected with WSN virus at an MOI of 5. At 6 h p.i., the cells were separated into nuclear (N) and cytoplasmic fractions (C). Each fraction was subjected to western blotting with a rabbit anti-NP pAb and a rabbit anti-PLSCR1 pAb for protein detection. (F) PLSCR1-overexpressing A549 cells or control A549 cells were treated with CHX to inhibit protein synthesis. The treated cells were infected with WSN virus at an MOI of 5, and were separated into nuclear and cytoplasmic fractions at 2 h p.i., followed by western blotting to detect the amount of NP in the nuclear and cytoplasmic fractions.
    Rabbit Anti Myc Pab, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti myc pab/product/Millipore
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    86
    Millipore cy3 labeled goat anti rabbit
    Immunohistochemical detection of Kv4.2W362F-FLAG in transfected SCG neurons. Isolated SCG neurons, transfected with EGFP alone ( A ) or with Kv4.2W362F-FLAG and EGFP ( B ) using the gene gun, were fixed and stained 24 hr later (see Materials and Methods). A , B , EGFP fluorescence ( left panels ) and <t>Cy3</t> fluorescence ( right panels ) images of the same field. Anti-FLAG staining is only evident in cultures transfected with Kv4.2W362F-FLAG expression (compare right panels in A and B ). In addition, EGFP expression correlates with Kv4.2W362F (compare left and right panels in B ). Scale bar, 50 μm.
    Cy3 Labeled Goat Anti Rabbit, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cy3 labeled goat anti rabbit/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cy3 labeled goat anti rabbit - by Bioz Stars, 2021-07
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    Image Search Results


    Hes5 and Pax6 expressions in injured and non-injured lungs

    Journal: Stem cells (Dayton, Ohio)

    Article Title: NOTCH1 is Required for Regeneration of Clara Cells During Repair of Airway Injury

    doi: 10.1002/stem.1059

    Figure Lengend Snippet: Hes5 and Pax6 expressions in injured and non-injured lungs

    Article Snippet: Antibodies against the following proteins were used: T1α (Developmental Studies Hybridoma Bank, Iowa City, Iowa), SP-B, SP-C, TTF-1 and CCSP (Seven Hill Bioreagents, Cincinnati, OH), NOTCH1 (Santa Cruz Biotechnology, INC. Santa Cruz, CA), β-tubulin (BioGenex, San Ramon, CA), PGP9.5 (AnaSpec, San Jose, CA), Ki67 (Thermo Scientific, Fremont, CA), HES5, CGRP and PAX6 (Millipore, Temecula, CA), activated NOTCH1 (Abcam, Cambridge, MA).

    Techniques:

    PLSCR1 inhibits the nuclear import of NP. (A, B) Empty retrovirus-transduced control A549 cells (A) or PLSCR1-overexpressing A549 cells (B) were infected with WSN virus at an MOI of 5. At 4, 6, 8, 10, and 12 h p.i., the infected cells were fixed and stained with mouse anti-NP mAb and rabbit anti-PLSCR1 pAb, followed by incubation with Alexa Fluor 488 donkey anti-rabbit IgG (H+L) (green) and Alexa Fluor 633 goat anti-mouse IgG (H+L) (red). The nuclei were stained with DAPI. (C) Quantitative analysis of NP localization in virus-infected cells. On the basis of the confocal microscopy in panels A and B, the localization of NP (indicative of vRNP) after nuclear import was categorized into four types, clear nuclear localization, simultaneous localization at the edge of the nucleus and the cytoplasm, predominant cytoplasmic localization, and close to the cytoplasmic membrane. The results shown are calculated from one hundred cells viewed under a confocal microscope with a 40X objective lens. (D). PLSCR1 inhibits the nuclear import of NP in transfected A549 cells. A549 cells were transfected with pCAGGS-WSNNP alone or were cotransfected with pCAGGS-WSNNP and pCAGGS-PLSCR1 and were assessed by confocal microscopy. NP was detected with a mouse anti-NP mAb and visualized with Alexa Fluor 633 (red). PLSCR1 was detected with a rabbit anti-PLSCR1 pAb and visualized with Alexa Fluor 488 (green). Yellow in the merged image indicates the colocalization of NP and PLSCR1. (E) PLSCR1-overexpressing or empty retrovirus-transduced control A549 cells were infected with WSN virus at an MOI of 5. At 6 h p.i., the cells were separated into nuclear (N) and cytoplasmic fractions (C). Each fraction was subjected to western blotting with a rabbit anti-NP pAb and a rabbit anti-PLSCR1 pAb for protein detection. (F) PLSCR1-overexpressing A549 cells or control A549 cells were treated with CHX to inhibit protein synthesis. The treated cells were infected with WSN virus at an MOI of 5, and were separated into nuclear and cytoplasmic fractions at 2 h p.i., followed by western blotting to detect the amount of NP in the nuclear and cytoplasmic fractions.

    Journal: PLoS Pathogens

    Article Title: Phospholipid scramblase 1 interacts with influenza A virus NP, impairing its nuclear import and thereby suppressing virus replication

    doi: 10.1371/journal.ppat.1006851

    Figure Lengend Snippet: PLSCR1 inhibits the nuclear import of NP. (A, B) Empty retrovirus-transduced control A549 cells (A) or PLSCR1-overexpressing A549 cells (B) were infected with WSN virus at an MOI of 5. At 4, 6, 8, 10, and 12 h p.i., the infected cells were fixed and stained with mouse anti-NP mAb and rabbit anti-PLSCR1 pAb, followed by incubation with Alexa Fluor 488 donkey anti-rabbit IgG (H+L) (green) and Alexa Fluor 633 goat anti-mouse IgG (H+L) (red). The nuclei were stained with DAPI. (C) Quantitative analysis of NP localization in virus-infected cells. On the basis of the confocal microscopy in panels A and B, the localization of NP (indicative of vRNP) after nuclear import was categorized into four types, clear nuclear localization, simultaneous localization at the edge of the nucleus and the cytoplasm, predominant cytoplasmic localization, and close to the cytoplasmic membrane. The results shown are calculated from one hundred cells viewed under a confocal microscope with a 40X objective lens. (D). PLSCR1 inhibits the nuclear import of NP in transfected A549 cells. A549 cells were transfected with pCAGGS-WSNNP alone or were cotransfected with pCAGGS-WSNNP and pCAGGS-PLSCR1 and were assessed by confocal microscopy. NP was detected with a mouse anti-NP mAb and visualized with Alexa Fluor 633 (red). PLSCR1 was detected with a rabbit anti-PLSCR1 pAb and visualized with Alexa Fluor 488 (green). Yellow in the merged image indicates the colocalization of NP and PLSCR1. (E) PLSCR1-overexpressing or empty retrovirus-transduced control A549 cells were infected with WSN virus at an MOI of 5. At 6 h p.i., the cells were separated into nuclear (N) and cytoplasmic fractions (C). Each fraction was subjected to western blotting with a rabbit anti-NP pAb and a rabbit anti-PLSCR1 pAb for protein detection. (F) PLSCR1-overexpressing A549 cells or control A549 cells were treated with CHX to inhibit protein synthesis. The treated cells were infected with WSN virus at an MOI of 5, and were separated into nuclear and cytoplasmic fractions at 2 h p.i., followed by western blotting to detect the amount of NP in the nuclear and cytoplasmic fractions.

    Article Snippet: The following primary antibodies were purchased from commercial resources: rabbit anti-V5 pAb (AB3792, Merck Millipore, Darmstadt, Germany); rabbit anti-GAPDH pAb (10494-1-AP), rabbit anti-importin β pAb (10077-1-AP), rabbit anti-LaminB1 pAb (12987-1-AP), rabbit anti-Mx1 pAb (13750-1-AP) and rabbit anti-PLSCR1 pAb (11582-1-AP) from Proteintech (Wuhan, China); mouse anti-actin mAb (sc-47778), mouse anti-p-Ser mAb (sc-81514) and mouse anti-p-Tyr mAb (sc-508) from Santa Cruz (Dallas, TX); mouse anti-Flag mAb (F3165), mouse anti-Myc mAb (M4439), mouse anti-V5 mAb (V8012), rabbit anti-Flag pAb (F7425) and rabbit anti-Myc pAb (C3965) from Sigma-Aldrich.

    Techniques: Infection, Staining, Incubation, Confocal Microscopy, Microscopy, Transfection, Western Blot

    PLSCR1 does not affect the phosphorylation status of NP and does not stimulate the IFN pathways. (A) PLSCR1-overexpressing or empty retrovirus-transduced control A549 cells were infected with WSN virus at an MOI of 5. Cell lysates were processed at 6 h and 8 h p.i., immunoprecipitated with a mouse anti-NP mAb, a mouse anti-p-Ser mAb, or a mouse anti-p-Tyr mAb, followed by western blotting with a rabbit anti-NP pAb to detect the level of total NP, serine-phosphorylated NP, and tyrosine-phosphorylated NP, respectively. (B) Replication of an NP-phosphorylation mutant virus in PLSCR1-overexpressing A549 cells. PLSCR1-overexpressing or empty retrovirus-transduced control A549 cells were infected with wild-type WSN virus or the phosphorylation mutant S9A/Y10F at an MOI of 0.1. Supernatants were collected at the indicated timepoints and titrated for infectious virus by means of plaque assay on MDCK cells. (C) Expression of Mx1 protein in PLSCR1-overexpressing or control A549 cells. PLSCR1-overexpressing or empty retrovirus-transduced control A549 cells were grown in 12-well plates, and were left untreated or were treated with IFN-α for 24 h. The cell lysates were then subjected to western blotting with a rabbit anti-Mx1 pAb for the detection of Mx1 protein. GAPDH, detected by a rabbit anti-GAPDH pAb, served as a negative control. (D) Expression of the ISRE luciferase reporter gene in HEK293T cells transfected with the PLSCR1-expressing construct or empty vector. HEK293T cells were transfected with the ISRE-Luc reporter plasmid, pRL-TK control plasmid, and the pCAGGS-PLSCR1 or empty pCAGGS plasmid for 20 h. The overexpression of PLSCR1 was confirmed by western blotting with a rabbit anti-PLSCR1 pAb. The luciferase activity of the transfected cells was analyzed by using the Dual-Luciferase reporter assay. After normalization with co-transfected Renilla luciferase activity, the relative firefly luciferase activity of PLSCR1-overexpressing cells was expressed as the fold-induction of the ISRE firefly luciferase activity compared to cells transfected with empty pCAGGS vector. **, P

    Journal: PLoS Pathogens

    Article Title: Phospholipid scramblase 1 interacts with influenza A virus NP, impairing its nuclear import and thereby suppressing virus replication

    doi: 10.1371/journal.ppat.1006851

    Figure Lengend Snippet: PLSCR1 does not affect the phosphorylation status of NP and does not stimulate the IFN pathways. (A) PLSCR1-overexpressing or empty retrovirus-transduced control A549 cells were infected with WSN virus at an MOI of 5. Cell lysates were processed at 6 h and 8 h p.i., immunoprecipitated with a mouse anti-NP mAb, a mouse anti-p-Ser mAb, or a mouse anti-p-Tyr mAb, followed by western blotting with a rabbit anti-NP pAb to detect the level of total NP, serine-phosphorylated NP, and tyrosine-phosphorylated NP, respectively. (B) Replication of an NP-phosphorylation mutant virus in PLSCR1-overexpressing A549 cells. PLSCR1-overexpressing or empty retrovirus-transduced control A549 cells were infected with wild-type WSN virus or the phosphorylation mutant S9A/Y10F at an MOI of 0.1. Supernatants were collected at the indicated timepoints and titrated for infectious virus by means of plaque assay on MDCK cells. (C) Expression of Mx1 protein in PLSCR1-overexpressing or control A549 cells. PLSCR1-overexpressing or empty retrovirus-transduced control A549 cells were grown in 12-well plates, and were left untreated or were treated with IFN-α for 24 h. The cell lysates were then subjected to western blotting with a rabbit anti-Mx1 pAb for the detection of Mx1 protein. GAPDH, detected by a rabbit anti-GAPDH pAb, served as a negative control. (D) Expression of the ISRE luciferase reporter gene in HEK293T cells transfected with the PLSCR1-expressing construct or empty vector. HEK293T cells were transfected with the ISRE-Luc reporter plasmid, pRL-TK control plasmid, and the pCAGGS-PLSCR1 or empty pCAGGS plasmid for 20 h. The overexpression of PLSCR1 was confirmed by western blotting with a rabbit anti-PLSCR1 pAb. The luciferase activity of the transfected cells was analyzed by using the Dual-Luciferase reporter assay. After normalization with co-transfected Renilla luciferase activity, the relative firefly luciferase activity of PLSCR1-overexpressing cells was expressed as the fold-induction of the ISRE firefly luciferase activity compared to cells transfected with empty pCAGGS vector. **, P

    Article Snippet: The following primary antibodies were purchased from commercial resources: rabbit anti-V5 pAb (AB3792, Merck Millipore, Darmstadt, Germany); rabbit anti-GAPDH pAb (10494-1-AP), rabbit anti-importin β pAb (10077-1-AP), rabbit anti-LaminB1 pAb (12987-1-AP), rabbit anti-Mx1 pAb (13750-1-AP) and rabbit anti-PLSCR1 pAb (11582-1-AP) from Proteintech (Wuhan, China); mouse anti-actin mAb (sc-47778), mouse anti-p-Ser mAb (sc-81514) and mouse anti-p-Tyr mAb (sc-508) from Santa Cruz (Dallas, TX); mouse anti-Flag mAb (F3165), mouse anti-Myc mAb (M4439), mouse anti-V5 mAb (V8012), rabbit anti-Flag pAb (F7425) and rabbit anti-Myc pAb (C3965) from Sigma-Aldrich.

    Techniques: Infection, Immunoprecipitation, Western Blot, Mutagenesis, Plaque Assay, Expressing, Negative Control, Luciferase, Transfection, Construct, Plasmid Preparation, Over Expression, Activity Assay, Reporter Assay

    NP interacts with PLSCR1 in mammalian cells. (A, B) Co-IP assay of V5-NP and Flag-PLSCR1 in HEK293T cells. HEK293T cells were transfected individually or in combination with plasmids expressing V5-WSNNP and Flag-PLSCR1. Cell lysates were immunoprecipitated with a mouse anti-V5 mAb (A) or a mouse anti-Flag mAb (B) and were subjected to western blotting with a rabbit anti-V5 pAb or a rabbit anti-Flag pAb to reveal the presence of NP and PLSCR1, respectively. (C, D) GST pull-down assay of NP and PLSCR1. Lysates of HEK293T cells transfected with the GST or GST-PLSCR1 construct were incubated with Glutathione Sepharose 4 Fast Flow and then mixed with lysates from cells transfected with pCAGGS or pCAGGS-WSNNP (C); HEK293T cell lysates containing exogenously expressed GST or GST-WSNNP were incubated with Glutathione Sepharose 4 Fast Flow and then mixed with lysates from cells transfected with pCAGGS or pCAGGS-PLSCR1 (D). After washing away the unbound proteins, equal volumes of proteins bound to the beads and the original cell lysates (5% of the input) were examined by western blotting using a rabbit anti-NP pAb, a rabbit anti-GST pAb, or a rabbit anti-PLSCR1 pAb. GST, GST-PLSCR1, or GST-WSNNP proteins in the eluates were detected by Coomassie blue (CB) staining. (E) The NP-PLSCR1 interaction does not rely on RNA binding. HEK293T cells were transfected individually or in combination with plasmids expressing V5-WSNNP and Flag-PLSCR1. Cell lysates treated with RNase A/T1 or left untreated were immunoprecipitated with a mouse anti-Flag mAb and were subjected to western blotting with a rabbit anti-V5 pAb or a rabbit anti-Flag pAb to reveal the presence of NP and PLSCR1, respectively. (F) PLSCR1 interacts with NP during natural viral infection. Confluent A549 cells grown in 6-well plates were mock infected with PBS or infected with WSN virus at an MOI of 5. At 6 h p.i., cell lysates were immunoprecipitated with a rabbit anti-PLSCR1 pAb and were subjected to western blotting with a mouse anti-NP mAb or a rabbit anti-PLSCR1 pAb to detect NP and PLSCR1, respectively. (G) Mapping of the PLSCR1-interacting domain within NP. Schematic presentation of influenza NP showing the different domains as well as the truncation mutants made in this study is on the left side. The interaction between PLSCR1 and the NP truncation mutants is shown on the right side. Lysates of HEK293T cells were pulled down with glutathione magnetic beads. The bound proteins were subjected to western blotting with a rabbit anti-Flag pAb or a rabbit anti-GST pAb to reveal the presence of PLSCR1 and NP, respectively. NES, nuclear export signal; NAS, nuclear accumulation signal.

    Journal: PLoS Pathogens

    Article Title: Phospholipid scramblase 1 interacts with influenza A virus NP, impairing its nuclear import and thereby suppressing virus replication

    doi: 10.1371/journal.ppat.1006851

    Figure Lengend Snippet: NP interacts with PLSCR1 in mammalian cells. (A, B) Co-IP assay of V5-NP and Flag-PLSCR1 in HEK293T cells. HEK293T cells were transfected individually or in combination with plasmids expressing V5-WSNNP and Flag-PLSCR1. Cell lysates were immunoprecipitated with a mouse anti-V5 mAb (A) or a mouse anti-Flag mAb (B) and were subjected to western blotting with a rabbit anti-V5 pAb or a rabbit anti-Flag pAb to reveal the presence of NP and PLSCR1, respectively. (C, D) GST pull-down assay of NP and PLSCR1. Lysates of HEK293T cells transfected with the GST or GST-PLSCR1 construct were incubated with Glutathione Sepharose 4 Fast Flow and then mixed with lysates from cells transfected with pCAGGS or pCAGGS-WSNNP (C); HEK293T cell lysates containing exogenously expressed GST or GST-WSNNP were incubated with Glutathione Sepharose 4 Fast Flow and then mixed with lysates from cells transfected with pCAGGS or pCAGGS-PLSCR1 (D). After washing away the unbound proteins, equal volumes of proteins bound to the beads and the original cell lysates (5% of the input) were examined by western blotting using a rabbit anti-NP pAb, a rabbit anti-GST pAb, or a rabbit anti-PLSCR1 pAb. GST, GST-PLSCR1, or GST-WSNNP proteins in the eluates were detected by Coomassie blue (CB) staining. (E) The NP-PLSCR1 interaction does not rely on RNA binding. HEK293T cells were transfected individually or in combination with plasmids expressing V5-WSNNP and Flag-PLSCR1. Cell lysates treated with RNase A/T1 or left untreated were immunoprecipitated with a mouse anti-Flag mAb and were subjected to western blotting with a rabbit anti-V5 pAb or a rabbit anti-Flag pAb to reveal the presence of NP and PLSCR1, respectively. (F) PLSCR1 interacts with NP during natural viral infection. Confluent A549 cells grown in 6-well plates were mock infected with PBS or infected with WSN virus at an MOI of 5. At 6 h p.i., cell lysates were immunoprecipitated with a rabbit anti-PLSCR1 pAb and were subjected to western blotting with a mouse anti-NP mAb or a rabbit anti-PLSCR1 pAb to detect NP and PLSCR1, respectively. (G) Mapping of the PLSCR1-interacting domain within NP. Schematic presentation of influenza NP showing the different domains as well as the truncation mutants made in this study is on the left side. The interaction between PLSCR1 and the NP truncation mutants is shown on the right side. Lysates of HEK293T cells were pulled down with glutathione magnetic beads. The bound proteins were subjected to western blotting with a rabbit anti-Flag pAb or a rabbit anti-GST pAb to reveal the presence of PLSCR1 and NP, respectively. NES, nuclear export signal; NAS, nuclear accumulation signal.

    Article Snippet: The following primary antibodies were purchased from commercial resources: rabbit anti-V5 pAb (AB3792, Merck Millipore, Darmstadt, Germany); rabbit anti-GAPDH pAb (10494-1-AP), rabbit anti-importin β pAb (10077-1-AP), rabbit anti-LaminB1 pAb (12987-1-AP), rabbit anti-Mx1 pAb (13750-1-AP) and rabbit anti-PLSCR1 pAb (11582-1-AP) from Proteintech (Wuhan, China); mouse anti-actin mAb (sc-47778), mouse anti-p-Ser mAb (sc-81514) and mouse anti-p-Tyr mAb (sc-508) from Santa Cruz (Dallas, TX); mouse anti-Flag mAb (F3165), mouse anti-Myc mAb (M4439), mouse anti-V5 mAb (V8012), rabbit anti-Flag pAb (F7425) and rabbit anti-Myc pAb (C3965) from Sigma-Aldrich.

    Techniques: Co-Immunoprecipitation Assay, Transfection, Expressing, Immunoprecipitation, Western Blot, Pull Down Assay, Construct, Incubation, Flow Cytometry, Staining, RNA Binding Assay, Infection, Magnetic Beads

    The formation of a complex comprising PLSCR1, NP, and importin α inhibits the incorporation of importin β into the complex. (A–D) PLSCR1 formed a complex with NP and different members of the importin α family: importin α1 (A), importin α3 (B), importin α5 (C), and importin α7 (D). HEK293T cells were transfected with plasmids expressing V5-WSNNP and Myc-tagged importin α proteins, together with gradual increasing amounts (0–0.6 μg) of Flag-PLSCR1. The cell lysates were immunoprecipitated with a mouse anti-Myc mAb, and the bound proteins were detected by western blotting with a rabbit anti-V5 pAb, a rabbit anti-Flag pAb, or a rabbit anti-Myc pAb to detect NP, PLSCR1, and importin α family members, respectively. (E) Validation of complex formation among NP, PLSCR1, and importin α1 by including MOV10 as a control. HEK293T cells were transfected with plasmids expressing V5-WSNNP and Myc-tagged importin α1, together with Flag-PLSCR1 or Flag-MOV10. The cell lysates were immunoprecipitated with a mouse anti-Myc mAb, and the bound proteins were detected by western blotting with a rabbit anti-V5 pAb, a rabbit anti-Flag pAb, or a rabbit anti-Myc pAb to detect NP, PLSCR1 or MOV10, and importin α1, respectively. (F) Complex formation among PLSCR1, NP, and importin α1 inhibited the incorporation of importin β into the complex. HEK293T cells were transfected with plasmids expressing V5-WSNNP, Myc-importin α1 and importin β, together with Flag-PLSCR1. The cell lysates were immunoprecipitated with a mouse anti-Myc mAb, and the bound proteins were detected by western blotting with rabbit pAb against V5, Myc or the Flag tag, or importin β.

    Journal: PLoS Pathogens

    Article Title: Phospholipid scramblase 1 interacts with influenza A virus NP, impairing its nuclear import and thereby suppressing virus replication

    doi: 10.1371/journal.ppat.1006851

    Figure Lengend Snippet: The formation of a complex comprising PLSCR1, NP, and importin α inhibits the incorporation of importin β into the complex. (A–D) PLSCR1 formed a complex with NP and different members of the importin α family: importin α1 (A), importin α3 (B), importin α5 (C), and importin α7 (D). HEK293T cells were transfected with plasmids expressing V5-WSNNP and Myc-tagged importin α proteins, together with gradual increasing amounts (0–0.6 μg) of Flag-PLSCR1. The cell lysates were immunoprecipitated with a mouse anti-Myc mAb, and the bound proteins were detected by western blotting with a rabbit anti-V5 pAb, a rabbit anti-Flag pAb, or a rabbit anti-Myc pAb to detect NP, PLSCR1, and importin α family members, respectively. (E) Validation of complex formation among NP, PLSCR1, and importin α1 by including MOV10 as a control. HEK293T cells were transfected with plasmids expressing V5-WSNNP and Myc-tagged importin α1, together with Flag-PLSCR1 or Flag-MOV10. The cell lysates were immunoprecipitated with a mouse anti-Myc mAb, and the bound proteins were detected by western blotting with a rabbit anti-V5 pAb, a rabbit anti-Flag pAb, or a rabbit anti-Myc pAb to detect NP, PLSCR1 or MOV10, and importin α1, respectively. (F) Complex formation among PLSCR1, NP, and importin α1 inhibited the incorporation of importin β into the complex. HEK293T cells were transfected with plasmids expressing V5-WSNNP, Myc-importin α1 and importin β, together with Flag-PLSCR1. The cell lysates were immunoprecipitated with a mouse anti-Myc mAb, and the bound proteins were detected by western blotting with rabbit pAb against V5, Myc or the Flag tag, or importin β.

    Article Snippet: The following primary antibodies were purchased from commercial resources: rabbit anti-V5 pAb (AB3792, Merck Millipore, Darmstadt, Germany); rabbit anti-GAPDH pAb (10494-1-AP), rabbit anti-importin β pAb (10077-1-AP), rabbit anti-LaminB1 pAb (12987-1-AP), rabbit anti-Mx1 pAb (13750-1-AP) and rabbit anti-PLSCR1 pAb (11582-1-AP) from Proteintech (Wuhan, China); mouse anti-actin mAb (sc-47778), mouse anti-p-Ser mAb (sc-81514) and mouse anti-p-Tyr mAb (sc-508) from Santa Cruz (Dallas, TX); mouse anti-Flag mAb (F3165), mouse anti-Myc mAb (M4439), mouse anti-V5 mAb (V8012), rabbit anti-Flag pAb (F7425) and rabbit anti-Myc pAb (C3965) from Sigma-Aldrich.

    Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot, FLAG-tag

    Rapamycin reduces progerin level in HGPS cells. A) Western blotting evaluation of lamin A/C and progerin in control (CONTROL) and Hutchinson Gilford progeria cells (HGPS). Whole lysates from control and HGPS cells untreated (−) or treated (+) with rapamycin (Rapamycin), MG132 or chloroquine-diphosphate (Cq) were subjected to lamin A/C, LC3-B2 and actin antibodies detection; B) densitometric analysis of lamin A and C immunolabeled bands detected in control fibroblasts; C) densitometric analysis of lamin A and C immunolabeled bands detected in HGPS fibroblasts; P=0.0358 for lamin A (rapa), P=0.0298 for lamin A (Mg); D) densitometric analysis of progerin immunoblotted bands; P=0.0390 for progerin (rapa); P=0.0458 for progerin (Mg); E) prelamin A, FLAG and LC3-B2 protein levels evaluation in HEK-293 cells expressing FLAG-tagged wild type prelamin A (LA-WT) or progerin (LA-Δ50). Immunolabeled bands observed in untreated (−) or rapamycin (Rapamycin) and chloroquine-diphosphate (Cq) treated (+) cells are shown; F) densitometric of FLAG immunoblotted bands; G) RT-PCR analysis of ZMPSTE24 and LMNA mRNA expression in untreated (Nt) and rapamycintreated HGPS cells (Rapa) and control (control); 2 −ΔΔCT values are reported relative to untreated control samples. P=0.0236 for LA-Δ50 (rapa); H) ratio between ZMPSTE24 and LMNA mRNA expression. Values are means of duplicate experiments ± S.D. In B, C, D and F densitometric analysis of triplicate experiments was performed, and the mean values ± S.D. are reported; asterisk indicates statistically significant difference with respect to lamin A or progerin densitometry in untreated samples; statistical significance was calculated by the Mann-Whitney test vs untreated HGPS samples, or cells expressing LMNA constructs.

    Journal: European Journal of Histochemistry : EJH

    Article Title: Autophagic degradation of farnesylated prelamin A as a therapeutic approach to lamin-linked progeria

    doi: 10.4081/ejh.2011.e36

    Figure Lengend Snippet: Rapamycin reduces progerin level in HGPS cells. A) Western blotting evaluation of lamin A/C and progerin in control (CONTROL) and Hutchinson Gilford progeria cells (HGPS). Whole lysates from control and HGPS cells untreated (−) or treated (+) with rapamycin (Rapamycin), MG132 or chloroquine-diphosphate (Cq) were subjected to lamin A/C, LC3-B2 and actin antibodies detection; B) densitometric analysis of lamin A and C immunolabeled bands detected in control fibroblasts; C) densitometric analysis of lamin A and C immunolabeled bands detected in HGPS fibroblasts; P=0.0358 for lamin A (rapa), P=0.0298 for lamin A (Mg); D) densitometric analysis of progerin immunoblotted bands; P=0.0390 for progerin (rapa); P=0.0458 for progerin (Mg); E) prelamin A, FLAG and LC3-B2 protein levels evaluation in HEK-293 cells expressing FLAG-tagged wild type prelamin A (LA-WT) or progerin (LA-Δ50). Immunolabeled bands observed in untreated (−) or rapamycin (Rapamycin) and chloroquine-diphosphate (Cq) treated (+) cells are shown; F) densitometric of FLAG immunoblotted bands; G) RT-PCR analysis of ZMPSTE24 and LMNA mRNA expression in untreated (Nt) and rapamycintreated HGPS cells (Rapa) and control (control); 2 −ΔΔCT values are reported relative to untreated control samples. P=0.0236 for LA-Δ50 (rapa); H) ratio between ZMPSTE24 and LMNA mRNA expression. Values are means of duplicate experiments ± S.D. In B, C, D and F densitometric analysis of triplicate experiments was performed, and the mean values ± S.D. are reported; asterisk indicates statistically significant difference with respect to lamin A or progerin densitometry in untreated samples; statistical significance was calculated by the Mann-Whitney test vs untreated HGPS samples, or cells expressing LMNA constructs.

    Article Snippet: Antibodies Antibodies employed for Western blot analysis or immunofluorescence labeling were: anti-lamin A/C, goat polyclonal (SC-6215, used at 1:1000 dilution for the Western blot analysis, Santa Cruz Biotechnology Inc., Segrate, MI, Italy); anti-prelamin A, goat polyclonal (SC-6214 used at 1:100 dilution for the immunofluorescence analysis, Santa Cruz); anti-prelamin A, rabbit polyclonal (antibody 1188-2, Diatheva, Fano, Italy), raised against the last 15 aminoacids of the prelamin A sequence including the farnesylated cysteine residue but not the SIM sequence; anti-LAP2α, rabbit polyclonal; anti-trimethyl-H3K9, rabbit polyclonal (Upstate, Lake Placid, NY, USA); anti-emerin, mouse monoclonal (Monosan, Uden, The Netherlands); anti-BAF, rabbit polyclonal (FL-89, Santa Cruz); anti-LC3 rabbit polyclonal antibody (ABR, Pierce); anti-FLAG, mouse monoclonal (M2, Sigma); anti-actin, goat polyclonal (Santa Cruz).

    Techniques: Western Blot, Immunolabeling, Expressing, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY, Construct

    Immunohistochemical detection of Kv4.2W362F-FLAG in transfected SCG neurons. Isolated SCG neurons, transfected with EGFP alone ( A ) or with Kv4.2W362F-FLAG and EGFP ( B ) using the gene gun, were fixed and stained 24 hr later (see Materials and Methods). A , B , EGFP fluorescence ( left panels ) and Cy3 fluorescence ( right panels ) images of the same field. Anti-FLAG staining is only evident in cultures transfected with Kv4.2W362F-FLAG expression (compare right panels in A and B ). In addition, EGFP expression correlates with Kv4.2W362F (compare left and right panels in B ). Scale bar, 50 μm.

    Journal: The Journal of Neuroscience

    Article Title: Elimination of the Fast Transient in Superior Cervical Ganglion Neurons with Expression of KV4.2W362F: Molecular Dissection ofIA

    doi: 10.1523/JNEUROSCI.20-14-05191.2000

    Figure Lengend Snippet: Immunohistochemical detection of Kv4.2W362F-FLAG in transfected SCG neurons. Isolated SCG neurons, transfected with EGFP alone ( A ) or with Kv4.2W362F-FLAG and EGFP ( B ) using the gene gun, were fixed and stained 24 hr later (see Materials and Methods). A , B , EGFP fluorescence ( left panels ) and Cy3 fluorescence ( right panels ) images of the same field. Anti-FLAG staining is only evident in cultures transfected with Kv4.2W362F-FLAG expression (compare right panels in A and B ). In addition, EGFP expression correlates with Kv4.2W362F (compare left and right panels in B ). Scale bar, 50 μm.

    Article Snippet: After washing, cultures were incubated with a Cy3-labeled goat anti-rabbit (or rabbit anti-mouse) IgG secondary antibody (Chemicon, Temecula, CA).

    Techniques: Immunohistochemistry, Transfection, Isolation, Staining, Fluorescence, Expressing