immunoprecipitation buffer  (Millipore)

 
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    Name:
    RIPA Buffer
    Description:
    Radioimmunoprecipitation assay buffer RIPA buffer is a lysis buffer used for rapid efficient cell lysis and solubilization of proteins from both adherent and suspension cultured mammalian cells
    Catalog Number:
    R0278
    Price:
    None
    Applications:
    RIPA Buffer enables efficient cell lysis and protein solubilization while avoiding protein degradation and interference with the proteins' immunoreactivity and biological activity. RIPA Buffer also results in low background in immunoprecipitation and molecular pull-down assays. Compatible with EZview(TM) Affinity Gels.
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    Structured Review

    Millipore immunoprecipitation buffer
    MNX1-AS1 interacts State3 and promotes phosphorylation of Stat3 by enhance the interaction between p-JAK and Stat3. (A) Silver staining of proteins bound to MNX1-AS1. The RNA pull-down assay was performed with MDA-MB-231 cell lysates. A specific band was identified as Stat3 by mass spectrometry. (B) Stat3 interacted with MNX1-AS1 was confirmed by RNA pull-down assay and Western blot. (C) MNX1-AS1 interacted with Stat3 was confirmed by RNA <t>immunoprecipitation</t> (RIP). Bar graphs represent the mean ± SD of experimental triplicates. (D) Silencing MNX1-AS1 reduced phosphorylation of Stat3 but had no effect on phosphorylation of JAK1/2 in MDA-MB-231 cells, as indicated by Western blot. (E,F) Silencing MNX1-AS1 reduced the interaction between Stat3 and p-JAK1/2. (G) Phosphorylation of Stat3 reduced by silencing MNX1-AS1 was rescued by Stat agonist colivelin 50 nM. (H) Viability of MDA-MB-231 cell o reduced by silencing MNX1-AS1 was rescued by Stat agonist colivelin 50 nM. Bar graphs represent the mean ± SD of experimental triplicates. (I) Colony formation of MDA-MB-231 cell reduced by silencing MNX1-AS1 was rescued by Stat agonist colivelin 50 nM. (J) Migration of MDA-MB-231 cell reduced by silencing MNX1-AS1 was rescued by Stat agonist colivelin 50 nM. (K) Invasion of MDA-MB-231 cell reduced by silencing MNX1-AS1 was rescued by Stat agonist colivelin 50 nM. (L) Apoptosis of MDA-MB-231 cell induced by silencing MNX1-AS1 was rescued by Stat agonist colivelin 50 nM. *** P
    Radioimmunoprecipitation assay buffer RIPA buffer is a lysis buffer used for rapid efficient cell lysis and solubilization of proteins from both adherent and suspension cultured mammalian cells
    https://www.bioz.com/result/immunoprecipitation buffer/product/Millipore
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    Images

    1) Product Images from "Long Non-Coding RNA MNX1-AS1 Promotes Progression of Triple Negative Breast Cancer by Enhancing Phosphorylation of Stat3"

    Article Title: Long Non-Coding RNA MNX1-AS1 Promotes Progression of Triple Negative Breast Cancer by Enhancing Phosphorylation of Stat3

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2020.01108

    MNX1-AS1 interacts State3 and promotes phosphorylation of Stat3 by enhance the interaction between p-JAK and Stat3. (A) Silver staining of proteins bound to MNX1-AS1. The RNA pull-down assay was performed with MDA-MB-231 cell lysates. A specific band was identified as Stat3 by mass spectrometry. (B) Stat3 interacted with MNX1-AS1 was confirmed by RNA pull-down assay and Western blot. (C) MNX1-AS1 interacted with Stat3 was confirmed by RNA immunoprecipitation (RIP). Bar graphs represent the mean ± SD of experimental triplicates. (D) Silencing MNX1-AS1 reduced phosphorylation of Stat3 but had no effect on phosphorylation of JAK1/2 in MDA-MB-231 cells, as indicated by Western blot. (E,F) Silencing MNX1-AS1 reduced the interaction between Stat3 and p-JAK1/2. (G) Phosphorylation of Stat3 reduced by silencing MNX1-AS1 was rescued by Stat agonist colivelin 50 nM. (H) Viability of MDA-MB-231 cell o reduced by silencing MNX1-AS1 was rescued by Stat agonist colivelin 50 nM. Bar graphs represent the mean ± SD of experimental triplicates. (I) Colony formation of MDA-MB-231 cell reduced by silencing MNX1-AS1 was rescued by Stat agonist colivelin 50 nM. (J) Migration of MDA-MB-231 cell reduced by silencing MNX1-AS1 was rescued by Stat agonist colivelin 50 nM. (K) Invasion of MDA-MB-231 cell reduced by silencing MNX1-AS1 was rescued by Stat agonist colivelin 50 nM. (L) Apoptosis of MDA-MB-231 cell induced by silencing MNX1-AS1 was rescued by Stat agonist colivelin 50 nM. *** P
    Figure Legend Snippet: MNX1-AS1 interacts State3 and promotes phosphorylation of Stat3 by enhance the interaction between p-JAK and Stat3. (A) Silver staining of proteins bound to MNX1-AS1. The RNA pull-down assay was performed with MDA-MB-231 cell lysates. A specific band was identified as Stat3 by mass spectrometry. (B) Stat3 interacted with MNX1-AS1 was confirmed by RNA pull-down assay and Western blot. (C) MNX1-AS1 interacted with Stat3 was confirmed by RNA immunoprecipitation (RIP). Bar graphs represent the mean ± SD of experimental triplicates. (D) Silencing MNX1-AS1 reduced phosphorylation of Stat3 but had no effect on phosphorylation of JAK1/2 in MDA-MB-231 cells, as indicated by Western blot. (E,F) Silencing MNX1-AS1 reduced the interaction between Stat3 and p-JAK1/2. (G) Phosphorylation of Stat3 reduced by silencing MNX1-AS1 was rescued by Stat agonist colivelin 50 nM. (H) Viability of MDA-MB-231 cell o reduced by silencing MNX1-AS1 was rescued by Stat agonist colivelin 50 nM. Bar graphs represent the mean ± SD of experimental triplicates. (I) Colony formation of MDA-MB-231 cell reduced by silencing MNX1-AS1 was rescued by Stat agonist colivelin 50 nM. (J) Migration of MDA-MB-231 cell reduced by silencing MNX1-AS1 was rescued by Stat agonist colivelin 50 nM. (K) Invasion of MDA-MB-231 cell reduced by silencing MNX1-AS1 was rescued by Stat agonist colivelin 50 nM. (L) Apoptosis of MDA-MB-231 cell induced by silencing MNX1-AS1 was rescued by Stat agonist colivelin 50 nM. *** P

    Techniques Used: Silver Staining, Pull Down Assay, Multiple Displacement Amplification, Mass Spectrometry, Western Blot, Immunoprecipitation, Migration

    2) Product Images from "Human DICER helicase domain recruits PKR and modulates its antiviral activity"

    Article Title: Human DICER helicase domain recruits PKR and modulates its antiviral activity

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1009549

    Identification of DICER domains involved in DICER-PKR interaction. A. Schematic representation of Human DICER proteins used in this study. The different conserved domains are shown in colored boxes. DUF283: Domain of Unknown Function; PAZ: PIWI ARGONAUTE ZWILLE domain; dsRBD: dsRNA-binding domain. hDICER WT is the full-length protein. hDICER N1 is deleted of the first N-terminal 495 amino acids. hDICER N3 is wholly deleted of the helicase domain. hDICER Hel. is the whole DICER’s helicase domain. hDICER ΔdsRBD is deleted of the C-terminal dsRBD. B. Western blot analysis of HA co-IP in mock NoDice 2.20 cells transfected with different versions of FHA:DICER proteins. Efficiency of immunoprecipitation was assessed using anti-HA and anti-DICER antibodies and co-IPs of TRBP, PKR and PACT were examined using appropriate antibodies. Expression of GFP in INPUT fraction was visualized as control of SINV-GFP infection. Ponceau staining of membranes is used as loading control. C. Western blot analysis of HA co-IP in NoDice 2.20 cells transfected with different versions of FHA:DICER proteins and infected with SINV-GFP (MOI of 2, 6 hpi). Efficiency of immunoprecipitation was assessed using an anti-Flag antibody and co-IPs of PKR, TRBP, p-PKR and PACT were examined using appropriate antibodies. Expression of GFP in INPUT fraction was visualized as control of SINV-GFP infection. Ponceau staining of membranes is used as loading control. The DICER Hel. band is indicated by a red asterisk. D. Plasmids expressing the different versions of DICER proteins fused to the N-terminal part of Venus and PKR:Venus C-ter plasmid were co-transfected in NoDiceΔPKR cells. Cells were treated as in Fig 3D . The different combinations are noted on the left side. The fluorescent signal was observed using an epifluorescence microscope. For each condition, the left panel corresponds to Venus signal and the right panel to the corresponding brightfield pictures. Scale bar: 100 μm. hpi: hours post-infection.
    Figure Legend Snippet: Identification of DICER domains involved in DICER-PKR interaction. A. Schematic representation of Human DICER proteins used in this study. The different conserved domains are shown in colored boxes. DUF283: Domain of Unknown Function; PAZ: PIWI ARGONAUTE ZWILLE domain; dsRBD: dsRNA-binding domain. hDICER WT is the full-length protein. hDICER N1 is deleted of the first N-terminal 495 amino acids. hDICER N3 is wholly deleted of the helicase domain. hDICER Hel. is the whole DICER’s helicase domain. hDICER ΔdsRBD is deleted of the C-terminal dsRBD. B. Western blot analysis of HA co-IP in mock NoDice 2.20 cells transfected with different versions of FHA:DICER proteins. Efficiency of immunoprecipitation was assessed using anti-HA and anti-DICER antibodies and co-IPs of TRBP, PKR and PACT were examined using appropriate antibodies. Expression of GFP in INPUT fraction was visualized as control of SINV-GFP infection. Ponceau staining of membranes is used as loading control. C. Western blot analysis of HA co-IP in NoDice 2.20 cells transfected with different versions of FHA:DICER proteins and infected with SINV-GFP (MOI of 2, 6 hpi). Efficiency of immunoprecipitation was assessed using an anti-Flag antibody and co-IPs of PKR, TRBP, p-PKR and PACT were examined using appropriate antibodies. Expression of GFP in INPUT fraction was visualized as control of SINV-GFP infection. Ponceau staining of membranes is used as loading control. The DICER Hel. band is indicated by a red asterisk. D. Plasmids expressing the different versions of DICER proteins fused to the N-terminal part of Venus and PKR:Venus C-ter plasmid were co-transfected in NoDiceΔPKR cells. Cells were treated as in Fig 3D . The different combinations are noted on the left side. The fluorescent signal was observed using an epifluorescence microscope. For each condition, the left panel corresponds to Venus signal and the right panel to the corresponding brightfield pictures. Scale bar: 100 μm. hpi: hours post-infection.

    Techniques Used: Binding Assay, Western Blot, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Expressing, Infection, Staining, Plasmid Preparation, Microscopy

    3) Product Images from "Apolipoprotein L2 contains a BH3-like domain but it does not behave as a BH3-only protein"

    Article Title: Apolipoprotein L2 contains a BH3-like domain but it does not behave as a BH3-only protein

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2014.237

    Immunoprecipitation of ApoL2 in HeLa cells. ( a ) Endogenous ApoL2 was immunoprecipitated (IP) and the presence of the indicated proteins was assayed by western blot. Blots from a single experiment representative of three independent experiments are shown. ( b ) HeLa cells were transfected with HA-Bcl-2 or empty vector. Anti-HA was used for immunoprecipitation and the presence of ApoL2, Bcl-2 and HA was assayed by western blot. Panel shown is representative of three independent experiments. Left and right panels were cropped from the same films
    Figure Legend Snippet: Immunoprecipitation of ApoL2 in HeLa cells. ( a ) Endogenous ApoL2 was immunoprecipitated (IP) and the presence of the indicated proteins was assayed by western blot. Blots from a single experiment representative of three independent experiments are shown. ( b ) HeLa cells were transfected with HA-Bcl-2 or empty vector. Anti-HA was used for immunoprecipitation and the presence of ApoL2, Bcl-2 and HA was assayed by western blot. Panel shown is representative of three independent experiments. Left and right panels were cropped from the same films

    Techniques Used: Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation

    4) Product Images from "Arabidopsis ACINUS is O-glycosylated and regulates transcription and alternative splicing of regulators of reproductive transitions"

    Article Title: Arabidopsis ACINUS is O-glycosylated and regulates transcription and alternative splicing of regulators of reproductive transitions

    Journal: Nature Communications

    doi: 10.1038/s41467-021-20929-7

    The acinus-2 pinin-1 double mutant is late flowering with increased FLC expression. a Rosette leaf numbers of WT, acinus-2 , pinin-1 , and acinus-2 pinin-1 at bolting stage grown in long day condition. Error bars indicate SD calculated from n > 12. Values represent mean ± SD calculated from at least 12 plants ( n > 12). Statistically significant differences to WT were determined by two-tailed t test. The P values for a, b, and c are 1.35E−6, 2.48E−3, and 5.15E−9. b FLC expression level relative to UBQ (At5g15400) in WT, acinus-2 , pinin-1 , and acinus-2 pinin-1 , determined by RT-qPCR in 12-day-old seedlings. Values represent mean ± SD calculated from three biological replicates ( n = 3). Statistically significant differences to WT were determined by two-tailed t test. The P values for a, b, and c are 5.15E−3, 3.32E−3, and 4.91E−3. c Analysis of AtACINUS-GFP association with the FLC locus by ChIP-PCR in 12-day-old AtACINUS-GFP/acinus-2 seedlings. WT serves as the negative control. Bars below the gene structure diagram represent regions analyzed by PCR (blue bars indicate regions enriched after immunoprecipitation). GFP IP shows PCR products using immunoprecipitated DNA. CO-FACTOR FOR NITRATE, REDUCTASE, AND XANTHINE DEHYDROGENASE 5 ( CNX5 ) serves as an internal control to show non-specific background DNA after immunoprecipitation. PCR reactions were set to 28 cycles.
    Figure Legend Snippet: The acinus-2 pinin-1 double mutant is late flowering with increased FLC expression. a Rosette leaf numbers of WT, acinus-2 , pinin-1 , and acinus-2 pinin-1 at bolting stage grown in long day condition. Error bars indicate SD calculated from n > 12. Values represent mean ± SD calculated from at least 12 plants ( n > 12). Statistically significant differences to WT were determined by two-tailed t test. The P values for a, b, and c are 1.35E−6, 2.48E−3, and 5.15E−9. b FLC expression level relative to UBQ (At5g15400) in WT, acinus-2 , pinin-1 , and acinus-2 pinin-1 , determined by RT-qPCR in 12-day-old seedlings. Values represent mean ± SD calculated from three biological replicates ( n = 3). Statistically significant differences to WT were determined by two-tailed t test. The P values for a, b, and c are 5.15E−3, 3.32E−3, and 4.91E−3. c Analysis of AtACINUS-GFP association with the FLC locus by ChIP-PCR in 12-day-old AtACINUS-GFP/acinus-2 seedlings. WT serves as the negative control. Bars below the gene structure diagram represent regions analyzed by PCR (blue bars indicate regions enriched after immunoprecipitation). GFP IP shows PCR products using immunoprecipitated DNA. CO-FACTOR FOR NITRATE, REDUCTASE, AND XANTHINE DEHYDROGENASE 5 ( CNX5 ) serves as an internal control to show non-specific background DNA after immunoprecipitation. PCR reactions were set to 28 cycles.

    Techniques Used: Mutagenesis, Expressing, Two Tailed Test, Quantitative RT-PCR, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Negative Control, Immunoprecipitation

    5) Product Images from "Mitogen-Activated Protein Kinase Signaling Mediates Opioid-induced Presynaptic NMDA Receptor Activation and Analgesic Tolerance"

    Article Title: Mitogen-Activated Protein Kinase Signaling Mediates Opioid-induced Presynaptic NMDA Receptor Activation and Analgesic Tolerance

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14628

    Flowchart diagrams show the timeline of experimental procedures used in the study. Rats were treated with morphine or vehicle for 8 days and used for spinal cord slice recording, behavioral tests (during co-treatment with intrathecal injection of 3 MAPK inhibitors), or co-immunoprecipitation assays. The number of animals used in each group was indicated in parenthesis.
    Figure Legend Snippet: Flowchart diagrams show the timeline of experimental procedures used in the study. Rats were treated with morphine or vehicle for 8 days and used for spinal cord slice recording, behavioral tests (during co-treatment with intrathecal injection of 3 MAPK inhibitors), or co-immunoprecipitation assays. The number of animals used in each group was indicated in parenthesis.

    Techniques Used: Injection, Immunoprecipitation

    6) Product Images from "The trypanosome-specific proteins FPRC and CIF4 regulate cytokinesis initiation by recruiting CIF1 to the cytokinesis initiation site"

    Article Title: The trypanosome-specific proteins FPRC and CIF4 regulate cytokinesis initiation by recruiting CIF1 to the cytokinesis initiation site

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA119.010538

    Identification of FPRC-proximal proteins by BioID. A , expression of FPRC–BirA*-HA in procyclic trypanosomes. FPRC was fused with a C-terminal BirA*-HA and ectopically expressed in a tetracycline-inducible manner. FPRC–BirA*-HA was detected by anti-HA antibody. TbPSA6 served as the loading control. B , localization of FPRC–BirA*-HA to the distal tips of FAZ filaments. FPRC-irA*-HA was detected by FITC-conjugated anti-HA mAb, and the FAZ was labeled by anti-CC2D polyclonal antibody. The open arrowhead and the solid arrowhead indicate FPRC–BirA*-HA signal at the new FAZ tip and old FAZ tip, respectively. Scale bar = 5 μm. C , affinity purification of biotinylated proteins from noninduced control cells and FPRC–BirA*-HA overexpression cells. Shown is the Western blot of the input samples and final elution samples detected by anti-HRP-streptavidin Western blotting. D , schematic of the structural motifs of seven known cytokinesis regulators and a new cytokinesis regulator, CIF4, identified by FPRC BioID. PB , Polo-box motif; Tpm , Tropomyosin-like domain. E , coimmunoprecipitation between PTP-tagged FPRC and 3HA-tagged CIF4. IP , immunoprecipitation. F , coimmunoprecipitation between CIF1 and 3HA-tagged CIF4 and FPRC. FAZ24–3HA served as a negative control.
    Figure Legend Snippet: Identification of FPRC-proximal proteins by BioID. A , expression of FPRC–BirA*-HA in procyclic trypanosomes. FPRC was fused with a C-terminal BirA*-HA and ectopically expressed in a tetracycline-inducible manner. FPRC–BirA*-HA was detected by anti-HA antibody. TbPSA6 served as the loading control. B , localization of FPRC–BirA*-HA to the distal tips of FAZ filaments. FPRC-irA*-HA was detected by FITC-conjugated anti-HA mAb, and the FAZ was labeled by anti-CC2D polyclonal antibody. The open arrowhead and the solid arrowhead indicate FPRC–BirA*-HA signal at the new FAZ tip and old FAZ tip, respectively. Scale bar = 5 μm. C , affinity purification of biotinylated proteins from noninduced control cells and FPRC–BirA*-HA overexpression cells. Shown is the Western blot of the input samples and final elution samples detected by anti-HRP-streptavidin Western blotting. D , schematic of the structural motifs of seven known cytokinesis regulators and a new cytokinesis regulator, CIF4, identified by FPRC BioID. PB , Polo-box motif; Tpm , Tropomyosin-like domain. E , coimmunoprecipitation between PTP-tagged FPRC and 3HA-tagged CIF4. IP , immunoprecipitation. F , coimmunoprecipitation between CIF1 and 3HA-tagged CIF4 and FPRC. FAZ24–3HA served as a negative control.

    Techniques Used: Expressing, Labeling, Affinity Purification, Over Expression, Western Blot, Immunoprecipitation, Negative Control

    7) Product Images from "The levels of H11/HspB8 DNA methylation in human melanoma tissues and xenografts are a critical molecular marker for 5'-Aza-2-deoxycytidine therapy"

    Article Title: The levels of H11/HspB8 DNA methylation in human melanoma tissues and xenografts are a critical molecular marker for 5'-Aza-2-deoxycytidine therapy

    Journal: Cancer investigation

    doi: 10.3109/07357907.2011.584588

    The H11/HspB8 mutant P173H does not bind TAK1 (A) Reciprocal pull-down assay of protein extracts from MeWo cells (express P173H) using antibodies to TAK1 (T), H11/HspB8 (H), or normal IgG (Ig) in immunoprecipitation (IP) and immunoblotting (IB). ( B ) Reciprocal pull-down assay of protein extracts from SKMEL-2 cells stably transfected with tet-regulated H11/HspB8 untreated or treated with Dox (5μg/ml; 3d) done as in (A) . Molecular weights are shown on the right. * indicates slower migrating phosphorylated TAK1 protein.
    Figure Legend Snippet: The H11/HspB8 mutant P173H does not bind TAK1 (A) Reciprocal pull-down assay of protein extracts from MeWo cells (express P173H) using antibodies to TAK1 (T), H11/HspB8 (H), or normal IgG (Ig) in immunoprecipitation (IP) and immunoblotting (IB). ( B ) Reciprocal pull-down assay of protein extracts from SKMEL-2 cells stably transfected with tet-regulated H11/HspB8 untreated or treated with Dox (5μg/ml; 3d) done as in (A) . Molecular weights are shown on the right. * indicates slower migrating phosphorylated TAK1 protein.

    Techniques Used: Mutagenesis, Pull Down Assay, Immunoprecipitation, Stable Transfection, Transfection

    H11/HspB8 methylation is inversely related to gene expression (A) Bisulfite treated DNA and mRNA isolated from cell suspensions of metastatic melanoma tissues, normal human melanocyte cultures (NHM) and melanoma cell lines SKMEL-2, MeWo and A2058 were assayed by MSP and QRT-PCR, respectively. MSP results are expressed as +/− based on the detection of methylated PCR products in 1.7% agarose gels. Data for QRT- PCR are expressed as ΔCt values calculated as described in Materials and Methods. Low ΔCt values indicate high expression. The ΔCt value for NHM (5.4) was used as cut-off point (dotted line) to assess the ΔCt values for the melanoma tissues and they are shown relative to the MSP results. ( B ) Bisulfite treated DNA from early passage melanoma cultures EK, NL, VO, RP, MS, LB, IH, and EI, established melanoma cell lines MeWo, A2058, A375, SKMEL2, LM and NHM was analyzed by MSQP and gene expression was determined by immunoprecipitation/immunoblotting with H11/ HspB8 antibody. Data are expressed as methylation and densitometric units, respectively. The inverse correlation between H11/HspB8 methylation and gene expression is statistically significant (p
    Figure Legend Snippet: H11/HspB8 methylation is inversely related to gene expression (A) Bisulfite treated DNA and mRNA isolated from cell suspensions of metastatic melanoma tissues, normal human melanocyte cultures (NHM) and melanoma cell lines SKMEL-2, MeWo and A2058 were assayed by MSP and QRT-PCR, respectively. MSP results are expressed as +/− based on the detection of methylated PCR products in 1.7% agarose gels. Data for QRT- PCR are expressed as ΔCt values calculated as described in Materials and Methods. Low ΔCt values indicate high expression. The ΔCt value for NHM (5.4) was used as cut-off point (dotted line) to assess the ΔCt values for the melanoma tissues and they are shown relative to the MSP results. ( B ) Bisulfite treated DNA from early passage melanoma cultures EK, NL, VO, RP, MS, LB, IH, and EI, established melanoma cell lines MeWo, A2058, A375, SKMEL2, LM and NHM was analyzed by MSQP and gene expression was determined by immunoprecipitation/immunoblotting with H11/ HspB8 antibody. Data are expressed as methylation and densitometric units, respectively. The inverse correlation between H11/HspB8 methylation and gene expression is statistically significant (p

    Techniques Used: Methylation, Expressing, Isolation, Quantitative RT-PCR, Polymerase Chain Reaction, Mass Spectrometry, Immunoprecipitation

    8) Product Images from "Processing of the Human Coronavirus 229E Replicase Polyproteins by the Virus-Encoded 3C-Like Proteinase: Identification of Proteolytic Products and Cleavage Sites Common to pp1a and pp1ab"

    Article Title: Processing of the Human Coronavirus 229E Replicase Polyproteins by the Virus-Encoded 3C-Like Proteinase: Identification of Proteolytic Products and Cleavage Sites Common to pp1a and pp1ab

    Journal: Journal of Virology

    doi:

    Detection of ORF 1a-encoded 3CL pro cleavage products in HCV 229E-infected cells. (A) Metabolically labeled lysates from mock-infected (M) (lanes 1, 3, 5, 7, 9, 11, 13, and 15) or HCV 229E-infected (I) (lanes 2, 4, 6, 8, 10, 12, 14, and 16) MRC-5 cells were analyzed by SDS–17.4% polyacrylamide gel electrophoresis after immunoprecipitation with the pp1a-specific rabbit antisera α-p5, α-p23, α-p12, and α-p16 or the corresponding preimmune sera. The cells were labeled from 5 to 12 h p.i. Either 180 μl (lanes 1 to 4) or 70 μl (lanes 5 to 16) was analyzed after immunoprecipitation with preimmune serum (lanes 1, 2, 5, 6, 9, 10, 13, and 14) or with the appropriate antiserum as indicated (lanes 3, 4, 7, 8, 11, 12, 15, and 16). (B) Metabolically labeled lysates (100 μl) from mock-infected (lanes 1 and 2) or HCV 229E-infected (lanes 3 and 4) MRC-5 cells were analyzed by SDS–17.4% polyacrylamide gel electrophoresis after immunoprecipitation with antiserum α-p16 (lanes 2 and 4) or the corresponding preimmune serum (lanes 1 and 3). The cells were labeled from 5 to 12 h p.i. Sizes of protein molecular mass markers (lanes PM) (CFA626 and CFA645; Amersham Pharmacia Biotech) and the processing products p5, p12, p23, and p16 are indicated.
    Figure Legend Snippet: Detection of ORF 1a-encoded 3CL pro cleavage products in HCV 229E-infected cells. (A) Metabolically labeled lysates from mock-infected (M) (lanes 1, 3, 5, 7, 9, 11, 13, and 15) or HCV 229E-infected (I) (lanes 2, 4, 6, 8, 10, 12, 14, and 16) MRC-5 cells were analyzed by SDS–17.4% polyacrylamide gel electrophoresis after immunoprecipitation with the pp1a-specific rabbit antisera α-p5, α-p23, α-p12, and α-p16 or the corresponding preimmune sera. The cells were labeled from 5 to 12 h p.i. Either 180 μl (lanes 1 to 4) or 70 μl (lanes 5 to 16) was analyzed after immunoprecipitation with preimmune serum (lanes 1, 2, 5, 6, 9, 10, 13, and 14) or with the appropriate antiserum as indicated (lanes 3, 4, 7, 8, 11, 12, 15, and 16). (B) Metabolically labeled lysates (100 μl) from mock-infected (lanes 1 and 2) or HCV 229E-infected (lanes 3 and 4) MRC-5 cells were analyzed by SDS–17.4% polyacrylamide gel electrophoresis after immunoprecipitation with antiserum α-p16 (lanes 2 and 4) or the corresponding preimmune serum (lanes 1 and 3). The cells were labeled from 5 to 12 h p.i. Sizes of protein molecular mass markers (lanes PM) (CFA626 and CFA645; Amersham Pharmacia Biotech) and the processing products p5, p12, p23, and p16 are indicated.

    Techniques Used: Infection, Metabolic Labelling, Labeling, Polyacrylamide Gel Electrophoresis, Immunoprecipitation

    9) Product Images from "Enhancer Analysis Unveils Genetic Interactions between TLX and SOX2 in Neural Stem Cells and In Vivo Reprogramming"

    Article Title: Enhancer Analysis Unveils Genetic Interactions between TLX and SOX2 in Neural Stem Cells and In Vivo Reprogramming

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2015.09.015

    Transcription Factors Regulating Tlx Enhancer Activity (A) A diagram showing locations of the consensus transcription factor binding sequences (BS). (B) Diminished enhancer activity with mutations in the SOX2- or MYT1-binding sequences. Constitutively expressed tdTomato was used as an internal control for electroporation. The ratios of GFP + cells over tdTomato + cells are indicated in the parentheses (n = 3 mice; mean ± SEM). The scale bar represents 50 μm. (C) MYT1 directly binds to the identified enhancer. Antibody-induced supershift in electrophoresis mobility shift assays (EMSA) is shown in the boxed region. Normal IgG was used as controls for EMSA and chromatin immunoprecipitation (ChIP) assays (mean ± SEM; n = 3 independent experiments for control IgG and MYT1 antibody). (D) SOX2 directly binds to the identified enhancer. The boxed region shows antibody-induced supershift of the probe. Normal IgG was used as controls for EMSA and ChIP (mean ± SEM; n = 3 independent experiments for control IgG and SOX2 antibody). See also Table S2 .
    Figure Legend Snippet: Transcription Factors Regulating Tlx Enhancer Activity (A) A diagram showing locations of the consensus transcription factor binding sequences (BS). (B) Diminished enhancer activity with mutations in the SOX2- or MYT1-binding sequences. Constitutively expressed tdTomato was used as an internal control for electroporation. The ratios of GFP + cells over tdTomato + cells are indicated in the parentheses (n = 3 mice; mean ± SEM). The scale bar represents 50 μm. (C) MYT1 directly binds to the identified enhancer. Antibody-induced supershift in electrophoresis mobility shift assays (EMSA) is shown in the boxed region. Normal IgG was used as controls for EMSA and chromatin immunoprecipitation (ChIP) assays (mean ± SEM; n = 3 independent experiments for control IgG and MYT1 antibody). (D) SOX2 directly binds to the identified enhancer. The boxed region shows antibody-induced supershift of the probe. Normal IgG was used as controls for EMSA and ChIP (mean ± SEM; n = 3 independent experiments for control IgG and SOX2 antibody). See also Table S2 .

    Techniques Used: Activity Assay, Binding Assay, Electroporation, Mouse Assay, Electrophoresis, Mobility Shift, Chromatin Immunoprecipitation

    10) Product Images from "Differential transactivation of the upstream aggrecan enhancer regulated by PAX1/9 depends on SOX9-driven transactivation"

    Article Title: Differential transactivation of the upstream aggrecan enhancer regulated by PAX1/9 depends on SOX9-driven transactivation

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-40810-4

    Binding of PAX1 to the 3′ region of the UE . ( a ) A 359-bp DNA sequence of the mouse UE . The SOX9-binding sites are underlined. ( b ) ChIP assays on extracts from AF cells. After immunoprecipitation of the cross-linked extracts with anti-PAX1 antibody, anti-SOX9 antibody, or normal IgG antibody, the DNA was subjected to PCR with primers that amplify a 116-bp fragment of the UE , 216-bp fragment of the I12E , 135-bp fragment of Nkx3 . 2-P , or 328-bp fragment of the Col2a1-E . Full-length gels are presented in Supplementary Fig. 7 . ( c , d ) Gel shift assays were performed using 20 fmol of biotin-labelled dsDNA probes, 20 pmol of unlabeled dsDNA fragments, and nuclear extracts (N.E.) of HEK293T cells transfected with pcDNA3 empty vector (N.E. Control) or pcDNA3-FLAG-Pax1 (N.E. FLAG-PAX1). The biotin-labelled and unlabeled DNA fragments correspond to positions 1–130, 118–259, and 249–359 of the UE . A shifted band was caused by interactions between FLAG-PAX1 and the biotin-labelled 249–359 of UE . The shifted band in ( c ) disappeared in the presence of the unlabeled 249–359 oligonucleotide or anti-FLAG antibody (Antibody). The shifted band in ( d ) disappeared in the presence of the unlabeled 315–359 oligonucleotide but remained detectable in the presence of the unlabeled 249–290 or 281–325 oligonucleotide. ( e , f ) Gel shift assays were performed using biotin-labelled oligonucleotides shown in Supplementary Table 1 and nuclear extracts (N.E.) of HEK293T cells transfected with pCAG empty vector (N.E Control), pCAG-Sox9 (N.E. SOX9), or pCAG-Pax1 (N.E. PAX1). For the assays in ( e , f ), 100 and 5 fmol of the biotin-labelled oligonucleotides were used, respectively. A total of 1.5 μL of N.E. was used for each binding reaction in ( f ). A shifted band is shown with an arrow. Non-specific bindings are shown with open arrowheads. s, shifted band.
    Figure Legend Snippet: Binding of PAX1 to the 3′ region of the UE . ( a ) A 359-bp DNA sequence of the mouse UE . The SOX9-binding sites are underlined. ( b ) ChIP assays on extracts from AF cells. After immunoprecipitation of the cross-linked extracts with anti-PAX1 antibody, anti-SOX9 antibody, or normal IgG antibody, the DNA was subjected to PCR with primers that amplify a 116-bp fragment of the UE , 216-bp fragment of the I12E , 135-bp fragment of Nkx3 . 2-P , or 328-bp fragment of the Col2a1-E . Full-length gels are presented in Supplementary Fig. 7 . ( c , d ) Gel shift assays were performed using 20 fmol of biotin-labelled dsDNA probes, 20 pmol of unlabeled dsDNA fragments, and nuclear extracts (N.E.) of HEK293T cells transfected with pcDNA3 empty vector (N.E. Control) or pcDNA3-FLAG-Pax1 (N.E. FLAG-PAX1). The biotin-labelled and unlabeled DNA fragments correspond to positions 1–130, 118–259, and 249–359 of the UE . A shifted band was caused by interactions between FLAG-PAX1 and the biotin-labelled 249–359 of UE . The shifted band in ( c ) disappeared in the presence of the unlabeled 249–359 oligonucleotide or anti-FLAG antibody (Antibody). The shifted band in ( d ) disappeared in the presence of the unlabeled 315–359 oligonucleotide but remained detectable in the presence of the unlabeled 249–290 or 281–325 oligonucleotide. ( e , f ) Gel shift assays were performed using biotin-labelled oligonucleotides shown in Supplementary Table 1 and nuclear extracts (N.E.) of HEK293T cells transfected with pCAG empty vector (N.E Control), pCAG-Sox9 (N.E. SOX9), or pCAG-Pax1 (N.E. PAX1). For the assays in ( e , f ), 100 and 5 fmol of the biotin-labelled oligonucleotides were used, respectively. A total of 1.5 μL of N.E. was used for each binding reaction in ( f ). A shifted band is shown with an arrow. Non-specific bindings are shown with open arrowheads. s, shifted band.

    Techniques Used: Binding Assay, Sequencing, Chromatin Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction, Electrophoretic Mobility Shift Assay, Transfection, Plasmid Preparation

    11) Product Images from "Transforming Growth Factor β‐Activated Kinase 1 Regulates Mesenchymal Stem Cell Proliferation Through Stabilization of Yap1/Taz Proteins"

    Article Title: Transforming Growth Factor β‐Activated Kinase 1 Regulates Mesenchymal Stem Cell Proliferation Through Stabilization of Yap1/Taz Proteins

    Journal: Stem Cells (Dayton, Ohio)

    doi: 10.1002/stem.3083

    Localization of TGFβ‐activated kinase 1 (Tak1) and interaction between Tak1‐Yap1/Taz. (A): Localization change of Tak1, Yap1, and Taz by 5zox treatment. Tbp is a TATA‐binding protein as a representative of nuclear localizing protein. αTubulin is a representative of cytoplasmic protein. (B): Immunoprecipitation (IP)‐Western blot (WB) showing interaction between Tak1 and Yap1/Taz. HA indicates IP fraction prepared with anti‐HA antibody. (C): IP‐WB showing affinity change between Tak1 and Yap1/Taz by 5zox treatment. IP fractions were prepared with anti‐HA antibody, which bound to HA‐Tak1. (D): Phosphorylation status of Yap1 and Tak1 under control and 5zox treatment condition. (E): IP‐based detection of ubiquitination status of Yap1 and Taz. Total Yap1 or Taz was collected from cell lysate of BMMSCs with anti‐HA antibody. Subsequently, the ubiquitinated Yap1 or Taz were detected with anti‐Ub antibody. Abbreviations: C, Cytoplasmic fraction; N, nuclear fraction of bone marrow‐derived mesenchymal stem cells (BMMSCs).
    Figure Legend Snippet: Localization of TGFβ‐activated kinase 1 (Tak1) and interaction between Tak1‐Yap1/Taz. (A): Localization change of Tak1, Yap1, and Taz by 5zox treatment. Tbp is a TATA‐binding protein as a representative of nuclear localizing protein. αTubulin is a representative of cytoplasmic protein. (B): Immunoprecipitation (IP)‐Western blot (WB) showing interaction between Tak1 and Yap1/Taz. HA indicates IP fraction prepared with anti‐HA antibody. (C): IP‐WB showing affinity change between Tak1 and Yap1/Taz by 5zox treatment. IP fractions were prepared with anti‐HA antibody, which bound to HA‐Tak1. (D): Phosphorylation status of Yap1 and Tak1 under control and 5zox treatment condition. (E): IP‐based detection of ubiquitination status of Yap1 and Taz. Total Yap1 or Taz was collected from cell lysate of BMMSCs with anti‐HA antibody. Subsequently, the ubiquitinated Yap1 or Taz were detected with anti‐Ub antibody. Abbreviations: C, Cytoplasmic fraction; N, nuclear fraction of bone marrow‐derived mesenchymal stem cells (BMMSCs).

    Techniques Used: Binding Assay, Immunoprecipitation, Western Blot, Derivative Assay

    12) Product Images from "New histone supply regulates replication fork speed and PCNA unloading"

    Article Title: New histone supply regulates replication fork speed and PCNA unloading

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201305017

    Nucleosome assembly is required for fork progression. Artificial blocking of Asf1–CAF-1–mediated nucleosome assembly by transient expression of the HIRA-B domain fused to an SV40 NLS. (A) Immunoprecipitation (IP) of FLAG-HA–tagged HIRA-B and HIRA-B mt carrying three point mutations disabling Asf1 binding. (B) Cell cycle profiles. GFP-spectrin was used to identify transfected cells by FACS. One representative experiment out of five biological replicas is shown. (C and D) EdU incorporation (C) and chromatin-bound RPA (D) detected by immunofluorescence in preextracted cells. Cells were cotransfected with H2B-GFP to identify transfected cells and treated 1 h with HU where indicated. Error bars indicate SDs of four biological replicas. Bars, 20 µm. (E) Single-cell analysis of DNA replication and DNA damage in U-2-OS cells transfected with full-length HIRA or the HIRA-B domain for 24 h. A dot plot of EdU and γ-H2AX intensities (left) and a scatter plot of γ-H2AX intensities (right) are shown. Cells treated 2 h with HU were included as a positive control. Cells were pulsed 15 min with EdU. Lines represent medians. n > 70. ***, P
    Figure Legend Snippet: Nucleosome assembly is required for fork progression. Artificial blocking of Asf1–CAF-1–mediated nucleosome assembly by transient expression of the HIRA-B domain fused to an SV40 NLS. (A) Immunoprecipitation (IP) of FLAG-HA–tagged HIRA-B and HIRA-B mt carrying three point mutations disabling Asf1 binding. (B) Cell cycle profiles. GFP-spectrin was used to identify transfected cells by FACS. One representative experiment out of five biological replicas is shown. (C and D) EdU incorporation (C) and chromatin-bound RPA (D) detected by immunofluorescence in preextracted cells. Cells were cotransfected with H2B-GFP to identify transfected cells and treated 1 h with HU where indicated. Error bars indicate SDs of four biological replicas. Bars, 20 µm. (E) Single-cell analysis of DNA replication and DNA damage in U-2-OS cells transfected with full-length HIRA or the HIRA-B domain for 24 h. A dot plot of EdU and γ-H2AX intensities (left) and a scatter plot of γ-H2AX intensities (right) are shown. Cells treated 2 h with HU were included as a positive control. Cells were pulsed 15 min with EdU. Lines represent medians. n > 70. ***, P

    Techniques Used: Blocking Assay, Expressing, Immunoprecipitation, Binding Assay, Transfection, FACS, Recombinase Polymerase Amplification, Immunofluorescence, Single-cell Analysis, Positive Control

    13) Product Images from "Varicella-Zoster Virus IE4 Protein Interacts with SR Proteins and Exports mRNAs through the TAP/NXF1 Pathway"

    Article Title: Varicella-Zoster Virus IE4 Protein Interacts with SR Proteins and Exports mRNAs through the TAP/NXF1 Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007882

    IE4 interacts with ASF/SF2, 9G8 and SRp20 through its arginine-rich Ra, Rb and Rc domains. (A) Nuclear extracts from co-transfected HeLa cells were immunoprecipitated with anti-GFP, anti-V5 or anti-HA antibodies as indicated. In Input (lanes 1 and 7), nuclear extracts were fractionated without immunoprecipitation. Nuclear extracts were treated (lane 6) or not (lane 5) with RNases A/T1 mix for 30 min at 37°C before immunoprecipitation. In lanes 3 and 9, immunoprecipitation was performed with beads without antibody as a negative control (−). The blots were probed with anti-HA (left panels) or anti-V5 (right panels) antibodies. (B) Total extracts from transfected HeLa cells were immunoprecipitated with anti-Flag or anti-V5 antibodies as indicated. In Input (lanes 1, 2, 5 and 6), total extracts were fractionated without immunoprecipitation. The blots were probed with anti-ASF/SF2 or anti-SRp20 (upper panels) and anti-V5 (lower panels) antibodies. The asterisk marks heavy chain IgG from the immunoprecipitation. (C) In vitro binding assay was performed by incubating GST-ASF/SF2 with in vitro -translated IE4-V5 or derivatives as indicated (lanes 2 to 7). Assay performed with GST alone constituted the negative control (−) (lane 1). In Input (lanes 8 to 14), [ 35 S]-methionine-labelled proteins were fractionated without binding assay. (D) In vitro -translated ASF/SF2-V5 was incubated with GST-IE4 derivatives as indicated (lanes 2 to 7). GST alone constituted the negative control (lane 1) and, in Input, ASF/SF2-V5 was loaded without binding assay (lane 8).
    Figure Legend Snippet: IE4 interacts with ASF/SF2, 9G8 and SRp20 through its arginine-rich Ra, Rb and Rc domains. (A) Nuclear extracts from co-transfected HeLa cells were immunoprecipitated with anti-GFP, anti-V5 or anti-HA antibodies as indicated. In Input (lanes 1 and 7), nuclear extracts were fractionated without immunoprecipitation. Nuclear extracts were treated (lane 6) or not (lane 5) with RNases A/T1 mix for 30 min at 37°C before immunoprecipitation. In lanes 3 and 9, immunoprecipitation was performed with beads without antibody as a negative control (−). The blots were probed with anti-HA (left panels) or anti-V5 (right panels) antibodies. (B) Total extracts from transfected HeLa cells were immunoprecipitated with anti-Flag or anti-V5 antibodies as indicated. In Input (lanes 1, 2, 5 and 6), total extracts were fractionated without immunoprecipitation. The blots were probed with anti-ASF/SF2 or anti-SRp20 (upper panels) and anti-V5 (lower panels) antibodies. The asterisk marks heavy chain IgG from the immunoprecipitation. (C) In vitro binding assay was performed by incubating GST-ASF/SF2 with in vitro -translated IE4-V5 or derivatives as indicated (lanes 2 to 7). Assay performed with GST alone constituted the negative control (−) (lane 1). In Input (lanes 8 to 14), [ 35 S]-methionine-labelled proteins were fractionated without binding assay. (D) In vitro -translated ASF/SF2-V5 was incubated with GST-IE4 derivatives as indicated (lanes 2 to 7). GST alone constituted the negative control (lane 1) and, in Input, ASF/SF2-V5 was loaded without binding assay (lane 8).

    Techniques Used: Transfection, Immunoprecipitation, Negative Control, In Vitro, Binding Assay, Incubation

    IE4 interacts with SRPK1 through its arginine-rich Ra and Rb domains. (A) Total extracts of transfected and mock- or VZV-infected MeWo cells were immunoprecipitated with anti-GFP, anti-IE4, anti-V5 or anti-Flag antibodies as indicated (lanes 3 to 6). In Input (lanes 1 and 2), total extracts were fractionated without immunoprecipitation. The blots were probed with anti-Flag or anti-IE4 antibodies. The asterisk marks heavy chain IgG from the immunoprecipitation. (B) Binding assay was performed by incubating HEK293 cells total extracts with GST-IE4 derivatives as indicated (lanes 2 to 7). Assay performed with GST alone constituted the negative control (lane 1). The blot was probed with anti-SRPK1 antibody. Coomassie Blue-stained gel is shown below.
    Figure Legend Snippet: IE4 interacts with SRPK1 through its arginine-rich Ra and Rb domains. (A) Total extracts of transfected and mock- or VZV-infected MeWo cells were immunoprecipitated with anti-GFP, anti-IE4, anti-V5 or anti-Flag antibodies as indicated (lanes 3 to 6). In Input (lanes 1 and 2), total extracts were fractionated without immunoprecipitation. The blots were probed with anti-Flag or anti-IE4 antibodies. The asterisk marks heavy chain IgG from the immunoprecipitation. (B) Binding assay was performed by incubating HEK293 cells total extracts with GST-IE4 derivatives as indicated (lanes 2 to 7). Assay performed with GST alone constituted the negative control (lane 1). The blot was probed with anti-SRPK1 antibody. Coomassie Blue-stained gel is shown below.

    Techniques Used: Transfection, Infection, Immunoprecipitation, Binding Assay, Negative Control, Staining

    IE4 interacts with TAP/NXF1 and Aly/REF. (A) In vitro binding assays were performed by incubating GST-TAP (lanes 2 to 7) or GST-REF (lanes 9 to 14) with in vitro -translated IE4-V5 or derivatives as indicated. Assays performed with GST alone constituted the negative control (−) (lanes 1 and 8). (B) In vitro -translated IE4 Nter-V5 was incubated with GST-TAP (lanes 4 and 5) or GST-REF (lanes 6 and 7). GST alone constituted the negative control (lanes 2 and 3). Complexes were treated (lanes 3, 5 and 7) or not (lanes 2, 4 and 6) with RNases A/T1 mix for 30 min at 37°C before SDS-PAGE. In Input (lane 1), IE4 Nter-V5 was loaded without binding assay. (C) Total extracts of VZV-infected MeWo cells were immunoprecipitated with anti-GFP (lane 2), anti-IE4 (lane 3), anti-Flag (lane 5), anti-Aly/REF or anti-TAP/NXF1 (lane 6) antibodies as indicated. In Input (lanes 1 and 4), total extracts were fractionated without immunoprecipitation. The blots were probed with anti-Aly/REF, anti-TAP/NXF1 or anti-IE4 antibodies. The asterisk marks heavy chain IgG from the immunoprecipitation. (D) Total extracts of VZV-infected MeWo cells were immunoprecipitated with anti-GFP (lanes 3 and 8), anti-Aly/REF (lanes 4 and 5) or anti-TAP/NXF1 (lanes 9 and 10) antibodies as indicated. In Input (lanes 1, 2, 6 and 7), total extracts were fractionated without immunoprecipitation. Total extracts were treated (lanes 5 and 10) or not (lanes 3, 4, 8 and 9) with RNases A/T1 mix for 30 min at 37°C before immunoprecipitation. The blots were probed with anti-IE4, anti-Aly/REF or anti-TAP/NXF1 antibodies.
    Figure Legend Snippet: IE4 interacts with TAP/NXF1 and Aly/REF. (A) In vitro binding assays were performed by incubating GST-TAP (lanes 2 to 7) or GST-REF (lanes 9 to 14) with in vitro -translated IE4-V5 or derivatives as indicated. Assays performed with GST alone constituted the negative control (−) (lanes 1 and 8). (B) In vitro -translated IE4 Nter-V5 was incubated with GST-TAP (lanes 4 and 5) or GST-REF (lanes 6 and 7). GST alone constituted the negative control (lanes 2 and 3). Complexes were treated (lanes 3, 5 and 7) or not (lanes 2, 4 and 6) with RNases A/T1 mix for 30 min at 37°C before SDS-PAGE. In Input (lane 1), IE4 Nter-V5 was loaded without binding assay. (C) Total extracts of VZV-infected MeWo cells were immunoprecipitated with anti-GFP (lane 2), anti-IE4 (lane 3), anti-Flag (lane 5), anti-Aly/REF or anti-TAP/NXF1 (lane 6) antibodies as indicated. In Input (lanes 1 and 4), total extracts were fractionated without immunoprecipitation. The blots were probed with anti-Aly/REF, anti-TAP/NXF1 or anti-IE4 antibodies. The asterisk marks heavy chain IgG from the immunoprecipitation. (D) Total extracts of VZV-infected MeWo cells were immunoprecipitated with anti-GFP (lanes 3 and 8), anti-Aly/REF (lanes 4 and 5) or anti-TAP/NXF1 (lanes 9 and 10) antibodies as indicated. In Input (lanes 1, 2, 6 and 7), total extracts were fractionated without immunoprecipitation. Total extracts were treated (lanes 5 and 10) or not (lanes 3, 4, 8 and 9) with RNases A/T1 mix for 30 min at 37°C before immunoprecipitation. The blots were probed with anti-IE4, anti-Aly/REF or anti-TAP/NXF1 antibodies.

    Techniques Used: In Vitro, Binding Assay, Negative Control, Incubation, SDS Page, Infection, Immunoprecipitation

    14) Product Images from "Loss of NPC1 function in a patient with a co-inherited novel insulin receptor mutation does not grossly modify the severity of the associated insulin resistance"

    Article Title: Loss of NPC1 function in a patient with a co-inherited novel insulin receptor mutation does not grossly modify the severity of the associated insulin resistance

    Journal: Journal of Inherited Metabolic Disease

    doi: 10.1007/s10545-010-9107-5

    The Proband has a heterozygous GTG deletion at the end of exon 20 of the INSR gene in genomic DNA ( a ), which is seen also in INSR cDNA ( b ). The nucleotide deletion causes deletion of Trp1220 (Trp1193 in the mature receptor), a strongly conserved residue ( c ) located in an alpha helix within the tyrosine kinase damin of the receptor. There was no detectable change in expression of the INSR in dermal fibroblasts from the proband as assessed by quantitative real time PCR ( d ) and immunoprecipitation/immunoblotting ( e ) for the insulin receptor beta subunit
    Figure Legend Snippet: The Proband has a heterozygous GTG deletion at the end of exon 20 of the INSR gene in genomic DNA ( a ), which is seen also in INSR cDNA ( b ). The nucleotide deletion causes deletion of Trp1220 (Trp1193 in the mature receptor), a strongly conserved residue ( c ) located in an alpha helix within the tyrosine kinase damin of the receptor. There was no detectable change in expression of the INSR in dermal fibroblasts from the proband as assessed by quantitative real time PCR ( d ) and immunoprecipitation/immunoblotting ( e ) for the insulin receptor beta subunit

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Immunoprecipitation

    15) Product Images from "The novel cyclophilin-D-interacting protein FASTKD1 protects cells against oxidative stress-induced cell death"

    Article Title: The novel cyclophilin-D-interacting protein FASTKD1 protects cells against oxidative stress-induced cell death

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00471.2018

    FASTKD1 interacts with the mitochondrial protein cyclophilin-D (CypD) and localizes to mitochondria. A : neonatal rat ventricular myocytes (NRVMs) were infected with β-galactosidase (βGal), FASTKD1-Myc, or CypD-FLAG adenoviruses for 48 h, and the lysates were blotted for either Myc or FLAG. Actin was used as a loading control. B : immunocytochemistry performed on NRVMs infected with either βGal or FASTKD1-Myc adenoviruses and then stained for Myc tag. Mitotracker Red (Mito) was used to identify mitochondria. C : NRVMs were infected with βGal, FASTKD1-Myc, CypD-FLAG, or FASTKD1-Myc plus CypD FLAG adenoviruses for 48 h. The lysates were then subjected to immunoprecipitation (IP) using a FLAG antibody, and the resultant complexes were blotted for Myc and FLAG. D : mouse embryonic fibroblasts (MEFs) were infected with βGal or FASTKD1-Myc for 48 h, and the lysates were blotted for Myc. The mitochondrial phosphate carrier (PiC) was used as a loading control. E : immunocytochemistry performed on MEFs infected with either βGal or FASTKD1-Myc adenoviruses and then stained for Myc tag. Mitotracker Red was used to stain mitochondria. All images are representative of experiments done with 4 independent MEF and NRVM isolates. Cells of both sexes were used.
    Figure Legend Snippet: FASTKD1 interacts with the mitochondrial protein cyclophilin-D (CypD) and localizes to mitochondria. A : neonatal rat ventricular myocytes (NRVMs) were infected with β-galactosidase (βGal), FASTKD1-Myc, or CypD-FLAG adenoviruses for 48 h, and the lysates were blotted for either Myc or FLAG. Actin was used as a loading control. B : immunocytochemistry performed on NRVMs infected with either βGal or FASTKD1-Myc adenoviruses and then stained for Myc tag. Mitotracker Red (Mito) was used to identify mitochondria. C : NRVMs were infected with βGal, FASTKD1-Myc, CypD-FLAG, or FASTKD1-Myc plus CypD FLAG adenoviruses for 48 h. The lysates were then subjected to immunoprecipitation (IP) using a FLAG antibody, and the resultant complexes were blotted for Myc and FLAG. D : mouse embryonic fibroblasts (MEFs) were infected with βGal or FASTKD1-Myc for 48 h, and the lysates were blotted for Myc. The mitochondrial phosphate carrier (PiC) was used as a loading control. E : immunocytochemistry performed on MEFs infected with either βGal or FASTKD1-Myc adenoviruses and then stained for Myc tag. Mitotracker Red was used to stain mitochondria. All images are representative of experiments done with 4 independent MEF and NRVM isolates. Cells of both sexes were used.

    Techniques Used: Infection, Immunocytochemistry, Staining, Immunoprecipitation

    16) Product Images from "Mutations inhibiting KDM4B drive ALT activation in ATRX-mutated glioblastomas"

    Article Title: Mutations inhibiting KDM4B drive ALT activation in ATRX-mutated glioblastomas

    Journal: Nature Communications

    doi: 10.1038/s41467-021-22543-z

    KDM4B −/− mouse ES cells show replication stress. a Protein immunoprecipitation with an anti-Flag antibody in ES cells expressing HA-H3.3 and either Flag-tagged KDM4A, KDM4B or KDM4C, followed by western blot analysis with an anti-HA antibody. b ChIP-sequencing analysis of KDM4 -A 34 , -B 33 and -C 33 with input sequencing. Data shows reads which aligned to telomeres, normalised for total read counts showing KDM4B binding to telomeres. c ChIP-qPCR analysis of KDM4B at telomeres in cell cycle-synchronised cells ( n = 3 independent experiments). d Western blot analyses of WT and KDM4B cells using antibodies against H3K9me3, H3K36me3, H3 and ACTIN. e ChIP-qPCR analyses of H3.3, total H3, H3K9me3, ATRX, HP1α. Level of H3K9me3 was normalised to total H3 levels (H3K9me3/H3) ( n = 3 independent experiments). f ChIP-qPCR analyses of BrdU incorporation at telomeres and Gapdh promoter in WT and Kdm4b −/− cells ( n = 4 independent experiments). g, h Immunofluorescence analyses of TERF1 (red; diluted at 1/500) and γH2AX (green; diluted at 1/1500) in WT and Kdm4b −/− cells with and without 1 mM APH treatment for 5 h. Arrows indicate presence of DNA damage (γH2AX) at telomeres (TERF1). Scale bars: 5 μm. Percentages of co-localised TERF1 and γH2AX foci in two independent Kdm4b −/− cell lines ( Kdm4b −/− #1 and #2) are shown in h . For each cell line, > 1500 telomeric or TERF1 foci were counted ( n = 4 independent experiments). Percentage of co-localised TERF1/γH2AX foci are determined as percentages of telomeric foci that co-stained with γH2AX over the total number of telomeric foci counted. c , e , f , h Data are presented as mean values ± SD. *indicates p
    Figure Legend Snippet: KDM4B −/− mouse ES cells show replication stress. a Protein immunoprecipitation with an anti-Flag antibody in ES cells expressing HA-H3.3 and either Flag-tagged KDM4A, KDM4B or KDM4C, followed by western blot analysis with an anti-HA antibody. b ChIP-sequencing analysis of KDM4 -A 34 , -B 33 and -C 33 with input sequencing. Data shows reads which aligned to telomeres, normalised for total read counts showing KDM4B binding to telomeres. c ChIP-qPCR analysis of KDM4B at telomeres in cell cycle-synchronised cells ( n = 3 independent experiments). d Western blot analyses of WT and KDM4B cells using antibodies against H3K9me3, H3K36me3, H3 and ACTIN. e ChIP-qPCR analyses of H3.3, total H3, H3K9me3, ATRX, HP1α. Level of H3K9me3 was normalised to total H3 levels (H3K9me3/H3) ( n = 3 independent experiments). f ChIP-qPCR analyses of BrdU incorporation at telomeres and Gapdh promoter in WT and Kdm4b −/− cells ( n = 4 independent experiments). g, h Immunofluorescence analyses of TERF1 (red; diluted at 1/500) and γH2AX (green; diluted at 1/1500) in WT and Kdm4b −/− cells with and without 1 mM APH treatment for 5 h. Arrows indicate presence of DNA damage (γH2AX) at telomeres (TERF1). Scale bars: 5 μm. Percentages of co-localised TERF1 and γH2AX foci in two independent Kdm4b −/− cell lines ( Kdm4b −/− #1 and #2) are shown in h . For each cell line, > 1500 telomeric or TERF1 foci were counted ( n = 4 independent experiments). Percentage of co-localised TERF1/γH2AX foci are determined as percentages of telomeric foci that co-stained with γH2AX over the total number of telomeric foci counted. c , e , f , h Data are presented as mean values ± SD. *indicates p

    Techniques Used: Immunoprecipitation, Expressing, Western Blot, Chromatin Immunoprecipitation, Sequencing, Binding Assay, Real-time Polymerase Chain Reaction, BrdU Incorporation Assay, Immunofluorescence, Staining

    17) Product Images from "RNA-Binding Protein MSI2 Binds to miR-301a-3p and Facilitates Its Distribution in Mitochondria of Endothelial Cells"

    Article Title: RNA-Binding Protein MSI2 Binds to miR-301a-3p and Facilitates Its Distribution in Mitochondria of Endothelial Cells

    Journal: Frontiers in Molecular Biosciences

    doi: 10.3389/fmolb.2020.609828

    MSI2 binds to miR-301a-3p. (A) Schematic representation of biotinylated miRNA pull-down assay followed by label-free quantitative proteomic analysis. 5′biotinylated miR-301a-3p or random sequence (control group) was incubated with streptavidin beads and whole cell extracts of HUVECs. After washing and elution, the pull-down proteins were analyzed by MS/MS. (B) Relative abundances of MSI2 protein identified in three replicate experiments of label-free quantitative proteomic analysis for the product of biotinylated miR-301a-3p pull-down [(Btn) miR-301a-3p]. Biotinylated random small sequence [(Btn) random] was used as a negative control. (C) Representative MS/MS spectrum of a parent ion of MSI2 protein in the label-free quantitative proteomic analysis. The peptide sequence identified by this spectrum was shown in the upper right side. (D) Western blotting analysis of MSI2 protein in the product of the biotinylated miR-301a-3p pull-down assay. WCE: whole cell extracts; Beads+Extract: the mixture of streptavidin–agarose beads and whole cell extracts of HUVECs. (E) miR-301a-3p level in the immunoprecipitation products obtained by incubating anti-MSI2 antibody with the HUVECs lysates. The result was reported as percentage of the input sample (% input). The normal IgG was used as control (IP IgG). (F) The representative sensorgram of bio-layer interferometry (BLI) analysis for the binding kinetics of MSI2 and 5′ biotinylated miR-301a-3p. The blue curves represented the measured responses for each tested concentration of MSI2 protein. The overlapped red curves showed the global fitting results of the binding data. Wilcoxon rank-sum test was used for statistical analysis in (E) . Median with interquartile range was shown for (E) . *** P
    Figure Legend Snippet: MSI2 binds to miR-301a-3p. (A) Schematic representation of biotinylated miRNA pull-down assay followed by label-free quantitative proteomic analysis. 5′biotinylated miR-301a-3p or random sequence (control group) was incubated with streptavidin beads and whole cell extracts of HUVECs. After washing and elution, the pull-down proteins were analyzed by MS/MS. (B) Relative abundances of MSI2 protein identified in three replicate experiments of label-free quantitative proteomic analysis for the product of biotinylated miR-301a-3p pull-down [(Btn) miR-301a-3p]. Biotinylated random small sequence [(Btn) random] was used as a negative control. (C) Representative MS/MS spectrum of a parent ion of MSI2 protein in the label-free quantitative proteomic analysis. The peptide sequence identified by this spectrum was shown in the upper right side. (D) Western blotting analysis of MSI2 protein in the product of the biotinylated miR-301a-3p pull-down assay. WCE: whole cell extracts; Beads+Extract: the mixture of streptavidin–agarose beads and whole cell extracts of HUVECs. (E) miR-301a-3p level in the immunoprecipitation products obtained by incubating anti-MSI2 antibody with the HUVECs lysates. The result was reported as percentage of the input sample (% input). The normal IgG was used as control (IP IgG). (F) The representative sensorgram of bio-layer interferometry (BLI) analysis for the binding kinetics of MSI2 and 5′ biotinylated miR-301a-3p. The blue curves represented the measured responses for each tested concentration of MSI2 protein. The overlapped red curves showed the global fitting results of the binding data. Wilcoxon rank-sum test was used for statistical analysis in (E) . Median with interquartile range was shown for (E) . *** P

    Techniques Used: Pull Down Assay, Sequencing, Incubation, Tandem Mass Spectroscopy, Negative Control, Western Blot, Immunoprecipitation, Binding Assay, Concentration Assay

    MSI2 exists in mitochondria and facilitates the distribution of miR-301a-3p in mitochondria. (A) Western blotting analysis of mitochondrial fractions of HUVECs for MSI2 and Cytochrome C. Mito: mitochondria; WCE: whole cell extracts. (B) Fluorescence colocalization of MSI2 and mitochondria in different types of cells. MSI2 was labeled with its Alexa Fluor 488-conjugated antibody (green). Mitochondria and nuclei were stained, respectively, with MitoTracker (red) and DAPI (blue). Yellow areas in the merged images represented the colocalization of MSI2 and mitochondria. R value represented the Mander's overlap coefficient between MSI2 and mitochondria calculated by Image-Pro Plus 6.0 software. Bar=10 μm. (C,D) The relative levels of miR-301a-3p in mitochondria (C) and cytosol (D) of HUVECs transfected with MSI2 siRNA compared to those in the control group. The 12S rRNA (mitochondria) and GAPDH (cytosol) were used as internal standards. (E,F) The relative levels of miR-301a-3p in mitochondria (E) and cytosol (F) of HUVECs transfected with MSI2 expression vector compared to those in the control group. (G) Western blotting analysis of MSI2 levels in the whole, cytosolic and mitochondrial fractions of HUVECs treated with H 2 O 2 for 0, 12, and 18 h. (H) Western blotting analysis of MSI2 levels in the whole, cytosolic and mitochondrial fractions of HUVECs treated with GO for 24 h. (I,J) Immunoprecipitation analysis of the binding of Ago2 and MSI2 in HUVECs mitochondria. The immune complexes were formed by incubating mitochondrial lysates with anti-MSI2 (MSI2 IP) and then immunoblotted with anti-Ago2 antibody (I) , or by incubating mitochondrial lysates with anti-Ago2 (Ago2 IP) and then immunoblotted with anti-MSI2 antibody (J) . Mitochondrial lysates were used as input sample and normal IgG was used as the negative control (IgG). Two-tailed Student's t -test was used for statistical analysis in (C–F,H) . One-way analysis of variance (ANOVA) was used for (G) . Data are presented as mean ± SEM. * P
    Figure Legend Snippet: MSI2 exists in mitochondria and facilitates the distribution of miR-301a-3p in mitochondria. (A) Western blotting analysis of mitochondrial fractions of HUVECs for MSI2 and Cytochrome C. Mito: mitochondria; WCE: whole cell extracts. (B) Fluorescence colocalization of MSI2 and mitochondria in different types of cells. MSI2 was labeled with its Alexa Fluor 488-conjugated antibody (green). Mitochondria and nuclei were stained, respectively, with MitoTracker (red) and DAPI (blue). Yellow areas in the merged images represented the colocalization of MSI2 and mitochondria. R value represented the Mander's overlap coefficient between MSI2 and mitochondria calculated by Image-Pro Plus 6.0 software. Bar=10 μm. (C,D) The relative levels of miR-301a-3p in mitochondria (C) and cytosol (D) of HUVECs transfected with MSI2 siRNA compared to those in the control group. The 12S rRNA (mitochondria) and GAPDH (cytosol) were used as internal standards. (E,F) The relative levels of miR-301a-3p in mitochondria (E) and cytosol (F) of HUVECs transfected with MSI2 expression vector compared to those in the control group. (G) Western blotting analysis of MSI2 levels in the whole, cytosolic and mitochondrial fractions of HUVECs treated with H 2 O 2 for 0, 12, and 18 h. (H) Western blotting analysis of MSI2 levels in the whole, cytosolic and mitochondrial fractions of HUVECs treated with GO for 24 h. (I,J) Immunoprecipitation analysis of the binding of Ago2 and MSI2 in HUVECs mitochondria. The immune complexes were formed by incubating mitochondrial lysates with anti-MSI2 (MSI2 IP) and then immunoblotted with anti-Ago2 antibody (I) , or by incubating mitochondrial lysates with anti-Ago2 (Ago2 IP) and then immunoblotted with anti-MSI2 antibody (J) . Mitochondrial lysates were used as input sample and normal IgG was used as the negative control (IgG). Two-tailed Student's t -test was used for statistical analysis in (C–F,H) . One-way analysis of variance (ANOVA) was used for (G) . Data are presented as mean ± SEM. * P

    Techniques Used: Western Blot, Fluorescence, Labeling, Staining, Software, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Binding Assay, Negative Control, Two Tailed Test

    18) Product Images from "The trypanosome-specific proteins FPRC and CIF4 regulate cytokinesis initiation by recruiting CIF1 to the cytokinesis initiation site"

    Article Title: The trypanosome-specific proteins FPRC and CIF4 regulate cytokinesis initiation by recruiting CIF1 to the cytokinesis initiation site

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA119.010538

    Identification of FPRC-proximal proteins by BioID. A , expression of FPRC–BirA*-HA in procyclic trypanosomes. FPRC was fused with a C-terminal BirA*-HA and ectopically expressed in a tetracycline-inducible manner. FPRC–BirA*-HA was detected by anti-HA antibody. TbPSA6 served as the loading control. B , localization of FPRC–BirA*-HA to the distal tips of FAZ filaments. FPRC-irA*-HA was detected by FITC-conjugated anti-HA mAb, and the FAZ was labeled by anti-CC2D polyclonal antibody. The open arrowhead and the solid arrowhead indicate FPRC–BirA*-HA signal at the new FAZ tip and old FAZ tip, respectively. Scale bar = 5 μm. C , affinity purification of biotinylated proteins from noninduced control cells and FPRC–BirA*-HA overexpression cells. Shown is the Western blot of the input samples and final elution samples detected by anti-HRP-streptavidin Western blotting. D , schematic of the structural motifs of seven known cytokinesis regulators and a new cytokinesis regulator, CIF4, identified by FPRC BioID. PB , Polo-box motif; Tpm , Tropomyosin-like domain. E , coimmunoprecipitation between PTP-tagged FPRC and 3HA-tagged CIF4. IP , immunoprecipitation. F , coimmunoprecipitation between CIF1 and 3HA-tagged CIF4 and FPRC. FAZ24–3HA served as a negative control.
    Figure Legend Snippet: Identification of FPRC-proximal proteins by BioID. A , expression of FPRC–BirA*-HA in procyclic trypanosomes. FPRC was fused with a C-terminal BirA*-HA and ectopically expressed in a tetracycline-inducible manner. FPRC–BirA*-HA was detected by anti-HA antibody. TbPSA6 served as the loading control. B , localization of FPRC–BirA*-HA to the distal tips of FAZ filaments. FPRC-irA*-HA was detected by FITC-conjugated anti-HA mAb, and the FAZ was labeled by anti-CC2D polyclonal antibody. The open arrowhead and the solid arrowhead indicate FPRC–BirA*-HA signal at the new FAZ tip and old FAZ tip, respectively. Scale bar = 5 μm. C , affinity purification of biotinylated proteins from noninduced control cells and FPRC–BirA*-HA overexpression cells. Shown is the Western blot of the input samples and final elution samples detected by anti-HRP-streptavidin Western blotting. D , schematic of the structural motifs of seven known cytokinesis regulators and a new cytokinesis regulator, CIF4, identified by FPRC BioID. PB , Polo-box motif; Tpm , Tropomyosin-like domain. E , coimmunoprecipitation between PTP-tagged FPRC and 3HA-tagged CIF4. IP , immunoprecipitation. F , coimmunoprecipitation between CIF1 and 3HA-tagged CIF4 and FPRC. FAZ24–3HA served as a negative control.

    Techniques Used: Expressing, Labeling, Affinity Purification, Over Expression, Western Blot, Immunoprecipitation, Negative Control

    19) Product Images from "CK2 Phosphorylates Sec31 and Regulates ER-To-Golgi Trafficking"

    Article Title: CK2 Phosphorylates Sec31 and Regulates ER-To-Golgi Trafficking

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0054382

    CK2 phosphorylates human Sec31. (A) FLAG-Sec31 expressed in HRK293 cells was immunoprecipitated and incubated with/without recombinant CK2 (recCK2) followed by western blotting with anti-phospho serine/threonine and anti-FLAG antibodies (IP’ed Sec31). The phospho-Sec31 levels were normalized to IP’ed Sec31 levels and expressed as the normalized ratio. (B) FLAG-Sec13 expressed in HRK293 cells was immunoprecipitated, treated and subjected to western blotting as described in (A). (C) Cells were first transfected with siRNA1 to CK2 alpha1 (RNAi +) or to eGFP (RNAi −). After 2 days incubation, cells were transfected with FLAG-tagged Sec31 transfection followed by immunoprecipitation and western blotting as described in (A). Total cell lysates were also analyzed for CK2 by western blotting to determine the depletion efficiency as shown in the bottom panel. (D) FLAG-Sec31 expressed in HRK293 cells treated with or without CK2 inhibitor was immunoprecipitated and subjected to western blotting as described in (A).
    Figure Legend Snippet: CK2 phosphorylates human Sec31. (A) FLAG-Sec31 expressed in HRK293 cells was immunoprecipitated and incubated with/without recombinant CK2 (recCK2) followed by western blotting with anti-phospho serine/threonine and anti-FLAG antibodies (IP’ed Sec31). The phospho-Sec31 levels were normalized to IP’ed Sec31 levels and expressed as the normalized ratio. (B) FLAG-Sec13 expressed in HRK293 cells was immunoprecipitated, treated and subjected to western blotting as described in (A). (C) Cells were first transfected with siRNA1 to CK2 alpha1 (RNAi +) or to eGFP (RNAi −). After 2 days incubation, cells were transfected with FLAG-tagged Sec31 transfection followed by immunoprecipitation and western blotting as described in (A). Total cell lysates were also analyzed for CK2 by western blotting to determine the depletion efficiency as shown in the bottom panel. (D) FLAG-Sec31 expressed in HRK293 cells treated with or without CK2 inhibitor was immunoprecipitated and subjected to western blotting as described in (A).

    Techniques Used: Immunoprecipitation, Incubation, Recombinant, Western Blot, Transfection

    Dephosphorylation of Sec31 at Serines 527 and 799 increases its affinity to Sec23. (A) HEK293 cells were co-transfected with indicated FLAG-tagged Sec31 mutants and GFP-tagged Sec23. Sec23 was immunoprecipitated with anti-GFP beads and subjected to western blotting with anti-FLAG and anti-GFP antibodies. The “input 10% Sec31” is a western blot of 10% aliquots of total cell lysates. The normalized ratio of Sec31 bound to Sec23 was shown below. W985A is a known Sec23-binding defect mutant [52] . The most right lane is a negative control with GFP. The graph below the blots shows the average of the quantification of three independent experiments with SD. (B) FLAG-Sec31 and GFP-Sec13 (instead of GFP-Sec23) were co-expressed and their interactions detected by co-immunoprecipitation and western blotting following the procedures described in Figure 4A . The lane on right most is a negative control with GFP.
    Figure Legend Snippet: Dephosphorylation of Sec31 at Serines 527 and 799 increases its affinity to Sec23. (A) HEK293 cells were co-transfected with indicated FLAG-tagged Sec31 mutants and GFP-tagged Sec23. Sec23 was immunoprecipitated with anti-GFP beads and subjected to western blotting with anti-FLAG and anti-GFP antibodies. The “input 10% Sec31” is a western blot of 10% aliquots of total cell lysates. The normalized ratio of Sec31 bound to Sec23 was shown below. W985A is a known Sec23-binding defect mutant [52] . The most right lane is a negative control with GFP. The graph below the blots shows the average of the quantification of three independent experiments with SD. (B) FLAG-Sec31 and GFP-Sec13 (instead of GFP-Sec23) were co-expressed and their interactions detected by co-immunoprecipitation and western blotting following the procedures described in Figure 4A . The lane on right most is a negative control with GFP.

    Techniques Used: De-Phosphorylation Assay, Transfection, Immunoprecipitation, Western Blot, Binding Assay, Mutagenesis, Negative Control

    20) Product Images from "DNA Polymerase ι Interacts with Both the TRAF-like and UBL1-2 Domains of USP7"

    Article Title: DNA Polymerase ι Interacts with Both the TRAF-like and UBL1-2 Domains of USP7

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2020.166733

    Pol ι associates with the TRAF and UBL1-2 domains of USP7. (a) Schematic illustrating USP7 truncation mutants employed in this figure. (b) Immunoprecipitation of the indicated USP7 truncations from HEK293T cells co-expressing WT FLAG Pol ι. Eluent and WCL (input) were immunoblotted as indicated. * = Myc antibody proteins. (c) WT FLAG Pol ι was co-expressed in 293T cells with WT Myc USP7, or a mutant lacking 19 aa from the C-terminal tail (Δ 19 aa Tail). WCL was then prepared and immunoblotted as indicated. (d) Immunoprecipitation of the indicated USP7 truncations from 293T cells co-expressing WT FLAG Pol ι. Eluent and WCL (input) were immunoblotted as indicated. * = Myc antibody proteins.
    Figure Legend Snippet: Pol ι associates with the TRAF and UBL1-2 domains of USP7. (a) Schematic illustrating USP7 truncation mutants employed in this figure. (b) Immunoprecipitation of the indicated USP7 truncations from HEK293T cells co-expressing WT FLAG Pol ι. Eluent and WCL (input) were immunoblotted as indicated. * = Myc antibody proteins. (c) WT FLAG Pol ι was co-expressed in 293T cells with WT Myc USP7, or a mutant lacking 19 aa from the C-terminal tail (Δ 19 aa Tail). WCL was then prepared and immunoblotted as indicated. (d) Immunoprecipitation of the indicated USP7 truncations from 293T cells co-expressing WT FLAG Pol ι. Eluent and WCL (input) were immunoblotted as indicated. * = Myc antibody proteins.

    Techniques Used: Immunoprecipitation, Expressing, Mutagenesis

    21) Product Images from "The Autocatalytic Release of a Putative RNA Virus Transcription Factor from Its Polyprotein Precursor Involves Two Paralogous Papain-like Proteases That Cleave the Same Peptide Bond *"

    Article Title: The Autocatalytic Release of a Putative RNA Virus Transcription Factor from Its Polyprotein Precursor Involves Two Paralogous Papain-like Proteases That Cleave the Same Peptide Bond *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M104097200

    Detection of an ORF1a-encoded 87-kDa cleavage product in HCoV-infected cells. Metabolically labeled lysates from 3 × 10 5 mock-infected ( M ) ( lanes 1 and 3 ) or HCoV-infected ( I ) ( lanes 2 and 4 ) MRC-5 cells were analyzed by SDS-polyacrylamide gel electrophoresis in a 10–17% acrylamide gradient gel after immunoprecipitation with α-H2 antiserum ( lanes 3 and 4 ) or the corresponding preimmune serum ( lanes 1 and 2 ). Antiserum α-H2 recognizes the HCoV ORF1a-encoded amino acids 112–322. The cells were labeled from 7 to 9.5-h postinfection with 100 µCi of [ 35 S]methionine per ml. Sizes of molecular mass markers (CFA 626; Amersham Pharmacia Biotech) with masses in kilodaltons as well as the 230- and 87-kDa processing products, p230 and p87, respectively, are indicated.
    Figure Legend Snippet: Detection of an ORF1a-encoded 87-kDa cleavage product in HCoV-infected cells. Metabolically labeled lysates from 3 × 10 5 mock-infected ( M ) ( lanes 1 and 3 ) or HCoV-infected ( I ) ( lanes 2 and 4 ) MRC-5 cells were analyzed by SDS-polyacrylamide gel electrophoresis in a 10–17% acrylamide gradient gel after immunoprecipitation with α-H2 antiserum ( lanes 3 and 4 ) or the corresponding preimmune serum ( lanes 1 and 2 ). Antiserum α-H2 recognizes the HCoV ORF1a-encoded amino acids 112–322. The cells were labeled from 7 to 9.5-h postinfection with 100 µCi of [ 35 S]methionine per ml. Sizes of molecular mass markers (CFA 626; Amersham Pharmacia Biotech) with masses in kilodaltons as well as the 230- and 87-kDa processing products, p230 and p87, respectively, are indicated.

    Techniques Used: Infection, Metabolic Labelling, Labeling, Polyacrylamide Gel Electrophoresis, Immunoprecipitation

    22) Product Images from "Phosphorylation by Cak1 Regulates the C-Terminal Domain Kinase Ctk1 in Saccharomyces cerevisiae"

    Article Title: Phosphorylation by Cak1 Regulates the C-Terminal Domain Kinase Ctk1 in Saccharomyces cerevisiae

    Journal:

    doi: 10.1128/MCB.25.10.3906-3913.2005

    Thr-338 phosphorylation is required for Ctk1 activity. (A) Ctk1-HA was immunoprecipitated from yeast strains expressing vector (−HA), wild-type ( WT ) Ctk1, Ctk1 T338A , and Ctk1 D324A (lanes 1 to 4). The expression levels and efficiencies of immunoprecipitation
    Figure Legend Snippet: Thr-338 phosphorylation is required for Ctk1 activity. (A) Ctk1-HA was immunoprecipitated from yeast strains expressing vector (−HA), wild-type ( WT ) Ctk1, Ctk1 T338A , and Ctk1 D324A (lanes 1 to 4). The expression levels and efficiencies of immunoprecipitation

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

    23) Product Images from "Dissecting the Molecular Pathway Involved in PLK2 Kinase-mediated α-Synuclein-selective Autophagic Degradation *"

    Article Title: Dissecting the Molecular Pathway Involved in PLK2 Kinase-mediated α-Synuclein-selective Autophagic Degradation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.759373

    α-Syn N-terminal region was required for its PLK2-mediated phosphorylation and degradation. A , Western blot illustrating the expression levels of α-syn and PLK2 in the presence of SPAR, which disrupts the formation of α-syn/PLK2 protein complex. B , quantification of α-syn protein levels showing that SPAR overexpression suppresses PLK2-mediated α-syn elimination ( n = 3). C , quantification of PLK2 protein levels showing that SPAR overexpression induced a significant accumulation of PLK2 ( n = 3). D , Western blot analysis of the total α-syn and Ser(P)-129 protein levels 24 h post transfection in HEK-239T cells. Cells were transiently transfected with 0.5 μg of α-syn Δ2–11 or 1 μg of α-syn Δ2–60 and 0.5 μg of PLK2 plasmids, and the total protein fraction was collected directly in 1X Laemmli buffer. E , histograms representing the quantification of α-syn protein levels, normalized against the actin expression, and showing that the N-terminal truncation Δ2–60, but not Δ2–11, affects α-syn degradation ( n = 3). F , histograms representing the quantification of Ser(P)-129 levels after PLK2 overexpression, normalized against total α-syn protein expression ( n = 3). The results show that Δ2–60 truncation induced a significant reduction of α-syn phosphorylation levels compared with α-syn Δ2–11. G , co-immunoprecipitation ( IP ) of PLK2 with N-terminal truncated α-syn (Δ2–11 or Δ2–60) showing that the deletion of the entire N-terminal regions (Δ2–60) is sufficient to block PLK2 and α-syn protein-protein transfection. *, p
    Figure Legend Snippet: α-Syn N-terminal region was required for its PLK2-mediated phosphorylation and degradation. A , Western blot illustrating the expression levels of α-syn and PLK2 in the presence of SPAR, which disrupts the formation of α-syn/PLK2 protein complex. B , quantification of α-syn protein levels showing that SPAR overexpression suppresses PLK2-mediated α-syn elimination ( n = 3). C , quantification of PLK2 protein levels showing that SPAR overexpression induced a significant accumulation of PLK2 ( n = 3). D , Western blot analysis of the total α-syn and Ser(P)-129 protein levels 24 h post transfection in HEK-239T cells. Cells were transiently transfected with 0.5 μg of α-syn Δ2–11 or 1 μg of α-syn Δ2–60 and 0.5 μg of PLK2 plasmids, and the total protein fraction was collected directly in 1X Laemmli buffer. E , histograms representing the quantification of α-syn protein levels, normalized against the actin expression, and showing that the N-terminal truncation Δ2–60, but not Δ2–11, affects α-syn degradation ( n = 3). F , histograms representing the quantification of Ser(P)-129 levels after PLK2 overexpression, normalized against total α-syn protein expression ( n = 3). The results show that Δ2–60 truncation induced a significant reduction of α-syn phosphorylation levels compared with α-syn Δ2–11. G , co-immunoprecipitation ( IP ) of PLK2 with N-terminal truncated α-syn (Δ2–11 or Δ2–60) showing that the deletion of the entire N-terminal regions (Δ2–60) is sufficient to block PLK2 and α-syn protein-protein transfection. *, p

    Techniques Used: Western Blot, Expressing, Over Expression, Transfection, Immunoprecipitation, Blocking Assay

    24) Product Images from "Amplification of CRKL induces transformation and EGFR inhibitor resistance in human non small cell lung cancers"

    Article Title: Amplification of CRKL induces transformation and EGFR inhibitor resistance in human non small cell lung cancers

    Journal: Cancer discovery

    doi: 10.1158/2159-8290.CD-11-0046

    CRKL-induced cell transformation requires SOS1-RAS-RAF (A) Overexpression of CRKL increased RAS activity. The levels of GTP-bound RAS in AALE cells overexpressing a control vector or CRKL were measured by a pull-down assay followed by immunoblotting for RAS. Total RAS levels in total lysates were used as loading control. Positive and negative technical controls were obtained by incubating the total lysates with non-hydrolyzable analog of GTP (GTPγS) or GDP, respectively, before pull-down assays. (B) Overexpression of CRKL increased in vitro BRAF kinase activity. The BRAF proteins in AALE cells expressing indicated constructs were isolated by immunoprecipitation. The kinase activity was assessed by incubating with substrate proteins (MEK1). Immunoblots of phospho-MEK1 and BRAF proteins in the isolated BRAF immune complexes after kinase activity assay are shown. (C) Immunoblot of phospho-S338-RAF1 in AALE cell lines overexpressing wildtype or mutant CRKL. (D) Interaction between CRKL and SOS1 in AALE cells overexpressing CRKL. CRKL immune complexes were isolated followed by immunoblotting for SOS1 or CRKL proteins in AALE cells expressing indicated constructs. (E) CRKL-induced anchorage independent growth required SOS1-RAS-BRAF/RAF1 signaling. Left, Immunoblots of SOS1, KRAS, BRAF, RAF1 or ARAF proteins in CRKL-overexpressing AALE cell lines expressing a control shRNA targeting GFP or each gene-specific shRNA. ShRNAs that suppressed more than 50% of target protein levels were marked in red color. Right, Anchorage independent growth of AALE cells expressing indicated constructs. Colony number indicates colonies greater than 0.2 mm in diameter 4 weeks after plating. Data represent mean + s.d. of six replicate determinations from two independent experiments. * indicates p
    Figure Legend Snippet: CRKL-induced cell transformation requires SOS1-RAS-RAF (A) Overexpression of CRKL increased RAS activity. The levels of GTP-bound RAS in AALE cells overexpressing a control vector or CRKL were measured by a pull-down assay followed by immunoblotting for RAS. Total RAS levels in total lysates were used as loading control. Positive and negative technical controls were obtained by incubating the total lysates with non-hydrolyzable analog of GTP (GTPγS) or GDP, respectively, before pull-down assays. (B) Overexpression of CRKL increased in vitro BRAF kinase activity. The BRAF proteins in AALE cells expressing indicated constructs were isolated by immunoprecipitation. The kinase activity was assessed by incubating with substrate proteins (MEK1). Immunoblots of phospho-MEK1 and BRAF proteins in the isolated BRAF immune complexes after kinase activity assay are shown. (C) Immunoblot of phospho-S338-RAF1 in AALE cell lines overexpressing wildtype or mutant CRKL. (D) Interaction between CRKL and SOS1 in AALE cells overexpressing CRKL. CRKL immune complexes were isolated followed by immunoblotting for SOS1 or CRKL proteins in AALE cells expressing indicated constructs. (E) CRKL-induced anchorage independent growth required SOS1-RAS-BRAF/RAF1 signaling. Left, Immunoblots of SOS1, KRAS, BRAF, RAF1 or ARAF proteins in CRKL-overexpressing AALE cell lines expressing a control shRNA targeting GFP or each gene-specific shRNA. ShRNAs that suppressed more than 50% of target protein levels were marked in red color. Right, Anchorage independent growth of AALE cells expressing indicated constructs. Colony number indicates colonies greater than 0.2 mm in diameter 4 weeks after plating. Data represent mean + s.d. of six replicate determinations from two independent experiments. * indicates p

    Techniques Used: Transformation Assay, Over Expression, Activity Assay, Plasmid Preparation, Pull Down Assay, In Vitro, Expressing, Construct, Isolation, Immunoprecipitation, Western Blot, Kinase Assay, Mutagenesis, shRNA

    25) Product Images from "DNA Polymerase ι Interacts with Both the TRAF-like and UBL1-2 Domains of USP7"

    Article Title: DNA Polymerase ι Interacts with Both the TRAF-like and UBL1-2 Domains of USP7

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2020.166733

    Pol ι associates with the TRAF and UBL1-2 domains of USP7. (a) Schematic illustrating USP7 truncation mutants employed in this figure. (b) Immunoprecipitation of the indicated USP7 truncations from HEK293T cells co-expressing WT FLAG Pol ι. Eluent and WCL (input) were immunoblotted as indicated. * = Myc antibody proteins. (c) WT FLAG Pol ι was co-expressed in 293T cells with WT Myc USP7, or a mutant lacking 19 aa from the C-terminal tail (Δ 19 aa Tail). WCL was then prepared and immunoblotted as indicated. (d) Immunoprecipitation of the indicated USP7 truncations from 293T cells co-expressing WT FLAG Pol ι. Eluent and WCL (input) were immunoblotted as indicated. * = Myc antibody proteins.
    Figure Legend Snippet: Pol ι associates with the TRAF and UBL1-2 domains of USP7. (a) Schematic illustrating USP7 truncation mutants employed in this figure. (b) Immunoprecipitation of the indicated USP7 truncations from HEK293T cells co-expressing WT FLAG Pol ι. Eluent and WCL (input) were immunoblotted as indicated. * = Myc antibody proteins. (c) WT FLAG Pol ι was co-expressed in 293T cells with WT Myc USP7, or a mutant lacking 19 aa from the C-terminal tail (Δ 19 aa Tail). WCL was then prepared and immunoblotted as indicated. (d) Immunoprecipitation of the indicated USP7 truncations from 293T cells co-expressing WT FLAG Pol ι. Eluent and WCL (input) were immunoblotted as indicated. * = Myc antibody proteins.

    Techniques Used: Immunoprecipitation, Expressing, Mutagenesis

    26) Product Images from "The Syk Kinase Promotes Mammary Epithelial Integrity and Inhibits Breast Cancer Invasion by Stabilizing the E-Cadherin/Catenin Complex"

    Article Title: The Syk Kinase Promotes Mammary Epithelial Integrity and Inhibits Breast Cancer Invasion by Stabilizing the E-Cadherin/Catenin Complex

    Journal: Cancers

    doi: 10.3390/cancers11121974

    Exogenous Syk expression increases E-cadherin and catenin phosphorylation and interaction. ( a ) Representative image of MCF7 cells that stably express GFP-Syk. Scale bar: 200 μm. Whole cell lysates (WCL) of parental and GFP-Syk MCF7 cells were analyzed by Western blotting (WB) with anti-Syk, -E-Cdh, -α-, -β-, and -p120-Ctn antibodies. β-tubulin was used as loading control. ( b ) Protein lysates of nontransfected and GFP-Syk-expressing MCF7 cells were immunoprecipitated (IP) with anti-E-Cdh, -α- and -β-Ctn antibodies and analyzed by Western blotting to detect E-Cdh/Ctn phosphorylation at the indicated tyrosine residues. ( c ) Protein lysates of HEK293T cells transiently transfected or not with a FLAG-Syk plasmid were immunoprecipitated (IP) using anti-E-Cdh, -α- and -β-Ctn or -Syk antibodies and analyzed by Western blotting (WB). ( d – f ) E-cadherin and catenin interaction was evaluated by co-immunoprecipitation (IP) followed by Western blotting (WB) using the indicated antibodies against E-Cdh, α-, and β-Ctn and the different phosphorylated forms and protein lysates from parental MCF7 cells (d), nontransfected and GFP-Syk-expressing MCF7 cells (e), and HEK293T cells transiently transfected or not with a FLAG-Syk plasmid (f).
    Figure Legend Snippet: Exogenous Syk expression increases E-cadherin and catenin phosphorylation and interaction. ( a ) Representative image of MCF7 cells that stably express GFP-Syk. Scale bar: 200 μm. Whole cell lysates (WCL) of parental and GFP-Syk MCF7 cells were analyzed by Western blotting (WB) with anti-Syk, -E-Cdh, -α-, -β-, and -p120-Ctn antibodies. β-tubulin was used as loading control. ( b ) Protein lysates of nontransfected and GFP-Syk-expressing MCF7 cells were immunoprecipitated (IP) with anti-E-Cdh, -α- and -β-Ctn antibodies and analyzed by Western blotting to detect E-Cdh/Ctn phosphorylation at the indicated tyrosine residues. ( c ) Protein lysates of HEK293T cells transiently transfected or not with a FLAG-Syk plasmid were immunoprecipitated (IP) using anti-E-Cdh, -α- and -β-Ctn or -Syk antibodies and analyzed by Western blotting (WB). ( d – f ) E-cadherin and catenin interaction was evaluated by co-immunoprecipitation (IP) followed by Western blotting (WB) using the indicated antibodies against E-Cdh, α-, and β-Ctn and the different phosphorylated forms and protein lysates from parental MCF7 cells (d), nontransfected and GFP-Syk-expressing MCF7 cells (e), and HEK293T cells transiently transfected or not with a FLAG-Syk plasmid (f).

    Techniques Used: Expressing, Stable Transfection, Western Blot, Immunoprecipitation, Transfection, Plasmid Preparation

    27) Product Images from "ERG-associated protein with SET domain (ESET)-Oct4 interaction regulates pluripotency and represses the trophectoderm lineage"

    Article Title: ERG-associated protein with SET domain (ESET)-Oct4 interaction regulates pluripotency and represses the trophectoderm lineage

    Journal: Epigenetics & Chromatin

    doi: 10.1186/1756-8935-2-12

    ERG-associated protein with SET domain (ESET)-mediated histone 3 lysine 9 trimethylation (H3K9me3) represses Cdx2 in embryonic stem (ES) cells . (a) Chromatin immunoprecipitation (ChIP) primers C1 to C10 used to detect enrichment of H3K9me3 on Cdx2 promoter (left). Primers O1 of Oct4 promoter served as a negative control (right). The numbers below the bars indicate distance from transcription start site (TSS) in base pairs (bp). (b) Quantitative polymerase chain reaction (Q-PCR) analysis of the enrichment of H3K9me3 at different positions along Cdx2 promoter region relative to the Oct4 promoter region after normalising against H3 ChIP and IgG controls. Primers C4 and C6 were not suitable for Q-PCR analysis. Error bars, standard deviation (SD) of three technical replicates. (c) Carrier ChIP semiquantitative PCR analysis of H3K9me3 at the Cdx2 promoter and major satellite regions of fluorescence-activated cell sorting (FACS)-sorted Eset knockdown ES cells relative to control cells after normalising against their respective input and IgG controls. Primers C6 and C7 were not suitable for carrier ChIP analysis. Error bars, SD of three independent experiments. (d) Q-PCR analysis of the levels of H3K9me3 at region C1 and C2 of the Cdx2 promoter in Eset knockdown ES cells relative to control cells after normalising against their respective input and IgG controls. Error bars, SD of three independent experiments. (e) Q-PCR analysis of the enrichment of ESET on different positions along the Cdx2 promoter relative to the Oct4 promoter after normalising against their respective input and IgG controls. Error bars, SD of three technical replicates.
    Figure Legend Snippet: ERG-associated protein with SET domain (ESET)-mediated histone 3 lysine 9 trimethylation (H3K9me3) represses Cdx2 in embryonic stem (ES) cells . (a) Chromatin immunoprecipitation (ChIP) primers C1 to C10 used to detect enrichment of H3K9me3 on Cdx2 promoter (left). Primers O1 of Oct4 promoter served as a negative control (right). The numbers below the bars indicate distance from transcription start site (TSS) in base pairs (bp). (b) Quantitative polymerase chain reaction (Q-PCR) analysis of the enrichment of H3K9me3 at different positions along Cdx2 promoter region relative to the Oct4 promoter region after normalising against H3 ChIP and IgG controls. Primers C4 and C6 were not suitable for Q-PCR analysis. Error bars, standard deviation (SD) of three technical replicates. (c) Carrier ChIP semiquantitative PCR analysis of H3K9me3 at the Cdx2 promoter and major satellite regions of fluorescence-activated cell sorting (FACS)-sorted Eset knockdown ES cells relative to control cells after normalising against their respective input and IgG controls. Primers C6 and C7 were not suitable for carrier ChIP analysis. Error bars, SD of three independent experiments. (d) Q-PCR analysis of the levels of H3K9me3 at region C1 and C2 of the Cdx2 promoter in Eset knockdown ES cells relative to control cells after normalising against their respective input and IgG controls. Error bars, SD of three independent experiments. (e) Q-PCR analysis of the enrichment of ESET on different positions along the Cdx2 promoter relative to the Oct4 promoter after normalising against their respective input and IgG controls. Error bars, SD of three technical replicates.

    Techniques Used: Chromatin Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Standard Deviation, Fluorescence, FACS

    Oct4 and ERG-associated protein with SET domain (ESET) synergistically repress Cdx2 . (a) Quantitative polymerase chain reaction (Q-PCR) analysis of the enrichment of Oct4 on different positions along the Cdx2 promoter relative to the least enriched region (C3) after normalising against their respective input and IgG controls. Error bars, standard deviation (SD) of three technical replicates. (b) Western blot shows upregulation of Cdx2 and downregulation of ESET upon depletion of Oct4 at day 2 of tetracycline (Tc) treatment of ZHBTc4 embryonic stem (ES) cells. (c) Q-PCR analysis of the levels of Oct4 and ESET enrichment at region C7 of the Cdx2 promoter in ZHBTc4 ES cells treated with Tc for 1 day relative to untreated cells after normalising against their respective input. Error bars, SD of three independent experiments. (d) Carrier chromatin immunoprecipitation (ChIP) Q-PCR analysis of the levels of histone 3 lysine 9 trimethylation (H3K9me3) at region C1 and C2 of the Cdx2 promoter in ZHBTc4 ES cells treated with Tc for the indicated days relative to untreated cells after normalising against their respective input and IgG controls. Error bars, SD of three independent experiments. (e) ES cell lysates were immunoprecipitated (IP) with anti-ESET antibody (kind gift of HH Ng; see text) under mild conditions in digitonin-containing buffer and subjected to western blotting (WB) with the antibodies indicated. Rabbit IgG was used as a negative control.
    Figure Legend Snippet: Oct4 and ERG-associated protein with SET domain (ESET) synergistically repress Cdx2 . (a) Quantitative polymerase chain reaction (Q-PCR) analysis of the enrichment of Oct4 on different positions along the Cdx2 promoter relative to the least enriched region (C3) after normalising against their respective input and IgG controls. Error bars, standard deviation (SD) of three technical replicates. (b) Western blot shows upregulation of Cdx2 and downregulation of ESET upon depletion of Oct4 at day 2 of tetracycline (Tc) treatment of ZHBTc4 embryonic stem (ES) cells. (c) Q-PCR analysis of the levels of Oct4 and ESET enrichment at region C7 of the Cdx2 promoter in ZHBTc4 ES cells treated with Tc for 1 day relative to untreated cells after normalising against their respective input. Error bars, SD of three independent experiments. (d) Carrier chromatin immunoprecipitation (ChIP) Q-PCR analysis of the levels of histone 3 lysine 9 trimethylation (H3K9me3) at region C1 and C2 of the Cdx2 promoter in ZHBTc4 ES cells treated with Tc for the indicated days relative to untreated cells after normalising against their respective input and IgG controls. Error bars, SD of three independent experiments. (e) ES cell lysates were immunoprecipitated (IP) with anti-ESET antibody (kind gift of HH Ng; see text) under mild conditions in digitonin-containing buffer and subjected to western blotting (WB) with the antibodies indicated. Rabbit IgG was used as a negative control.

    Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Standard Deviation, Western Blot, Chromatin Immunoprecipitation, Immunoprecipitation, Negative Control

    28) Product Images from "Targeting YTHDF1 effectively re-sensitizes cisplatin-resistant colon cancer cells by modulating GLS-mediated glutamine metabolism"

    Article Title: Targeting YTHDF1 effectively re-sensitizes cisplatin-resistant colon cancer cells by modulating GLS-mediated glutamine metabolism

    Journal: Molecular Therapy Oncolytics

    doi: 10.1016/j.omto.2021.01.001

    YTHDF1 directly binds with GLS1 to promote its protein translation (A and B) LoVo and DLD-1 cells were transfected with control, wild-type (WT) YTHDF1, or YTH domain mutant YTHDF1 for 48 h (A), and glutamine uptake and GLS activity (B) were detected. (C) Predicted YTHDF1 binding motif on the 3′ UTR of GLS1 mRNA. (D) RNA immunoprecipitation (RIP) was performed in LoVo and DLD-1 cells using anti-IgG control or anti-YTHDF1 antibody. (E and F) GLS1 abundance in the immunoprecipitated fraction was measured by agarose gel electrophoresis and (E) qRT-PCR. (F) An RNA pull-down assay was performed in LoVo and DLD-1 cells. The biotin-labeled 3′ UTR of WT or binding motif mutant GLS1 was incubated with proteins extracted from cells. The YTHDF1 protein, which was pulled down by the GLS1 binding motif, was detected by western blot. β-Actin was used as a negative control. (G) LoVo and DLD-1 cells were transfected with control shRNA or YTHDF1 shRNA for 48 h, and RIP experiments were performed using an anti-IgG control or anti-YTHDF1 antibody. GLS1 and β-actin mRNA abundance levels in the immunoprecipitated fraction were measured by agarose gel electrophoresis and (H) qRT-PCR. (I and J) Reporter constructs containing the WT or the binding motif mutant (Mut) GLS1 3′ UTR were co-transfected with control shRNA or GLS1 shRNA into LoVo and DLD-1 cells. Luciferase activities were measured using a Dual-Luciferase reporter assay kit. (K) LoVo and DLD-1 cells were treated with 10 μg/mL CHX for 0 and 6 h, and the relative GLS1 protein expression was measured by western blot. (L) LoVo and DLD-1 cells were treated with 50 nM MG-132 for 0 and 6 h, and the relative GLS1 protein expression was measured by western blot. β-Actin was used as an internal control. Data are presented as mean ± SD. ∗p
    Figure Legend Snippet: YTHDF1 directly binds with GLS1 to promote its protein translation (A and B) LoVo and DLD-1 cells were transfected with control, wild-type (WT) YTHDF1, or YTH domain mutant YTHDF1 for 48 h (A), and glutamine uptake and GLS activity (B) were detected. (C) Predicted YTHDF1 binding motif on the 3′ UTR of GLS1 mRNA. (D) RNA immunoprecipitation (RIP) was performed in LoVo and DLD-1 cells using anti-IgG control or anti-YTHDF1 antibody. (E and F) GLS1 abundance in the immunoprecipitated fraction was measured by agarose gel electrophoresis and (E) qRT-PCR. (F) An RNA pull-down assay was performed in LoVo and DLD-1 cells. The biotin-labeled 3′ UTR of WT or binding motif mutant GLS1 was incubated with proteins extracted from cells. The YTHDF1 protein, which was pulled down by the GLS1 binding motif, was detected by western blot. β-Actin was used as a negative control. (G) LoVo and DLD-1 cells were transfected with control shRNA or YTHDF1 shRNA for 48 h, and RIP experiments were performed using an anti-IgG control or anti-YTHDF1 antibody. GLS1 and β-actin mRNA abundance levels in the immunoprecipitated fraction were measured by agarose gel electrophoresis and (H) qRT-PCR. (I and J) Reporter constructs containing the WT or the binding motif mutant (Mut) GLS1 3′ UTR were co-transfected with control shRNA or GLS1 shRNA into LoVo and DLD-1 cells. Luciferase activities were measured using a Dual-Luciferase reporter assay kit. (K) LoVo and DLD-1 cells were treated with 10 μg/mL CHX for 0 and 6 h, and the relative GLS1 protein expression was measured by western blot. (L) LoVo and DLD-1 cells were treated with 50 nM MG-132 for 0 and 6 h, and the relative GLS1 protein expression was measured by western blot. β-Actin was used as an internal control. Data are presented as mean ± SD. ∗p

    Techniques Used: Transfection, Mutagenesis, Activity Assay, Binding Assay, Immunoprecipitation, Agarose Gel Electrophoresis, Quantitative RT-PCR, Pull Down Assay, Labeling, Incubation, Western Blot, Negative Control, shRNA, Construct, Luciferase, Reporter Assay, Expressing

    29) Product Images from "Membrane-anchored ubiquitin ligase complex is required for the turnover of lysosomal membrane proteins"

    Article Title: Membrane-anchored ubiquitin ligase complex is required for the turnover of lysosomal membrane proteins

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201505062

    Biochemical evidence for the vacuolar localization of the Dsc complex and a model for the regulation of vacuolar membrane proteins. (A) Subcellular fractionation of WT yeast cells. The whole-cell lysate (T) was separated into P13, P100, and S100 fractions by differential centrifugation and probed with the indicated antibodies. (B) Purified vacuole membrane fraction was compared with the whole-cell lysate (total) by Western blot analysis. Samples that contain an equal amount of Vph1 were loaded in each lane. Approximately 60% of Ubx3 was estimated to localize to the vacuole membrane. (C) Immunoprecipitation experiments from the WT vacuole membrane fraction using preimmune, Dsc2, and Dsc3 antibodies. The immunoprecipitation reaction was probed with the indicated antibodies. (D) Immunoprecipitation experiment from the UBX3-FLAG vacuole membrane fraction using the M2 Flag antibody. The immunoprecipitation reaction was probed with indicated antibodies. (E) Silver staining analysis of the eluates from Fig. 7 D . (F) Two distinct E3 ligase systems converge on the vacuole membrane to regulate different vacuolar membrane transporters via the vReD pathway.
    Figure Legend Snippet: Biochemical evidence for the vacuolar localization of the Dsc complex and a model for the regulation of vacuolar membrane proteins. (A) Subcellular fractionation of WT yeast cells. The whole-cell lysate (T) was separated into P13, P100, and S100 fractions by differential centrifugation and probed with the indicated antibodies. (B) Purified vacuole membrane fraction was compared with the whole-cell lysate (total) by Western blot analysis. Samples that contain an equal amount of Vph1 were loaded in each lane. Approximately 60% of Ubx3 was estimated to localize to the vacuole membrane. (C) Immunoprecipitation experiments from the WT vacuole membrane fraction using preimmune, Dsc2, and Dsc3 antibodies. The immunoprecipitation reaction was probed with the indicated antibodies. (D) Immunoprecipitation experiment from the UBX3-FLAG vacuole membrane fraction using the M2 Flag antibody. The immunoprecipitation reaction was probed with indicated antibodies. (E) Silver staining analysis of the eluates from Fig. 7 D . (F) Two distinct E3 ligase systems converge on the vacuole membrane to regulate different vacuolar membrane transporters via the vReD pathway.

    Techniques Used: Fractionation, Centrifugation, Purification, Western Blot, Immunoprecipitation, Silver Staining

    30) Product Images from "Interactions between lysyl oxidases and ADAMTS proteins suggest a novel crosstalk between two extracellular matrix families"

    Article Title: Interactions between lysyl oxidases and ADAMTS proteins suggest a novel crosstalk between two extracellular matrix families

    Journal: Matrix biology : journal of the International Society for Matrix Biology

    doi: 10.1016/j.matbio.2018.05.003

    LOX forms a complex with ADAMTSL2 and ADAMTS10 A. Co-immunoprecipitation of 4 day conditioned medium from stably transfected HEK293 cells expressing LOX plus ADAMTSL2, ADAMTSL2 alone (negative control), LOX plus ADAMTS10, or ADAMTS10 alone (negative control). B. Co-immunoprecipitation of stably transfected HEK293 cells expressing LOX plus ADAMTSL2 or LOX plus ADAMTS10 or parental HEK293 lysate (negative control). LOX migrates as two bands at ~50 kDa. ADAMTSL2 at ~150 kDa, and ADAMTS10 at 150 kDa. Membranes (A,B) were incubated with anti-myc (top) and anti-V5 (bottom). C. Proximity ligation assays performed on HEK293 cells expressing LOX+ADAMTSL2 (I, I′ IV, IV′), LOX+ADAMTS10 (II, II′) and LOX+P85 (III, III′). Anti-LOX and anti-myc antibodies were used to target LOX and ADAMTSL2/ADAMTS10 or P85, respectively. Signal amplification, marked by red signal (top, I-IV) or shown in grayscale (bottom, I′–IV′) is observed only in cells expressing LOX and an ADAMTS protein. In panel IV (control), no primary antibodies we added.
    Figure Legend Snippet: LOX forms a complex with ADAMTSL2 and ADAMTS10 A. Co-immunoprecipitation of 4 day conditioned medium from stably transfected HEK293 cells expressing LOX plus ADAMTSL2, ADAMTSL2 alone (negative control), LOX plus ADAMTS10, or ADAMTS10 alone (negative control). B. Co-immunoprecipitation of stably transfected HEK293 cells expressing LOX plus ADAMTSL2 or LOX plus ADAMTS10 or parental HEK293 lysate (negative control). LOX migrates as two bands at ~50 kDa. ADAMTSL2 at ~150 kDa, and ADAMTS10 at 150 kDa. Membranes (A,B) were incubated with anti-myc (top) and anti-V5 (bottom). C. Proximity ligation assays performed on HEK293 cells expressing LOX+ADAMTSL2 (I, I′ IV, IV′), LOX+ADAMTS10 (II, II′) and LOX+P85 (III, III′). Anti-LOX and anti-myc antibodies were used to target LOX and ADAMTSL2/ADAMTS10 or P85, respectively. Signal amplification, marked by red signal (top, I-IV) or shown in grayscale (bottom, I′–IV′) is observed only in cells expressing LOX and an ADAMTS protein. In panel IV (control), no primary antibodies we added.

    Techniques Used: Immunoprecipitation, Stable Transfection, Transfection, Expressing, Negative Control, Incubation, Ligation, Amplification

    ADAMTSL2 and ADAMTS10 interact with LOXL2 and LOXL3 A,B. Co-immunoprecipitation from 4 day conditioned medium (A) or the cell lysate (B) from HEK293 cells stably transfected with ADAMTSL2 plus LOXL3 or LOXL3 alone. C . Co-immunoprecipitation of 4 day conditioned medium from stably transfected HEK293 cells expressing either LOXL2 plus ADAMTSL2, LOXL2 plus ADAMTS10, or LOXL2 alone.
    Figure Legend Snippet: ADAMTSL2 and ADAMTS10 interact with LOXL2 and LOXL3 A,B. Co-immunoprecipitation from 4 day conditioned medium (A) or the cell lysate (B) from HEK293 cells stably transfected with ADAMTSL2 plus LOXL3 or LOXL3 alone. C . Co-immunoprecipitation of 4 day conditioned medium from stably transfected HEK293 cells expressing either LOXL2 plus ADAMTSL2, LOXL2 plus ADAMTS10, or LOXL2 alone.

    Techniques Used: Immunoprecipitation, Stable Transfection, Transfection, Expressing

    LOX complexes with ADAMTSL4 A . A yeast-two-hybrid (Y2H) screen using LOX as the bait identified three distinct ADAMTSL4 clones (clones 1-3). The selective interaction domain (SID) indicates the consensus interacting region of ADAMTSL4 determined by the overlap of the clones. B . Diploid yeast cells containing both a bait vector (LOX) and prey vector (ADAMTSL4 clone 1) were grown on nutrient permissive or selective plates along with negative controls of empty bait vector, empty prey vector and empty bait and prey vectors. The left-hand panel represents yeast colonies grown on (-)Leu (-)Trp permissive medium to maintain the growth of yeast containing both vectors, while the right-hand panel shows a replicate of the same plate on selective medium. Note that only colonies expressing both LOX and ADAMTSL4 grow on selective medium demonstrating that the two proteins interact. C. Autoradiograph of 35 S-labeled proteins transcribed in the TNT system. Co-immunoprecipitation of lysates expressing Lox plus ADAMTSL4 or Lox plus P85. A band corresponding to Lox is observed slightly under 50 kDa (bottom panel), while that corresponding for ADAMTSL4 is slightly under 150 kDa (top panel). P85 has a molecular mass of 85 kDa. In the TNT reactions where both proteins were translated (as observed in the input lane, right) bands corresponding to both ADAMTSL4 and LOX are observed upon pull down of either one of the proteins. In contrast, P85, which was co-translated with LOX (right input lane), is not observed following LOX pulldown.
    Figure Legend Snippet: LOX complexes with ADAMTSL4 A . A yeast-two-hybrid (Y2H) screen using LOX as the bait identified three distinct ADAMTSL4 clones (clones 1-3). The selective interaction domain (SID) indicates the consensus interacting region of ADAMTSL4 determined by the overlap of the clones. B . Diploid yeast cells containing both a bait vector (LOX) and prey vector (ADAMTSL4 clone 1) were grown on nutrient permissive or selective plates along with negative controls of empty bait vector, empty prey vector and empty bait and prey vectors. The left-hand panel represents yeast colonies grown on (-)Leu (-)Trp permissive medium to maintain the growth of yeast containing both vectors, while the right-hand panel shows a replicate of the same plate on selective medium. Note that only colonies expressing both LOX and ADAMTSL4 grow on selective medium demonstrating that the two proteins interact. C. Autoradiograph of 35 S-labeled proteins transcribed in the TNT system. Co-immunoprecipitation of lysates expressing Lox plus ADAMTSL4 or Lox plus P85. A band corresponding to Lox is observed slightly under 50 kDa (bottom panel), while that corresponding for ADAMTSL4 is slightly under 150 kDa (top panel). P85 has a molecular mass of 85 kDa. In the TNT reactions where both proteins were translated (as observed in the input lane, right) bands corresponding to both ADAMTSL4 and LOX are observed upon pull down of either one of the proteins. In contrast, P85, which was co-translated with LOX (right input lane), is not observed following LOX pulldown.

    Techniques Used: Clone Assay, Plasmid Preparation, Expressing, Autoradiography, Labeling, Immunoprecipitation

    31) Product Images from "Excess Podocyte Semaphorin-3A Leads to Glomerular Disease Involving PlexinA1–Nephrin Interaction"

    Article Title: Excess Podocyte Semaphorin-3A Leads to Glomerular Disease Involving PlexinA1–Nephrin Interaction

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2013.06.022

    Sema3a signaling receptor plexinA 1 interacts with nephrin. A : Representative Western blots and quantitation indicate no significant changes in plexinA 1 or neuropilin 1 expression in whole kidneys after doxycycline induction. B : Immunofluorescence reveals colocalization of plexinA 1 and nephrin in cultured podocytes. C : Co-immunoprecipitation reveals association of endogenous plexinA 1 and FLAG-nephrin. Lane 1, cultured podocytes; lane 2, whole kidney. D : Reciprocal nephrin and plexinA 1 coimmunoprecipitation reveals nephrin–plexinA 1 interaction in transfected HEK cells. E and F : GST binding assay ( E ) indicates direct interaction of purified FLAG-plexinA 1 with nephrin cytoplasmic domain (GST-CD-nephrin, approximately 60 kDa; red arrowhead ); the GST-control is approximately 25 kDa ( black arrowhead ). Overlay assay ( F ) indicates that plexinA 1 –nephrin interaction is direct. Purified FLAG-plexinA 1 binds increasing amounts of GST-CD-nephrin blotted on cellulose membrane, as detected by FLAG immunoblotting; the GST Western blot confirms equal loading. Data are representative of at least three independent experiments. IP, immunoprecipitation; RS, rabbit serum; WB, Western blot; WCL, whole-cell lysate. Original magnification, ×400 ( B ).
    Figure Legend Snippet: Sema3a signaling receptor plexinA 1 interacts with nephrin. A : Representative Western blots and quantitation indicate no significant changes in plexinA 1 or neuropilin 1 expression in whole kidneys after doxycycline induction. B : Immunofluorescence reveals colocalization of plexinA 1 and nephrin in cultured podocytes. C : Co-immunoprecipitation reveals association of endogenous plexinA 1 and FLAG-nephrin. Lane 1, cultured podocytes; lane 2, whole kidney. D : Reciprocal nephrin and plexinA 1 coimmunoprecipitation reveals nephrin–plexinA 1 interaction in transfected HEK cells. E and F : GST binding assay ( E ) indicates direct interaction of purified FLAG-plexinA 1 with nephrin cytoplasmic domain (GST-CD-nephrin, approximately 60 kDa; red arrowhead ); the GST-control is approximately 25 kDa ( black arrowhead ). Overlay assay ( F ) indicates that plexinA 1 –nephrin interaction is direct. Purified FLAG-plexinA 1 binds increasing amounts of GST-CD-nephrin blotted on cellulose membrane, as detected by FLAG immunoblotting; the GST Western blot confirms equal loading. Data are representative of at least three independent experiments. IP, immunoprecipitation; RS, rabbit serum; WB, Western blot; WCL, whole-cell lysate. Original magnification, ×400 ( B ).

    Techniques Used: Western Blot, Quantitation Assay, Expressing, Immunofluorescence, Cell Culture, Immunoprecipitation, Transfection, Binding Assay, Purification, Overlay Assay

    32) Product Images from "Dissecting the Molecular Pathway Involved in PLK2 Kinase-mediated α-Synuclein-selective Autophagic Degradation *"

    Article Title: Dissecting the Molecular Pathway Involved in PLK2 Kinase-mediated α-Synuclein-selective Autophagic Degradation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.759373

    α-Syn N-terminal region was required for its PLK2-mediated phosphorylation and degradation. A , Western blot illustrating the expression levels of α-syn and PLK2 in the presence of SPAR, which disrupts the formation of α-syn/PLK2 protein complex. B , quantification of α-syn protein levels showing that SPAR overexpression suppresses PLK2-mediated α-syn elimination ( n = 3). C , quantification of PLK2 protein levels showing that SPAR overexpression induced a significant accumulation of PLK2 ( n = 3). D , Western blot analysis of the total α-syn and Ser(P)-129 protein levels 24 h post transfection in HEK-239T cells. Cells were transiently transfected with 0.5 μg of α-syn Δ2–11 or 1 μg of α-syn Δ2–60 and 0.5 μg of PLK2 plasmids, and the total protein fraction was collected directly in 1X Laemmli buffer. E , histograms representing the quantification of α-syn protein levels, normalized against the actin expression, and showing that the N-terminal truncation Δ2–60, but not Δ2–11, affects α-syn degradation ( n = 3). F , histograms representing the quantification of Ser(P)-129 levels after PLK2 overexpression, normalized against total α-syn protein expression ( n = 3). The results show that Δ2–60 truncation induced a significant reduction of α-syn phosphorylation levels compared with α-syn Δ2–11. G , co-immunoprecipitation ( IP ) of PLK2 with N-terminal truncated α-syn (Δ2–11 or Δ2–60) showing that the deletion of the entire N-terminal regions (Δ2–60) is sufficient to block PLK2 and α-syn protein-protein transfection. *, p
    Figure Legend Snippet: α-Syn N-terminal region was required for its PLK2-mediated phosphorylation and degradation. A , Western blot illustrating the expression levels of α-syn and PLK2 in the presence of SPAR, which disrupts the formation of α-syn/PLK2 protein complex. B , quantification of α-syn protein levels showing that SPAR overexpression suppresses PLK2-mediated α-syn elimination ( n = 3). C , quantification of PLK2 protein levels showing that SPAR overexpression induced a significant accumulation of PLK2 ( n = 3). D , Western blot analysis of the total α-syn and Ser(P)-129 protein levels 24 h post transfection in HEK-239T cells. Cells were transiently transfected with 0.5 μg of α-syn Δ2–11 or 1 μg of α-syn Δ2–60 and 0.5 μg of PLK2 plasmids, and the total protein fraction was collected directly in 1X Laemmli buffer. E , histograms representing the quantification of α-syn protein levels, normalized against the actin expression, and showing that the N-terminal truncation Δ2–60, but not Δ2–11, affects α-syn degradation ( n = 3). F , histograms representing the quantification of Ser(P)-129 levels after PLK2 overexpression, normalized against total α-syn protein expression ( n = 3). The results show that Δ2–60 truncation induced a significant reduction of α-syn phosphorylation levels compared with α-syn Δ2–11. G , co-immunoprecipitation ( IP ) of PLK2 with N-terminal truncated α-syn (Δ2–11 or Δ2–60) showing that the deletion of the entire N-terminal regions (Δ2–60) is sufficient to block PLK2 and α-syn protein-protein transfection. *, p

    Techniques Used: Western Blot, Expressing, Over Expression, Transfection, Immunoprecipitation, Blocking Assay

    33) Product Images from "Phosphorylated Human Keratinocyte Ornithine Decarboxylase Is Preferentially Associated with Insoluble Cellular Proteins"

    Article Title: Phosphorylated Human Keratinocyte Ornithine Decarboxylase Is Preferentially Associated with Insoluble Cellular Proteins

    Journal: Molecular Biology of the Cell

    doi:

    Immunoprecipitation of ODC from Subcellular Fractions
    Figure Legend Snippet: Immunoprecipitation of ODC from Subcellular Fractions

    Techniques Used: Immunoprecipitation

    34) Product Images from "Dietary apigenin potentiates the inhibitory effect of interferon-α on cancer cell viability through inhibition of 26S proteasome-mediated interferon receptor degradation"

    Article Title: Dietary apigenin potentiates the inhibitory effect of interferon-α on cancer cell viability through inhibition of 26S proteasome-mediated interferon receptor degradation

    Journal: Food & Nutrition Research

    doi: 10.3402/fnr.v60.31288

    Apigenin inhibits IFN-α-induced degradation of IFNAR1. (a) HEK293A cells were treated with 20 µM cycloheximde (CHX) for 2 h, and then with apigenin (1 µM, 10 µM, 25 µM) for 12 h before the treatment with IFN-α (1×10 4 U/mL) for an additional 2 h. The cell lysates were immunoblotted with anti-IFNAR1 antibody. GAPDH staining is shown as a loading control. (b) HeLa cells were transfected with pCMV-ubiquitin plasmid for 48 h and incubated with MG132 (20 µM) or apigenin (20 µM) for 12 h before treatment with IFN-α (1×10 4 U/mL) for another 2 h. Cell lysates were immunoprecipitated with the IFNAR1 antibody. Immunoblotting was performed using ubiquitin antibody. GAPDH antibody staining represents 5% of the total cell lysates used in immunoprecipitation. (c) HeLa cells were treated with 20 µM CHX, IFN-α (1×10 4 U/mL) or apigenin (20 µM) for the indicated time. The cell lysates were immunoblotted with anti-IFNAR1 antibodies. GAPDH staining is shown as a loading control.
    Figure Legend Snippet: Apigenin inhibits IFN-α-induced degradation of IFNAR1. (a) HEK293A cells were treated with 20 µM cycloheximde (CHX) for 2 h, and then with apigenin (1 µM, 10 µM, 25 µM) for 12 h before the treatment with IFN-α (1×10 4 U/mL) for an additional 2 h. The cell lysates were immunoblotted with anti-IFNAR1 antibody. GAPDH staining is shown as a loading control. (b) HeLa cells were transfected with pCMV-ubiquitin plasmid for 48 h and incubated with MG132 (20 µM) or apigenin (20 µM) for 12 h before treatment with IFN-α (1×10 4 U/mL) for another 2 h. Cell lysates were immunoprecipitated with the IFNAR1 antibody. Immunoblotting was performed using ubiquitin antibody. GAPDH antibody staining represents 5% of the total cell lysates used in immunoprecipitation. (c) HeLa cells were treated with 20 µM CHX, IFN-α (1×10 4 U/mL) or apigenin (20 µM) for the indicated time. The cell lysates were immunoblotted with anti-IFNAR1 antibodies. GAPDH staining is shown as a loading control.

    Techniques Used: Staining, Transfection, Plasmid Preparation, Incubation, Immunoprecipitation

    35) Product Images from "METTL3-mediated m6A modification of ZBTB4 mRNA is involved in the smoking-induced EMT in cancer of the lung"

    Article Title: METTL3-mediated m6A modification of ZBTB4 mRNA is involved in the smoking-induced EMT in cancer of the lung

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2020.12.001

    Silencing of ZBTB4 by a METTL3-m 6 A-YTHDF2-dependent mechanism T-HBE cells were transfected with METTL3 siRNA or siRNA con, pcD METTL3, or pcD con and YTHDF2 siRNA or siRNA con. (A) Methylated RNA immunoprecipitation (meRIP)-qPCR was applied to assess the m 6 A levels for ZBTB4 in CSE-HBE cells (mean ± SD, n = 3). (B) meRIP-qPCR was applied to assess the m 6 A levels for ZBTB4 in T-HBE cells (mean ± SD, n = 3). (C) Western blots were performed, and (D) relative protein levels (mean ± SD, n = 3) of METTL3 and ZBTB4 were determined. (E) Peaks indicating the relative abundance of m 6 A sites in ZBTB4 mRNA. (F) WT or m 6 A consensus sequence mutant ZBTB4 3′ UTR was fused with a firefly luciferase reporter. Mutations of m 6 A consensus sequences were generated by replacing A with G. (G) Relative activities of the WT and Mut luciferase reporters in T-HBE cells (mean ± SD, n = 3). (H) western blots were performed, and (I) relative protein levels (mean ± SD, n = 3) of YTHDF2 and ZBTB4 were determined. (J) RIP assays were performed in T-HBE cells to detect the direct binding between the ZBTB4 mRNA and YTHDF2 protein (mean ± SD, n = 3). (K) Relative activities of the WT and Mut luciferase reporters in T-HBE cells (mean ± SD, n = 3). ∗p
    Figure Legend Snippet: Silencing of ZBTB4 by a METTL3-m 6 A-YTHDF2-dependent mechanism T-HBE cells were transfected with METTL3 siRNA or siRNA con, pcD METTL3, or pcD con and YTHDF2 siRNA or siRNA con. (A) Methylated RNA immunoprecipitation (meRIP)-qPCR was applied to assess the m 6 A levels for ZBTB4 in CSE-HBE cells (mean ± SD, n = 3). (B) meRIP-qPCR was applied to assess the m 6 A levels for ZBTB4 in T-HBE cells (mean ± SD, n = 3). (C) Western blots were performed, and (D) relative protein levels (mean ± SD, n = 3) of METTL3 and ZBTB4 were determined. (E) Peaks indicating the relative abundance of m 6 A sites in ZBTB4 mRNA. (F) WT or m 6 A consensus sequence mutant ZBTB4 3′ UTR was fused with a firefly luciferase reporter. Mutations of m 6 A consensus sequences were generated by replacing A with G. (G) Relative activities of the WT and Mut luciferase reporters in T-HBE cells (mean ± SD, n = 3). (H) western blots were performed, and (I) relative protein levels (mean ± SD, n = 3) of YTHDF2 and ZBTB4 were determined. (J) RIP assays were performed in T-HBE cells to detect the direct binding between the ZBTB4 mRNA and YTHDF2 protein (mean ± SD, n = 3). (K) Relative activities of the WT and Mut luciferase reporters in T-HBE cells (mean ± SD, n = 3). ∗p

    Techniques Used: Transfection, Methylation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Western Blot, Sequencing, Mutagenesis, Luciferase, Generated, Binding Assay

    36) Product Images from "The novel cyclophilin-D-interacting protein FASTKD1 protects cells against oxidative stress-induced cell death"

    Article Title: The novel cyclophilin-D-interacting protein FASTKD1 protects cells against oxidative stress-induced cell death

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00471.2018

    FASTKD1 interacts with the mitochondrial protein cyclophilin-D (CypD) and localizes to mitochondria. A : neonatal rat ventricular myocytes (NRVMs) were infected with β-galactosidase (βGal), FASTKD1-Myc, or CypD-FLAG adenoviruses for 48 h, and the lysates were blotted for either Myc or FLAG. Actin was used as a loading control. B : immunocytochemistry performed on NRVMs infected with either βGal or FASTKD1-Myc adenoviruses and then stained for Myc tag. Mitotracker Red (Mito) was used to identify mitochondria. C : NRVMs were infected with βGal, FASTKD1-Myc, CypD-FLAG, or FASTKD1-Myc plus CypD FLAG adenoviruses for 48 h. The lysates were then subjected to immunoprecipitation (IP) using a FLAG antibody, and the resultant complexes were blotted for Myc and FLAG. D : mouse embryonic fibroblasts (MEFs) were infected with βGal or FASTKD1-Myc for 48 h, and the lysates were blotted for Myc. The mitochondrial phosphate carrier (PiC) was used as a loading control. E : immunocytochemistry performed on MEFs infected with either βGal or FASTKD1-Myc adenoviruses and then stained for Myc tag. Mitotracker Red was used to stain mitochondria. All images are representative of experiments done with 4 independent MEF and NRVM isolates. Cells of both sexes were used.
    Figure Legend Snippet: FASTKD1 interacts with the mitochondrial protein cyclophilin-D (CypD) and localizes to mitochondria. A : neonatal rat ventricular myocytes (NRVMs) were infected with β-galactosidase (βGal), FASTKD1-Myc, or CypD-FLAG adenoviruses for 48 h, and the lysates were blotted for either Myc or FLAG. Actin was used as a loading control. B : immunocytochemistry performed on NRVMs infected with either βGal or FASTKD1-Myc adenoviruses and then stained for Myc tag. Mitotracker Red (Mito) was used to identify mitochondria. C : NRVMs were infected with βGal, FASTKD1-Myc, CypD-FLAG, or FASTKD1-Myc plus CypD FLAG adenoviruses for 48 h. The lysates were then subjected to immunoprecipitation (IP) using a FLAG antibody, and the resultant complexes were blotted for Myc and FLAG. D : mouse embryonic fibroblasts (MEFs) were infected with βGal or FASTKD1-Myc for 48 h, and the lysates were blotted for Myc. The mitochondrial phosphate carrier (PiC) was used as a loading control. E : immunocytochemistry performed on MEFs infected with either βGal or FASTKD1-Myc adenoviruses and then stained for Myc tag. Mitotracker Red was used to stain mitochondria. All images are representative of experiments done with 4 independent MEF and NRVM isolates. Cells of both sexes were used.

    Techniques Used: Infection, Immunocytochemistry, Staining, Immunoprecipitation

    37) Product Images from "Syndecan‐4 influences mammalian myoblast proliferation by modulating myostatin signalling and G1/S transition"

    Article Title: Syndecan‐4 influences mammalian myoblast proliferation by modulating myostatin signalling and G1/S transition

    Journal: Febs Letters

    doi: 10.1002/1873-3468.13227

    Characterization of SDC 4–promyostatin interaction. (A) Co‐immunoprecipitations (co‐ IP s) were carried out with rabbit antiserum to the C‐terminal part of myostatin ( AB 3239) in un‐injured (control, day 0, d0) and injured (3 days after notexin injury, d3) soleus muscle homogenates. Different volumes (20 and 7 μL) of the eluted immunocomplex were loaded in case of the injured sample. The blots were reacted with antibodies to SDC 4 raised in goat. Myostatin co‐immunoprecipitated SDC 4 in both cases. Input lanes represent the total homogenates; 10% of the total protein amount used in co‐ IP was loaded. (B) Co‐ IP assays were performed with anti‐ SDC 4 antibodies, and the blots were reacted with anti‐myostatin antisera ( AB 3239). The negative control was incubated only with the secondary antibody. For the input lanes, 10% of the total protein amount used in co‐ IP was loaded. The additional band at ~ 42 kD a in d0 input can be a processing intermediate of promyostatin. (C) Heparan sulfate chains were digested in injured samples (d3; sample 1 and 2) with heparinase II enzyme following immunoprecipitation with goat anti‐ SDC 4 antibody. Different amounts (25 and 5 μL) of the eluted volume of the heparinase II digested immunoprecipitate of sample 1 were loaded. We could not detect promyostatin in the immunoprecipitate after heparinase digestion. For the input lanes, 7.5% of the total protein amount used in co‐ IP s were loaded. Note, that both promyostatin and mature myostatin were detected in the supernatant (digestion buffer) after heparinase digestion.
    Figure Legend Snippet: Characterization of SDC 4–promyostatin interaction. (A) Co‐immunoprecipitations (co‐ IP s) were carried out with rabbit antiserum to the C‐terminal part of myostatin ( AB 3239) in un‐injured (control, day 0, d0) and injured (3 days after notexin injury, d3) soleus muscle homogenates. Different volumes (20 and 7 μL) of the eluted immunocomplex were loaded in case of the injured sample. The blots were reacted with antibodies to SDC 4 raised in goat. Myostatin co‐immunoprecipitated SDC 4 in both cases. Input lanes represent the total homogenates; 10% of the total protein amount used in co‐ IP was loaded. (B) Co‐ IP assays were performed with anti‐ SDC 4 antibodies, and the blots were reacted with anti‐myostatin antisera ( AB 3239). The negative control was incubated only with the secondary antibody. For the input lanes, 10% of the total protein amount used in co‐ IP was loaded. The additional band at ~ 42 kD a in d0 input can be a processing intermediate of promyostatin. (C) Heparan sulfate chains were digested in injured samples (d3; sample 1 and 2) with heparinase II enzyme following immunoprecipitation with goat anti‐ SDC 4 antibody. Different amounts (25 and 5 μL) of the eluted volume of the heparinase II digested immunoprecipitate of sample 1 were loaded. We could not detect promyostatin in the immunoprecipitate after heparinase digestion. For the input lanes, 7.5% of the total protein amount used in co‐ IP s were loaded. Note, that both promyostatin and mature myostatin were detected in the supernatant (digestion buffer) after heparinase digestion.

    Techniques Used: Co-Immunoprecipitation Assay, Immunoprecipitation, Negative Control, Incubation

    38) Product Images from "Ankyrin Repeat Proteins of Orf Virus Influence the Cellular Hypoxia Response Pathway"

    Article Title: Ankyrin Repeat Proteins of Orf Virus Influence the Cellular Hypoxia Response Pathway

    Journal: Journal of Virology

    doi: 10.1128/JVI.01430-16

    ORFV ANK proteins interact with endogenous FIH in an ANK domain-dependent manner. (A) Immunoprecipitation assays of HeLa cells transiently transfected with the indicated Flag-tagged ORFV ANK protein or the vector-only control (N) and immunoprecipitated
    Figure Legend Snippet: ORFV ANK proteins interact with endogenous FIH in an ANK domain-dependent manner. (A) Immunoprecipitation assays of HeLa cells transiently transfected with the indicated Flag-tagged ORFV ANK protein or the vector-only control (N) and immunoprecipitated

    Techniques Used: Immunoprecipitation, Transfection, Plasmid Preparation

    39) Product Images from "Developmentally Regulated Recruitment of Transcription Factors and Chromatin Modification Activities to Chicken Lysozyme cis-Regulatory Elements In Vivo"

    Article Title: Developmentally Regulated Recruitment of Transcription Factors and Chromatin Modification Activities to Chicken Lysozyme cis-Regulatory Elements In Vivo

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.12.4386-4400.2003

    Chromatin immunoprecipitation assay examining transcription factor binding to the lysozyme 5′ regulatory region with antibodies against CBP in HD50 MEP, HD11 and LPS-treated HD11 cells as indicated. The relative enrichment was calculated by normalizing the PCR signals to that of the −10.07 kb primer and the β-actin control primer as described in the text. The data are plotted as mean value of at least two independent chromatin immunoprecipitation assays and three independent amplifications.
    Figure Legend Snippet: Chromatin immunoprecipitation assay examining transcription factor binding to the lysozyme 5′ regulatory region with antibodies against CBP in HD50 MEP, HD11 and LPS-treated HD11 cells as indicated. The relative enrichment was calculated by normalizing the PCR signals to that of the −10.07 kb primer and the β-actin control primer as described in the text. The data are plotted as mean value of at least two independent chromatin immunoprecipitation assays and three independent amplifications.

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction

    Chromatin immunoprecipitation assay examining transcription factor binding to the lysozyme 5′ regulatory region with antibodies against C/EBPβ (NF-M) in HD50 MEP, HD11, and LPS-treated HD11 cells as indicated. The relative enrichment was calculated by normalizing the PCR signals to that of the −10.07-kb primer and the β-actin control primer as described in the text. The data are plotted as mean value of at least two independent chromatin immunoprecipitation assays and three independent amplifications.
    Figure Legend Snippet: Chromatin immunoprecipitation assay examining transcription factor binding to the lysozyme 5′ regulatory region with antibodies against C/EBPβ (NF-M) in HD50 MEP, HD11, and LPS-treated HD11 cells as indicated. The relative enrichment was calculated by normalizing the PCR signals to that of the −10.07-kb primer and the β-actin control primer as described in the text. The data are plotted as mean value of at least two independent chromatin immunoprecipitation assays and three independent amplifications.

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction

    Chromatin immunoprecipitation assay examining transcription factor binding to the lysozyme 5′ regulatory region with antibodies against Fli-1 (A) or NF1 (B) in HD50 MEP, HD37, HD11, and LPS-treated HD11 cells. The relative enrichment was calculated by normalizing the PCR signals to that of the −10.07-kb primer and the β-actin control primer as described in the text. The hatched bars in the lower panel refer to a control experiment in HD50 MEP cells in which chromatin was not cross-linked. The data are plotted as mean value of at least two independent chromatin immunoprecipitation assays and three independent amplifications.
    Figure Legend Snippet: Chromatin immunoprecipitation assay examining transcription factor binding to the lysozyme 5′ regulatory region with antibodies against Fli-1 (A) or NF1 (B) in HD50 MEP, HD37, HD11, and LPS-treated HD11 cells. The relative enrichment was calculated by normalizing the PCR signals to that of the −10.07-kb primer and the β-actin control primer as described in the text. The hatched bars in the lower panel refer to a control experiment in HD50 MEP cells in which chromatin was not cross-linked. The data are plotted as mean value of at least two independent chromatin immunoprecipitation assays and three independent amplifications.

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction

    Chromatin immunoprecipitation assay examining the histone H3 modification status in HD37, HD50 MEP, HD11, and LPS-treated HD11 cells as indicated. (A) Chromatin immunoprecipitation assay with antibodies recognizing histone H3 lysine 9 acetylation. The relative enrichment was calculated by normalizing the PCR signals to that of a β-actin control primer as described in the text. Note therefore that the ratio of β-actin to −10.07-kb signal intensity is constant. (B) Chromatin immunoprecipitation assay with antibodies recognizing methylated histone H3 lysine 9. In this case the relative enrichment or depletion was calculated by normalizing the PCR signals to that of the −10.07-kb primer as described in the text. The levels of histone H3 methylation at the boundaries of the DNase I-sensitive domain at −10.07 kb in the different cell lines are the same, as determined by normalization to signals obtained with the β-actin primer. The β-actin primer yielded signals that were two- to threefold lower that those obtained with the −10.07-kb primer (data not shown). The data are plotted as mean value of at least two independent chromatin immunoprecipitation assays and three independent amplifications.
    Figure Legend Snippet: Chromatin immunoprecipitation assay examining the histone H3 modification status in HD37, HD50 MEP, HD11, and LPS-treated HD11 cells as indicated. (A) Chromatin immunoprecipitation assay with antibodies recognizing histone H3 lysine 9 acetylation. The relative enrichment was calculated by normalizing the PCR signals to that of a β-actin control primer as described in the text. Note therefore that the ratio of β-actin to −10.07-kb signal intensity is constant. (B) Chromatin immunoprecipitation assay with antibodies recognizing methylated histone H3 lysine 9. In this case the relative enrichment or depletion was calculated by normalizing the PCR signals to that of the −10.07-kb primer as described in the text. The levels of histone H3 methylation at the boundaries of the DNase I-sensitive domain at −10.07 kb in the different cell lines are the same, as determined by normalization to signals obtained with the β-actin primer. The β-actin primer yielded signals that were two- to threefold lower that those obtained with the −10.07-kb primer (data not shown). The data are plotted as mean value of at least two independent chromatin immunoprecipitation assays and three independent amplifications.

    Techniques Used: Chromatin Immunoprecipitation, Modification, Polymerase Chain Reaction, Methylation

    Related Articles

    Immunoprecipitation:

    Article Title: Olfactomedin 4 deficiency promotes prostate neoplastic progression and is associated with upregulation of the hedgehog-signaling pathway
    Article Snippet: For transient transfection, the cells (5 × 105 ) were plated in 6-well plates and transfection performed with pCMV-6-AC-GFP tag-vector or pCMV-6-OLFM4-GFP tag plasmids, using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s instructions. .. Coimmunoprecipitation assays For coimmunoprecipitation assays, PC-3 cells were harvested and lysed in immunoprecipitation lysis buffer . .. Culture media were harvested from 48-h cell cultures in T75 flasks.

    Article Title: The Poxviral RING Protein p28 Is a Ubiquitin Ligase That Targets Ubiquitin to Viral Replication Factories
    Article Snippet: To examine if p28 was ubiquitinated, we generated recombinant VV-WR expressing FLAG-p28, FLAG-M143R, HA-ubiquitin, or His-ubiquitin according to standard procedures ( ). .. CV-1 cells were infected with VV-FLAG-p28 or VV-FLAG-M143R for 16 h at an MOI of 5 in the presence or absence of VV-HA-ubiquitin, and FLAG-tagged p28 or M143R was immunoprecipitated using immunoprecipitation (RIPA) buffer and anti-FLAG antibody (M2; Sigma). .. Immunoblotting the precipitates with anti-FLAG antibody revealed, as expected, a predominant band in the 28-kDa range in cells infected with VV-FLAG-p28 and VV-FLAG-M143R but not in VV-HA-ubiquitin (Fig. ).

    Article Title: Arabidopsis ACINUS is O-glycosylated and regulates transcription and alternative splicing of regulators of reproductive transitions
    Article Snippet: Briefly, proteins were extracted in 10 mL of MOPS buffer (100 mmol/L MOPS, pH 7.6, 150 mmol/L NaCl, 1% (v/v) Triton X-100, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 2× Complete protease inhibitor cocktail, and PhosStop cocktail (Roche)), centrifuged, and filtered through two layers of Miracloth. .. The flow through was incubated with 20 µg of anti-AtACINUS antibody for 1 h at 4 °C, then 50 µL of protein A agarose beads were added and incubated for another hour, followed by four 2-min washes with immunoprecipitation buffer. ..

    Article Title: Long Non-Coding RNA MNX1-AS1 Promotes Progression of Triple Negative Breast Cancer by Enhancing Phosphorylation of Stat3
    Article Snippet: Magnetic beads were preincubated with 5 μg of IP-grade antibody for 30 min at room temperature with rotation. .. The supernatant was added to bead-antibody complexes in immunoprecipitation buffer and incubated at 4°C overnight. .. Finally, the RNA was purified and quantified by qRT-PCR.

    Article Title: Apolipoprotein L2 contains a BH3-like domain but it does not behave as a BH3-only protein
    Article Snippet: A total of 1400 μ g of cell extract were incubated overnight in 1 ml of immunoprecipitation buffer with the antibody-coupled beads. .. The next day, beads were washed five times with immunoprecipitation buffer and eluted with 60 μ l of immunoprecipitation buffer containing 2% SDS. ..

    Lysis:

    Article Title: Olfactomedin 4 deficiency promotes prostate neoplastic progression and is associated with upregulation of the hedgehog-signaling pathway
    Article Snippet: For transient transfection, the cells (5 × 105 ) were plated in 6-well plates and transfection performed with pCMV-6-AC-GFP tag-vector or pCMV-6-OLFM4-GFP tag plasmids, using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s instructions. .. Coimmunoprecipitation assays For coimmunoprecipitation assays, PC-3 cells were harvested and lysed in immunoprecipitation lysis buffer . .. Culture media were harvested from 48-h cell cultures in T75 flasks.

    Infection:

    Article Title: The Poxviral RING Protein p28 Is a Ubiquitin Ligase That Targets Ubiquitin to Viral Replication Factories
    Article Snippet: To examine if p28 was ubiquitinated, we generated recombinant VV-WR expressing FLAG-p28, FLAG-M143R, HA-ubiquitin, or His-ubiquitin according to standard procedures ( ). .. CV-1 cells were infected with VV-FLAG-p28 or VV-FLAG-M143R for 16 h at an MOI of 5 in the presence or absence of VV-HA-ubiquitin, and FLAG-tagged p28 or M143R was immunoprecipitated using immunoprecipitation (RIPA) buffer and anti-FLAG antibody (M2; Sigma). .. Immunoblotting the precipitates with anti-FLAG antibody revealed, as expected, a predominant band in the 28-kDa range in cells infected with VV-FLAG-p28 and VV-FLAG-M143R but not in VV-HA-ubiquitin (Fig. ).

    Incubation:

    Article Title: Arabidopsis ACINUS is O-glycosylated and regulates transcription and alternative splicing of regulators of reproductive transitions
    Article Snippet: Briefly, proteins were extracted in 10 mL of MOPS buffer (100 mmol/L MOPS, pH 7.6, 150 mmol/L NaCl, 1% (v/v) Triton X-100, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 2× Complete protease inhibitor cocktail, and PhosStop cocktail (Roche)), centrifuged, and filtered through two layers of Miracloth. .. The flow through was incubated with 20 µg of anti-AtACINUS antibody for 1 h at 4 °C, then 50 µL of protein A agarose beads were added and incubated for another hour, followed by four 2-min washes with immunoprecipitation buffer. ..

    Article Title: Long Non-Coding RNA MNX1-AS1 Promotes Progression of Triple Negative Breast Cancer by Enhancing Phosphorylation of Stat3
    Article Snippet: Magnetic beads were preincubated with 5 μg of IP-grade antibody for 30 min at room temperature with rotation. .. The supernatant was added to bead-antibody complexes in immunoprecipitation buffer and incubated at 4°C overnight. .. Finally, the RNA was purified and quantified by qRT-PCR.

    Radio Immunoprecipitation:

    Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1
    Article Snippet: Neddylation assay MCF7 cells plated on 15 cm dishes were transfected using FuGENE HD with 8 μ g pcDNA3-HA-NEDD8 from Addgene (Cambridge, MA, USA), 8 μ g pCMV6-KSR1 or 8 μ g pCMV6-vector constructs as indicated. .. After 24 h, cells were lysed in radio-immunoprecipitation assay (RIPA) buffer (Sigma Aldrich) supplemented with protease inhibitors. .. Total protein was quantified by the BCA assay (Pierce).

    Article Title: 3,4-Dihydroxyphenylacetaldehyde Is More Efficient than Dopamine in Oligomerizing and Quinonizing α-Synuclein
    Article Snippet: To assess Cu(II) effects on DOPAL-quinonized proteins in MO3.13 cells, after 24 hours of incubation with medium containing tolcapone the cells were treated with DOPAL (100 µ M) and Cu(II) (10 and 30 µ M) for 5 hours at 37°C in a CO2 incubator. .. The cells were then lysed with radioimmunoprecipitation assay buffer (Millipore, Temecula, CA) containing one tablet per 10 ml of cOmplete Mini Protease Inhibitor (Roche Diagnostics, Indianapolis, IN). ..

    Protease Inhibitor:

    Article Title: 3,4-Dihydroxyphenylacetaldehyde Is More Efficient than Dopamine in Oligomerizing and Quinonizing α-Synuclein
    Article Snippet: To assess Cu(II) effects on DOPAL-quinonized proteins in MO3.13 cells, after 24 hours of incubation with medium containing tolcapone the cells were treated with DOPAL (100 µ M) and Cu(II) (10 and 30 µ M) for 5 hours at 37°C in a CO2 incubator. .. The cells were then lysed with radioimmunoprecipitation assay buffer (Millipore, Temecula, CA) containing one tablet per 10 ml of cOmplete Mini Protease Inhibitor (Roche Diagnostics, Indianapolis, IN). ..

    Article Title: Extracellular vesicles from human airway basal cells respond to cigarette smoke extract and affect vascular endothelial cells
    Article Snippet: As a negative control, BEGM without BPE was incubated for 48 h in flasks without cells and then processed by means of the same procedure used to isolate EVs; the product of this was also tested for VEGFA by use of the same ELISA. .. The ELISA was performed both with and without exposure of the EVs and negative control to RIPA buffer (Sigma-Aldrich) with Complete Protease Inhibitor Cocktail (Roche, Mannheim, Germany), which was used to lyse the EVs. .. Analysis of VEGFR2 signaling protein activation in HUVECs In total, 1.5 × 105 HUVECs were seeded into a 12-well plate using an endothelial cell growth medium (Medium 199; Sigma-Aldrich) with 20% (v/v) FBS, 20 μg/mL endothelial cell growth supplement (Hallway), 1% (v/v) antibiotics (Hallway), and 20 units/mL heparin (Sigma-Aldrich).

    Enzyme-linked Immunosorbent Assay:

    Article Title: Extracellular vesicles from human airway basal cells respond to cigarette smoke extract and affect vascular endothelial cells
    Article Snippet: As a negative control, BEGM without BPE was incubated for 48 h in flasks without cells and then processed by means of the same procedure used to isolate EVs; the product of this was also tested for VEGFA by use of the same ELISA. .. The ELISA was performed both with and without exposure of the EVs and negative control to RIPA buffer (Sigma-Aldrich) with Complete Protease Inhibitor Cocktail (Roche, Mannheim, Germany), which was used to lyse the EVs. .. Analysis of VEGFR2 signaling protein activation in HUVECs In total, 1.5 × 105 HUVECs were seeded into a 12-well plate using an endothelial cell growth medium (Medium 199; Sigma-Aldrich) with 20% (v/v) FBS, 20 μg/mL endothelial cell growth supplement (Hallway), 1% (v/v) antibiotics (Hallway), and 20 units/mL heparin (Sigma-Aldrich).

    Negative Control:

    Article Title: Extracellular vesicles from human airway basal cells respond to cigarette smoke extract and affect vascular endothelial cells
    Article Snippet: As a negative control, BEGM without BPE was incubated for 48 h in flasks without cells and then processed by means of the same procedure used to isolate EVs; the product of this was also tested for VEGFA by use of the same ELISA. .. The ELISA was performed both with and without exposure of the EVs and negative control to RIPA buffer (Sigma-Aldrich) with Complete Protease Inhibitor Cocktail (Roche, Mannheim, Germany), which was used to lyse the EVs. .. Analysis of VEGFR2 signaling protein activation in HUVECs In total, 1.5 × 105 HUVECs were seeded into a 12-well plate using an endothelial cell growth medium (Medium 199; Sigma-Aldrich) with 20% (v/v) FBS, 20 μg/mL endothelial cell growth supplement (Hallway), 1% (v/v) antibiotics (Hallway), and 20 units/mL heparin (Sigma-Aldrich).

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  • 93
    Millipore anti sp1 antibody
    Ras overexpression increases association of RbAp46 with HDAC1 at the <t>SP1</t> site of RECK promoter in 7–4 cells. (A) The 7–4 cells were transiently transfected with plasmid DNA pcDNA-RbAp46 (0.2 μg) or RbAp46-specific siRNA (55.6 nM) in the presence of IPTG for 48 hr. After treatment, co-immunoprecipitation was conducted using anti-RbAp46, anti-HDAC1 antibodies, or anti-Sp1 antibody. (B) The cells were co-transfected with 0.2 μg of plasmid DNA of pGL3-RECK and pGL3-Sp1 mutant in the presence or absence of pcDNA-RbAp46 plasmid. RECK promoter activity was measured at 48 hr post-transfection. **: statistical significance at p
    Anti Sp1 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore co immunoprecipitation buffer
    Patch 1 and Patch 2 residues of the AP2-μ2 subunit that bind to PIP 2 lipid are not critical for the binding of the AP2 complex to CXCR2 A) Non-silencing (NS), HEK-293-μ2-KD cells and HEK-293-μ2-KD cells with transiently over-expressed AP2-μ2 mutants (P1, P1+P2, P1P2T and WT) were serum starved, stimulated with 100 ng /ml CXCL8 and cross-linked with DSP. The cells were lysed and a CXCR2 <t>co-immunoprecipitation</t> assay was performed. The CXCR2 associated proteins were eluted with 50 mM DTT and separated by 10%SDS-PAGE. The CXCR2 associated AP2 complex was probed with an anti-β2 antibody. Experiments were repeated 3 times and the mean band densities as normalized to co-immunoprecipitation with CXCR2 ±S. D. are shown. B) HA-AP2-μ2 associates with endogenous α2 subunit of AP-2. HEK-293-μ2-KD cells with transiently over-expressed HA-AP2-μ2 mutants (P1, P1+P2, T, P1P2T and WT), were lysed subjected to Western blot analysis for α2 and HA-μ2 subunits (upper panel). Each HA-tagged μ2 subunit was immunoprecipitated with anti-HA-agarose and blotted for endogenous α2 and for HA-μ2 (upper panel). A representative blot from 3 individual experiments is shown. C) Functional AP-2 complexes successfully incorporate transiently expressed HA-AP2-μ2 as shown by a reciprocal co-immunoprecipitation. A reciprocal co-immunoprecipitation of the endogenous AP2-β2 from 1.5 mg of total lysate shows that the functional AP-2 complexes contain both endogenous AP2-α2 and overexpressed HA-AP2-μ2. One thirtieth (1/30) of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. The top panel shows co-immunoprecipitation and the bottom panel shows the lysates. A representative blot from 2 individual experiments is shown.
    Co Immunoprecipitation Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore gst lysis buffer
    (A) Endogenous MYCN and <t>p53</t> co-IP. Nuclear extracts from the neuroblastoma cell line IMR-32 treated with Nutlin-3a were co-immunoprecipitated using anti-p53 (Ab-7) antibody or IgG (negative control). Western blots of immunoprecipitated proteins were performed using anti-p53 (DO-1), anti- MYCN (B8.4.B), or anti-Max (C-17) antibodies. (B) Endogenous MYC and p53 co-IP. HeLa cells treated with Nutlin-3a were co-immunoprecipitated using anti-p53 antibody or negative control IgG. Immunoprecipitated proteins were analyzed by Western blotting, using with anti-p53 (DO-1), anti- MYC (N262), and anti-MAX (C-17) antibodies. (C) in vitro <t>GST-C-MYC</t> pull-down. Crude nuclear protein extract from transient p53 over-expressing HEK-293T cells was incubated overnight with full-length GST-MYC or GST control proteins immobilized on glutathione-agarose beads. Pull-down samples were immunoblotted with the anti-p53 antibody. Membrane Ponceau S staining is shown as a loading control. (D) MYCN and p53 in vitro pull-down. Purified recombinant MYCN-6×His, GST-p53 (full length), and GST-control proteins were loaded as input samples. Recombinant MYCN- 6×His protein was incubated with GST-p53 or GST-control proteins immobilized on glutathione-agarose beads. GST proteins were pulled down and associated MYCN was detected by Western Blotting. Stain-Free total protein staining was used as a loading control. (E) Recombinant p53 and MYCN co-immunoprecipitation. The p53-null, non-small cell lung carcinoma cell line H-1299 was transiently transfected with plasmids overexpressing p53-GFP and MYCN-3×Flag. Crude nuclear protein extract collected from cells cultured under different transfection conditions were immunoprecipitated (IP) with either anti-p53 (Ab-7) or anti-FLAG (M2) antibody, and Western blots were performed using either anti-FLAG (M2) or anti-p53 (DO-1) antibody. (F) MYCN interacts with tetrameric form of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE2-(C) cells were incubated with GST alone and a series GST-p53 purified proteins: p53-WT (dimeric-tetrameric), p53-L344A (dimeric only) and p53-L344P (monomeric only). Input and pull-down samples were immunoblotted using anti-MYCN antibody and Ponceau staining was used as loading control.
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    Millipore mg132
    Proteasome inhibition rescues Siah1 overexpression phenotype. Confocal stacks of heat shocked (HS) Tg[ XOPS :GFP] (wildtype), Tg[ hsp70 :siah1]/Tg[ XOPS :GFP] (siah1), and Tg[ hsp70 :siah1ΔRING]/Tg[ XOPS :GFP], (siah1ΔRING) embryos treated with DMSO or <t>MG132</t> were collected analyzed in 3D for GFP fluorescence ( A-D ). Treatment with MG132 prevented a significant decrease in GFP+ rod cells compared to DMSO in siah1 HS embryos ( E ). Confocal stacks of heat shocked (HS) Tg[ TαC :GFP] (wildtype), Tg[ hsp70 :siah1]/Tg[ TαC :GFP] (siah1), and Tg[ hsp70 :siah1ΔRING]/Tg[ TαC :GFP], (siah1ΔRING) embryos treated with DMSO or MG132 were analyzed in 3D for GFP fluorescence ( F-I ). Treatment with MG132 prevented a significant decrease in GFP+ cone cells compared to DMSO in siah1 HS embryos ( J ). Scale bar = 50 μm.
    Mg132, 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
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    Image Search Results


    Ras overexpression increases association of RbAp46 with HDAC1 at the SP1 site of RECK promoter in 7–4 cells. (A) The 7–4 cells were transiently transfected with plasmid DNA pcDNA-RbAp46 (0.2 μg) or RbAp46-specific siRNA (55.6 nM) in the presence of IPTG for 48 hr. After treatment, co-immunoprecipitation was conducted using anti-RbAp46, anti-HDAC1 antibodies, or anti-Sp1 antibody. (B) The cells were co-transfected with 0.2 μg of plasmid DNA of pGL3-RECK and pGL3-Sp1 mutant in the presence or absence of pcDNA-RbAp46 plasmid. RECK promoter activity was measured at 48 hr post-transfection. **: statistical significance at p

    Journal: BMC Cancer

    Article Title: Ras induces experimental lung metastasis through up-regulation of RbAp46 to suppress RECK promoter activity

    doi: 10.1186/s12885-015-1155-7

    Figure Lengend Snippet: Ras overexpression increases association of RbAp46 with HDAC1 at the SP1 site of RECK promoter in 7–4 cells. (A) The 7–4 cells were transiently transfected with plasmid DNA pcDNA-RbAp46 (0.2 μg) or RbAp46-specific siRNA (55.6 nM) in the presence of IPTG for 48 hr. After treatment, co-immunoprecipitation was conducted using anti-RbAp46, anti-HDAC1 antibodies, or anti-Sp1 antibody. (B) The cells were co-transfected with 0.2 μg of plasmid DNA of pGL3-RECK and pGL3-Sp1 mutant in the presence or absence of pcDNA-RbAp46 plasmid. RECK promoter activity was measured at 48 hr post-transfection. **: statistical significance at p

    Article Snippet: After sonication, the resulting soluble chromatin was diluted 1:10 with ChIP dilution buffer and immunoprecipitated by anti-Sp1 antibody (Millipore, Billerica, MA, USA), anti-RbAp46 antibody (Abcam, Cambridge, MA, USA) or control IgG.

    Techniques: Over Expression, Transfection, Plasmid Preparation, Immunoprecipitation, Mutagenesis, Activity Assay

    A schematic hypothetical model shows that RbAp46 is a Ras up-regulated gene which participates in Ras-induced experimental lung metastasis through binding with HDAC1 and Sp1 to suppress RECK expression followed by MMP-9 activation and metastasis.

    Journal: BMC Cancer

    Article Title: Ras induces experimental lung metastasis through up-regulation of RbAp46 to suppress RECK promoter activity

    doi: 10.1186/s12885-015-1155-7

    Figure Lengend Snippet: A schematic hypothetical model shows that RbAp46 is a Ras up-regulated gene which participates in Ras-induced experimental lung metastasis through binding with HDAC1 and Sp1 to suppress RECK expression followed by MMP-9 activation and metastasis.

    Article Snippet: After sonication, the resulting soluble chromatin was diluted 1:10 with ChIP dilution buffer and immunoprecipitated by anti-Sp1 antibody (Millipore, Billerica, MA, USA), anti-RbAp46 antibody (Abcam, Cambridge, MA, USA) or control IgG.

    Techniques: Binding Assay, Expressing, Activation Assay

    Patch 1 and Patch 2 residues of the AP2-μ2 subunit that bind to PIP 2 lipid are not critical for the binding of the AP2 complex to CXCR2 A) Non-silencing (NS), HEK-293-μ2-KD cells and HEK-293-μ2-KD cells with transiently over-expressed AP2-μ2 mutants (P1, P1+P2, P1P2T and WT) were serum starved, stimulated with 100 ng /ml CXCL8 and cross-linked with DSP. The cells were lysed and a CXCR2 co-immunoprecipitation assay was performed. The CXCR2 associated proteins were eluted with 50 mM DTT and separated by 10%SDS-PAGE. The CXCR2 associated AP2 complex was probed with an anti-β2 antibody. Experiments were repeated 3 times and the mean band densities as normalized to co-immunoprecipitation with CXCR2 ±S. D. are shown. B) HA-AP2-μ2 associates with endogenous α2 subunit of AP-2. HEK-293-μ2-KD cells with transiently over-expressed HA-AP2-μ2 mutants (P1, P1+P2, T, P1P2T and WT), were lysed subjected to Western blot analysis for α2 and HA-μ2 subunits (upper panel). Each HA-tagged μ2 subunit was immunoprecipitated with anti-HA-agarose and blotted for endogenous α2 and for HA-μ2 (upper panel). A representative blot from 3 individual experiments is shown. C) Functional AP-2 complexes successfully incorporate transiently expressed HA-AP2-μ2 as shown by a reciprocal co-immunoprecipitation. A reciprocal co-immunoprecipitation of the endogenous AP2-β2 from 1.5 mg of total lysate shows that the functional AP-2 complexes contain both endogenous AP2-α2 and overexpressed HA-AP2-μ2. One thirtieth (1/30) of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. The top panel shows co-immunoprecipitation and the bottom panel shows the lysates. A representative blot from 2 individual experiments is shown.

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Adaptor Protein2 (AP2) orchestrates CXCR2-mediated cell migration

    doi: 10.1111/tra.12154

    Figure Lengend Snippet: Patch 1 and Patch 2 residues of the AP2-μ2 subunit that bind to PIP 2 lipid are not critical for the binding of the AP2 complex to CXCR2 A) Non-silencing (NS), HEK-293-μ2-KD cells and HEK-293-μ2-KD cells with transiently over-expressed AP2-μ2 mutants (P1, P1+P2, P1P2T and WT) were serum starved, stimulated with 100 ng /ml CXCL8 and cross-linked with DSP. The cells were lysed and a CXCR2 co-immunoprecipitation assay was performed. The CXCR2 associated proteins were eluted with 50 mM DTT and separated by 10%SDS-PAGE. The CXCR2 associated AP2 complex was probed with an anti-β2 antibody. Experiments were repeated 3 times and the mean band densities as normalized to co-immunoprecipitation with CXCR2 ±S. D. are shown. B) HA-AP2-μ2 associates with endogenous α2 subunit of AP-2. HEK-293-μ2-KD cells with transiently over-expressed HA-AP2-μ2 mutants (P1, P1+P2, T, P1P2T and WT), were lysed subjected to Western blot analysis for α2 and HA-μ2 subunits (upper panel). Each HA-tagged μ2 subunit was immunoprecipitated with anti-HA-agarose and blotted for endogenous α2 and for HA-μ2 (upper panel). A representative blot from 3 individual experiments is shown. C) Functional AP-2 complexes successfully incorporate transiently expressed HA-AP2-μ2 as shown by a reciprocal co-immunoprecipitation. A reciprocal co-immunoprecipitation of the endogenous AP2-β2 from 1.5 mg of total lysate shows that the functional AP-2 complexes contain both endogenous AP2-α2 and overexpressed HA-AP2-μ2. One thirtieth (1/30) of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. The top panel shows co-immunoprecipitation and the bottom panel shows the lysates. A representative blot from 2 individual experiments is shown.

    Article Snippet: The cells were lysed in ice-cold co-immunoprecipitation buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA) containing proteinase inhibitor cocktail I and phosphatase inhibitor cocktails 3 and 2 (Sigma/Aldrich, St. Louis, MO).

    Techniques: Binding Assay, Co-Immunoprecipitation Assay, SDS Page, Immunoprecipitation, Western Blot, Functional Assay

    AP2 is essential for CXCR2-mediated chemotaxis, but β-arrestin1 is dispensable A) Top panel: LLKIL motif in CTDs of human CXC chemokine receptors is conserved. The CTDs of CXCR2 (45 residues), CXCR1 (44 residues), CXCR3 (49 residues) and CXCR4 (47 residues) were aligned with CLUSTALW (1.83) multiple sequence alignment program. The LLKIL functional motif of CXCR2 and similar putative motifs in other CXC receptor CTDs are in bold. Also, the serine residues known to be phosphorylated in CXCR2 CTD in response to CXCL8 stimulation are in bold. Bottom panel: The mutations in CXCR2 important for binding of AP2 and β-arrestin are illustrated. B) Decreased association of CXCR2 mutants with AP2 and/or β-arrestin1 after stimulation with CXCL8. dHL-60 cells stably expressing CXCR2-WT, 4A or CXCR2-4A/IL mutants were stimulated with or without CXCL8. CXCR2 was immunoprecipitated with anti-CXCR2 antibody, and blotted for AP2-β2 subunit or β-arrestin1. The blot was stripped and re-blotted for CXCR2. The relative values of fold increase in response to CXCL8 stimulation for each cell line calculated from 3 independent experiments is shown under the western blots (fold ± S.E.M.). One tenth of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. C) CXCL8-mediated internalization of CXCR2 is abolished in 4A/IL mutant of CXCR2, but only partially attenuated in 4A-CXCR2 mutant. The internalization of CXCR2 was performed by following the internalization of 125 I-CXCL8 in dHL60-CXCR2 cells stably expressing CXCR2-WT, 4A or 4A/IL mutant. Error bars are S.E.M and the experiments were repeated 3 times with duplicates for each treatment. ANOVA: 2 min – 4A vs. 4A/IL, p

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Adaptor Protein2 (AP2) orchestrates CXCR2-mediated cell migration

    doi: 10.1111/tra.12154

    Figure Lengend Snippet: AP2 is essential for CXCR2-mediated chemotaxis, but β-arrestin1 is dispensable A) Top panel: LLKIL motif in CTDs of human CXC chemokine receptors is conserved. The CTDs of CXCR2 (45 residues), CXCR1 (44 residues), CXCR3 (49 residues) and CXCR4 (47 residues) were aligned with CLUSTALW (1.83) multiple sequence alignment program. The LLKIL functional motif of CXCR2 and similar putative motifs in other CXC receptor CTDs are in bold. Also, the serine residues known to be phosphorylated in CXCR2 CTD in response to CXCL8 stimulation are in bold. Bottom panel: The mutations in CXCR2 important for binding of AP2 and β-arrestin are illustrated. B) Decreased association of CXCR2 mutants with AP2 and/or β-arrestin1 after stimulation with CXCL8. dHL-60 cells stably expressing CXCR2-WT, 4A or CXCR2-4A/IL mutants were stimulated with or without CXCL8. CXCR2 was immunoprecipitated with anti-CXCR2 antibody, and blotted for AP2-β2 subunit or β-arrestin1. The blot was stripped and re-blotted for CXCR2. The relative values of fold increase in response to CXCL8 stimulation for each cell line calculated from 3 independent experiments is shown under the western blots (fold ± S.E.M.). One tenth of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. C) CXCL8-mediated internalization of CXCR2 is abolished in 4A/IL mutant of CXCR2, but only partially attenuated in 4A-CXCR2 mutant. The internalization of CXCR2 was performed by following the internalization of 125 I-CXCL8 in dHL60-CXCR2 cells stably expressing CXCR2-WT, 4A or 4A/IL mutant. Error bars are S.E.M and the experiments were repeated 3 times with duplicates for each treatment. ANOVA: 2 min – 4A vs. 4A/IL, p

    Article Snippet: The cells were lysed in ice-cold co-immunoprecipitation buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA) containing proteinase inhibitor cocktail I and phosphatase inhibitor cocktails 3 and 2 (Sigma/Aldrich, St. Louis, MO).

    Techniques: Chemotaxis Assay, Sequencing, Functional Assay, Binding Assay, Stable Transfection, Expressing, Immunoprecipitation, Western Blot, Mutagenesis

    (A) Endogenous MYCN and p53 co-IP. Nuclear extracts from the neuroblastoma cell line IMR-32 treated with Nutlin-3a were co-immunoprecipitated using anti-p53 (Ab-7) antibody or IgG (negative control). Western blots of immunoprecipitated proteins were performed using anti-p53 (DO-1), anti- MYCN (B8.4.B), or anti-Max (C-17) antibodies. (B) Endogenous MYC and p53 co-IP. HeLa cells treated with Nutlin-3a were co-immunoprecipitated using anti-p53 antibody or negative control IgG. Immunoprecipitated proteins were analyzed by Western blotting, using with anti-p53 (DO-1), anti- MYC (N262), and anti-MAX (C-17) antibodies. (C) in vitro GST-C-MYC pull-down. Crude nuclear protein extract from transient p53 over-expressing HEK-293T cells was incubated overnight with full-length GST-MYC or GST control proteins immobilized on glutathione-agarose beads. Pull-down samples were immunoblotted with the anti-p53 antibody. Membrane Ponceau S staining is shown as a loading control. (D) MYCN and p53 in vitro pull-down. Purified recombinant MYCN-6×His, GST-p53 (full length), and GST-control proteins were loaded as input samples. Recombinant MYCN- 6×His protein was incubated with GST-p53 or GST-control proteins immobilized on glutathione-agarose beads. GST proteins were pulled down and associated MYCN was detected by Western Blotting. Stain-Free total protein staining was used as a loading control. (E) Recombinant p53 and MYCN co-immunoprecipitation. The p53-null, non-small cell lung carcinoma cell line H-1299 was transiently transfected with plasmids overexpressing p53-GFP and MYCN-3×Flag. Crude nuclear protein extract collected from cells cultured under different transfection conditions were immunoprecipitated (IP) with either anti-p53 (Ab-7) or anti-FLAG (M2) antibody, and Western blots were performed using either anti-FLAG (M2) or anti-p53 (DO-1) antibody. (F) MYCN interacts with tetrameric form of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE2-(C) cells were incubated with GST alone and a series GST-p53 purified proteins: p53-WT (dimeric-tetrameric), p53-L344A (dimeric only) and p53-L344P (monomeric only). Input and pull-down samples were immunoblotted using anti-MYCN antibody and Ponceau staining was used as loading control.

    Journal: Oncotarget

    Article Title: MYCN acts as a direct co-regulator of p53 in MYCN amplified neuroblastoma

    doi: 10.18632/oncotarget.24859

    Figure Lengend Snippet: (A) Endogenous MYCN and p53 co-IP. Nuclear extracts from the neuroblastoma cell line IMR-32 treated with Nutlin-3a were co-immunoprecipitated using anti-p53 (Ab-7) antibody or IgG (negative control). Western blots of immunoprecipitated proteins were performed using anti-p53 (DO-1), anti- MYCN (B8.4.B), or anti-Max (C-17) antibodies. (B) Endogenous MYC and p53 co-IP. HeLa cells treated with Nutlin-3a were co-immunoprecipitated using anti-p53 antibody or negative control IgG. Immunoprecipitated proteins were analyzed by Western blotting, using with anti-p53 (DO-1), anti- MYC (N262), and anti-MAX (C-17) antibodies. (C) in vitro GST-C-MYC pull-down. Crude nuclear protein extract from transient p53 over-expressing HEK-293T cells was incubated overnight with full-length GST-MYC or GST control proteins immobilized on glutathione-agarose beads. Pull-down samples were immunoblotted with the anti-p53 antibody. Membrane Ponceau S staining is shown as a loading control. (D) MYCN and p53 in vitro pull-down. Purified recombinant MYCN-6×His, GST-p53 (full length), and GST-control proteins were loaded as input samples. Recombinant MYCN- 6×His protein was incubated with GST-p53 or GST-control proteins immobilized on glutathione-agarose beads. GST proteins were pulled down and associated MYCN was detected by Western Blotting. Stain-Free total protein staining was used as a loading control. (E) Recombinant p53 and MYCN co-immunoprecipitation. The p53-null, non-small cell lung carcinoma cell line H-1299 was transiently transfected with plasmids overexpressing p53-GFP and MYCN-3×Flag. Crude nuclear protein extract collected from cells cultured under different transfection conditions were immunoprecipitated (IP) with either anti-p53 (Ab-7) or anti-FLAG (M2) antibody, and Western blots were performed using either anti-FLAG (M2) or anti-p53 (DO-1) antibody. (F) MYCN interacts with tetrameric form of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE2-(C) cells were incubated with GST alone and a series GST-p53 purified proteins: p53-WT (dimeric-tetrameric), p53-L344A (dimeric only) and p53-L344P (monomeric only). Input and pull-down samples were immunoblotted using anti-MYCN antibody and Ponceau staining was used as loading control.

    Article Snippet: GST-p53 cells were lysed in GST lysis buffer (1% Triton, 1 μg/μl lysozyme, 0.5 mM EDTA, and 1 mM PMSF in phosphate buffered saline), purified and immobilized on glutathione-agarose beads (Sigma Aldrich).

    Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Negative Control, Western Blot, In Vitro, Expressing, Incubation, Staining, Purification, Recombinant, Transfection, Cell Culture, Amplification

    (A) Graphical representations of p53 and MYCN proteins. p53 (upper panel) and MYCN (lower panel) protein domains and truncation constructs. p53 protein domains: Trans Activation Domain (TAD), SRC Homology 3 domain (SH3), DNA binding domain, Nuclear Localization Signal (NLS), Tetramerization domain (TET), Regulatory domain (REG). MYCN protein domains: MYC boxes (MB), the basic region helix loop helix (BR-HLH), and the leucine zipper. The GST protein fragments are indicated with bars, and numbers refer to amino-acid positions. p53 and MYCN protein fragments were cloned in frame with the N-terminal GST in a pGEX-2T vector. GST-p53 and GST-MYCN fragments were cloned, expressed in BL-21 E.Coli strain and purified using gluthatione-agarose beads. (B) MYCN interacts with the C-terminus of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE-(2)-c cells were incubated with the different p53 truncations or GST alone (negative control) immobilized onto glutathione-agarose beads. Input and pull-down samples were immunoblotted using anti-MYCN and anti-MAX antibodies. Stain-Free total protein staining was used as the loading control. (C) GST pull-down assay of MYCN truncations. Crude nuclear protein extract from transiently transfected p53-overexpressing HEK-293T cells was incubated with different MYCN-GST fragments immobilized on glutathione-agarose beads. GST alone was used as a negative control. Input and pull-down samples were immunoblotted using anti-p53 (DO-1) antibody. Ponceau staining was used as a loading control.

    Journal: Oncotarget

    Article Title: MYCN acts as a direct co-regulator of p53 in MYCN amplified neuroblastoma

    doi: 10.18632/oncotarget.24859

    Figure Lengend Snippet: (A) Graphical representations of p53 and MYCN proteins. p53 (upper panel) and MYCN (lower panel) protein domains and truncation constructs. p53 protein domains: Trans Activation Domain (TAD), SRC Homology 3 domain (SH3), DNA binding domain, Nuclear Localization Signal (NLS), Tetramerization domain (TET), Regulatory domain (REG). MYCN protein domains: MYC boxes (MB), the basic region helix loop helix (BR-HLH), and the leucine zipper. The GST protein fragments are indicated with bars, and numbers refer to amino-acid positions. p53 and MYCN protein fragments were cloned in frame with the N-terminal GST in a pGEX-2T vector. GST-p53 and GST-MYCN fragments were cloned, expressed in BL-21 E.Coli strain and purified using gluthatione-agarose beads. (B) MYCN interacts with the C-terminus of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE-(2)-c cells were incubated with the different p53 truncations or GST alone (negative control) immobilized onto glutathione-agarose beads. Input and pull-down samples were immunoblotted using anti-MYCN and anti-MAX antibodies. Stain-Free total protein staining was used as the loading control. (C) GST pull-down assay of MYCN truncations. Crude nuclear protein extract from transiently transfected p53-overexpressing HEK-293T cells was incubated with different MYCN-GST fragments immobilized on glutathione-agarose beads. GST alone was used as a negative control. Input and pull-down samples were immunoblotted using anti-p53 (DO-1) antibody. Ponceau staining was used as a loading control.

    Article Snippet: GST-p53 cells were lysed in GST lysis buffer (1% Triton, 1 μg/μl lysozyme, 0.5 mM EDTA, and 1 mM PMSF in phosphate buffered saline), purified and immobilized on glutathione-agarose beads (Sigma Aldrich).

    Techniques: Construct, Activation Assay, Binding Assay, Clone Assay, Plasmid Preparation, Purification, Amplification, Incubation, Negative Control, Staining, Pull Down Assay, Transfection

    Proteasome inhibition rescues Siah1 overexpression phenotype. Confocal stacks of heat shocked (HS) Tg[ XOPS :GFP] (wildtype), Tg[ hsp70 :siah1]/Tg[ XOPS :GFP] (siah1), and Tg[ hsp70 :siah1ΔRING]/Tg[ XOPS :GFP], (siah1ΔRING) embryos treated with DMSO or MG132 were collected analyzed in 3D for GFP fluorescence ( A-D ). Treatment with MG132 prevented a significant decrease in GFP+ rod cells compared to DMSO in siah1 HS embryos ( E ). Confocal stacks of heat shocked (HS) Tg[ TαC :GFP] (wildtype), Tg[ hsp70 :siah1]/Tg[ TαC :GFP] (siah1), and Tg[ hsp70 :siah1ΔRING]/Tg[ TαC :GFP], (siah1ΔRING) embryos treated with DMSO or MG132 were analyzed in 3D for GFP fluorescence ( F-I ). Treatment with MG132 prevented a significant decrease in GFP+ cone cells compared to DMSO in siah1 HS embryos ( J ). Scale bar = 50 μm.

    Journal: bioRxiv

    Article Title: Proteasome-mediated regulation of Cdhr1a by Siah1 modulates photoreceptor development and survival in zebrafish

    doi: 10.1101/2020.05.15.098350

    Figure Lengend Snippet: Proteasome inhibition rescues Siah1 overexpression phenotype. Confocal stacks of heat shocked (HS) Tg[ XOPS :GFP] (wildtype), Tg[ hsp70 :siah1]/Tg[ XOPS :GFP] (siah1), and Tg[ hsp70 :siah1ΔRING]/Tg[ XOPS :GFP], (siah1ΔRING) embryos treated with DMSO or MG132 were collected analyzed in 3D for GFP fluorescence ( A-D ). Treatment with MG132 prevented a significant decrease in GFP+ rod cells compared to DMSO in siah1 HS embryos ( E ). Confocal stacks of heat shocked (HS) Tg[ TαC :GFP] (wildtype), Tg[ hsp70 :siah1]/Tg[ TαC :GFP] (siah1), and Tg[ hsp70 :siah1ΔRING]/Tg[ TαC :GFP], (siah1ΔRING) embryos treated with DMSO or MG132 were analyzed in 3D for GFP fluorescence ( F-I ). Treatment with MG132 prevented a significant decrease in GFP+ cone cells compared to DMSO in siah1 HS embryos ( J ). Scale bar = 50 μm.

    Article Snippet: MG132 treatment30 embryos were transferred at 52 hpf (3 hours post heat shock) into a 35 mm petri dish containing 5 mL E3 embryo media plus 12.5 mM MG132 (Sigma-Aldrich), or an equal volume of vehicle (DMSO) until 72 hpf.

    Techniques: Inhibition, Over Expression, Fluorescence

    Proteasome inhibition can rescue the decrease in rod photoreceptors and increased apoptosis resulting from Siah1 overactivation. Retinal cryosections from wildtype, Tg[ hsp70 :siah1] (siah1), and Tg[ hsp70 :siah1ΔRING] (siah1ΔRING) embryos heat shocked (HS) and treated with DMSO or MG132, were analyzed using IHC for cell death using TUNEL staining ( A-E ) as well as mature and immature rods using 4C12 antibodies ( G-K ). Number of TUNEL positive cells measured significantly higher in siah1 HS +DMSO embryos compared to all other treatments ( F ). Treatment with MG132 significantly decreased cell death in siah1 HS embryos compared to DMSO to an average comparable to wildtype ( F ). Numbers of mature and immature rod photoreceptors were significantly decreased in Siah1+ DMSO HS embryos treated with DMSO but not with MG132 ( L ). DNA was stained with DAPI (blue). Scale bar = 50μm. L: lens, ONL: outer nuclear layer, INL: Inner nuclear layer, GCL: ganglion cell layer, D: Dorsal and V: Ventral.

    Journal: bioRxiv

    Article Title: Proteasome-mediated regulation of Cdhr1a by Siah1 modulates photoreceptor development and survival in zebrafish

    doi: 10.1101/2020.05.15.098350

    Figure Lengend Snippet: Proteasome inhibition can rescue the decrease in rod photoreceptors and increased apoptosis resulting from Siah1 overactivation. Retinal cryosections from wildtype, Tg[ hsp70 :siah1] (siah1), and Tg[ hsp70 :siah1ΔRING] (siah1ΔRING) embryos heat shocked (HS) and treated with DMSO or MG132, were analyzed using IHC for cell death using TUNEL staining ( A-E ) as well as mature and immature rods using 4C12 antibodies ( G-K ). Number of TUNEL positive cells measured significantly higher in siah1 HS +DMSO embryos compared to all other treatments ( F ). Treatment with MG132 significantly decreased cell death in siah1 HS embryos compared to DMSO to an average comparable to wildtype ( F ). Numbers of mature and immature rod photoreceptors were significantly decreased in Siah1+ DMSO HS embryos treated with DMSO but not with MG132 ( L ). DNA was stained with DAPI (blue). Scale bar = 50μm. L: lens, ONL: outer nuclear layer, INL: Inner nuclear layer, GCL: ganglion cell layer, D: Dorsal and V: Ventral.

    Article Snippet: MG132 treatment30 embryos were transferred at 52 hpf (3 hours post heat shock) into a 35 mm petri dish containing 5 mL E3 embryo media plus 12.5 mM MG132 (Sigma-Aldrich), or an equal volume of vehicle (DMSO) until 72 hpf.

    Techniques: Inhibition, Immunohistochemistry, TUNEL Assay, Staining

    Siah1 targets Cdhr1a for proteasomal degradation. Alignment of CDHR1 degron motif sequence from different vertebrates: Xenopus, chicken, mouse, rat, human and zebrafish outlining overall protein sequence as well as motif conservation ( A ). Western blot analysis of cdhr1a protein stability in response to Siah activity. cdhr1a-FLAG signal is significantly decreased by co-transfection of siah1-myc, but not siah1ΔR-myc or upon MG132 treatment. Alpha/betta tubulin was used as a loading control ( B ). Co-immunoprecipitation of cdhr1a-FLAG co-transfected with siah1-myc or siah1ΔR-myc probed for FLAG (green), MYC (red). Cdhr1a-FLAG is able to pull down both siah1 and siah1ΔRING. ( C ). Western blot analysis of Siah1 targeting specificity. cdhr1a-FLAG signal is significantly decreased by co-transfection of siah1-myc. Signal of cdhr1a LMA -FLAG, a cdhr1a variant encoding a non-recognized degron motif, does not decrease upon co-transfection of siah1-myc. Alpha/betta tubulin was used as a loading control ( D ).

    Journal: bioRxiv

    Article Title: Proteasome-mediated regulation of Cdhr1a by Siah1 modulates photoreceptor development and survival in zebrafish

    doi: 10.1101/2020.05.15.098350

    Figure Lengend Snippet: Siah1 targets Cdhr1a for proteasomal degradation. Alignment of CDHR1 degron motif sequence from different vertebrates: Xenopus, chicken, mouse, rat, human and zebrafish outlining overall protein sequence as well as motif conservation ( A ). Western blot analysis of cdhr1a protein stability in response to Siah activity. cdhr1a-FLAG signal is significantly decreased by co-transfection of siah1-myc, but not siah1ΔR-myc or upon MG132 treatment. Alpha/betta tubulin was used as a loading control ( B ). Co-immunoprecipitation of cdhr1a-FLAG co-transfected with siah1-myc or siah1ΔR-myc probed for FLAG (green), MYC (red). Cdhr1a-FLAG is able to pull down both siah1 and siah1ΔRING. ( C ). Western blot analysis of Siah1 targeting specificity. cdhr1a-FLAG signal is significantly decreased by co-transfection of siah1-myc. Signal of cdhr1a LMA -FLAG, a cdhr1a variant encoding a non-recognized degron motif, does not decrease upon co-transfection of siah1-myc. Alpha/betta tubulin was used as a loading control ( D ).

    Article Snippet: MG132 treatment30 embryos were transferred at 52 hpf (3 hours post heat shock) into a 35 mm petri dish containing 5 mL E3 embryo media plus 12.5 mM MG132 (Sigma-Aldrich), or an equal volume of vehicle (DMSO) until 72 hpf.

    Techniques: Sequencing, Western Blot, Activity Assay, Cotransfection, Immunoprecipitation, Transfection, Variant Assay