caspase lysis buffer  (Roche)


Bioz Verified Symbol Roche is a verified supplier
Bioz Manufacturer Symbol Roche manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 88

    Structured Review

    Roche caspase lysis buffer
    Nutlin prevents <t>caspase</t> activation and γH2AX accumulation in response to Wee1 inhibitor and/or gemcitabine A . U2OS cells were treated with 8μM Nutlin for 24 hrs, followed by treatment with 1μM Wee1 inhibitor, 300nM gemcitabine, and/or 8μM Nutlin in the absence and presence of 50μM ZVAD-FMK for another 24 hrs. Cells were harvested and immunoblot analysis was performed to detect poly-ADP ribose polymerase (PARP) and γH2AX. B ., C . U2OS cells were treated as in (A). The cells were then fixed and stained for γH2AX by immunofluorescence. Detection and analysis was performed using automated immunofluorescence microscopy (BD Pathway). Figure panel (B) shows images of γH2AX staining for each treatment condition. Quantitation of γH2AX intensities was done using the BD pathway analysis tool and depicted in figure panel (C). Error bars represent the SD, n=3. D . U2OS cells were treated with 8μM Nutlin for 24 hrs, followed by treatment with 1μM Wee1 inhibitor, 300nM gemcitabine, 8μM Nutlin in the absence and presence ( Supplementary Figure 1 ) of 50μM ZVAD-FMK for another 24 hrs. The cells were harvested and lysed for caspase activity assay. Fluorescent intensity measurements were obtained for each treatment. The activity (arbitrary units of fluorescence/min) was calculated for each treatment at the linear part of the curve (cf. Supplementary Figure 1 ). Error bars represent the S.D, n=3.
    Caspase Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 5645 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/caspase lysis buffer/product/Roche
    Average 88 stars, based on 5645 article reviews
    Price from $9.99 to $1999.99
    caspase lysis buffer - by Bioz Stars, 2020-09
    88/100 stars

    Images

    1) Product Images from "Mdm2 inhibition confers protection of p53-proficient cells from the cytotoxic effects of Wee1 inhibitors"

    Article Title: Mdm2 inhibition confers protection of p53-proficient cells from the cytotoxic effects of Wee1 inhibitors

    Journal: Oncotarget

    doi:

    Nutlin prevents caspase activation and γH2AX accumulation in response to Wee1 inhibitor and/or gemcitabine A . U2OS cells were treated with 8μM Nutlin for 24 hrs, followed by treatment with 1μM Wee1 inhibitor, 300nM gemcitabine, and/or 8μM Nutlin in the absence and presence of 50μM ZVAD-FMK for another 24 hrs. Cells were harvested and immunoblot analysis was performed to detect poly-ADP ribose polymerase (PARP) and γH2AX. B ., C . U2OS cells were treated as in (A). The cells were then fixed and stained for γH2AX by immunofluorescence. Detection and analysis was performed using automated immunofluorescence microscopy (BD Pathway). Figure panel (B) shows images of γH2AX staining for each treatment condition. Quantitation of γH2AX intensities was done using the BD pathway analysis tool and depicted in figure panel (C). Error bars represent the SD, n=3. D . U2OS cells were treated with 8μM Nutlin for 24 hrs, followed by treatment with 1μM Wee1 inhibitor, 300nM gemcitabine, 8μM Nutlin in the absence and presence ( Supplementary Figure 1 ) of 50μM ZVAD-FMK for another 24 hrs. The cells were harvested and lysed for caspase activity assay. Fluorescent intensity measurements were obtained for each treatment. The activity (arbitrary units of fluorescence/min) was calculated for each treatment at the linear part of the curve (cf. Supplementary Figure 1 ). Error bars represent the S.D, n=3.
    Figure Legend Snippet: Nutlin prevents caspase activation and γH2AX accumulation in response to Wee1 inhibitor and/or gemcitabine A . U2OS cells were treated with 8μM Nutlin for 24 hrs, followed by treatment with 1μM Wee1 inhibitor, 300nM gemcitabine, and/or 8μM Nutlin in the absence and presence of 50μM ZVAD-FMK for another 24 hrs. Cells were harvested and immunoblot analysis was performed to detect poly-ADP ribose polymerase (PARP) and γH2AX. B ., C . U2OS cells were treated as in (A). The cells were then fixed and stained for γH2AX by immunofluorescence. Detection and analysis was performed using automated immunofluorescence microscopy (BD Pathway). Figure panel (B) shows images of γH2AX staining for each treatment condition. Quantitation of γH2AX intensities was done using the BD pathway analysis tool and depicted in figure panel (C). Error bars represent the SD, n=3. D . U2OS cells were treated with 8μM Nutlin for 24 hrs, followed by treatment with 1μM Wee1 inhibitor, 300nM gemcitabine, 8μM Nutlin in the absence and presence ( Supplementary Figure 1 ) of 50μM ZVAD-FMK for another 24 hrs. The cells were harvested and lysed for caspase activity assay. Fluorescent intensity measurements were obtained for each treatment. The activity (arbitrary units of fluorescence/min) was calculated for each treatment at the linear part of the curve (cf. Supplementary Figure 1 ). Error bars represent the S.D, n=3.

    Techniques Used: Activation Assay, Staining, Immunofluorescence, Microscopy, Quantitation Assay, Caspase Activity Assay, Activity Assay, Fluorescence

    2) Product Images from "LncRNA-SARCC suppresses renal cell carcinoma (RCC) progression via altering the androgen receptor(AR)/miRNA-143-3p signals"

    Article Title: LncRNA-SARCC suppresses renal cell carcinoma (RCC) progression via altering the androgen receptor(AR)/miRNA-143-3p signals

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2017.74

    LncRNA-SARCC is physically associated with and negatively correlated with AR. ( a ) Immunoblot of AR expression in a series of RCC cell lines or immortalized proximal tubule epithelial cell line from normal adult human kidney (HK2, A498, SW839, 769-P, ACHN, 786-O, OSRC-2, Caki-1 and Caki-2), with prostate cancer cell line C4-2 as positive control. ( b ) A schematic illustration of the procedure used to discover and define LncRNAs binding to AR in RCC tissues. ( c ) RIP assays for the potential LncRNA candidates endogenously associated with AR in SW839 cells. Total RNA was subjected to qRT-PCR assays. ( d ) Primary RCC and adjacent non-cancerous renal tissues were subjected to RNA FISH and analyzed by ultraviolet light excitation using a fluorescence microscope. ( e ) qRT-PCR for LncRNA-SARCC, GAPDH and U1 from RNA extracted from cytoplasmic and nuclear fractions. ( f ) SW839 and OSRC-2 cells were cross-linked with/without 4% paraformaldehyde before RIP assays were carried out. ( g ) RIP assay in 10 nM DHT-treated SW839 cells at the indicated time points. ( h ) RNAs corresponding to different fragments of LncRNA-SARCC were biotinylated and incubated with SW839 cell extracts, targeted with streptavidin beads and washed. Associated AR protein was detected by WB and compared between full length LncRNA-SARCC and its antisense RNA. ( i and j ) qRT-PCR assays (left panels) for the shRNA-SARCC mRNA level in stable SW839 ( i ) and OSRC-2 ( j ) cell clones. AR protein and luciferase level were measured by WB (middle panels) and luciferase reporter assay (right panels). ( k and l ) qRT-PCR assays for the oe-SARCC mRNA levels in stable SW839 ( k ) and OSRC-2 ( l ) cell clones. AR protein and luciferase level were measured by WB (middle panels) and luciferase reporter assay (right panels). ( m ) Immunofluorescence staining of AR. SW839-control and SW839-LncRNA-SARCC cells were hormone-starved for 3 days and the SW839-control cells treated with DMSO or 1 nM R1881, whereas the SW839-LncRNA-SARCC cells were treated with R1881 for 24 h before subjected to immunostaining using an anti-AR antibody. ( n ) SW839 cells expressing control shRNA or LncRNA-SARCC shRNA were treated with 20 mg/ml cycloheximide (CHX) for the indicated time periods and cell lysates analyzed by WB. ( o ) SW839 cells expressing mock or oe-LncRNA-SARCC were treated as in ( n ) and analyzed by WB. ( p ) SW839 cells expressing shRNA-control (−) or shRNA-SARCC (+) were cultured with/without 5 mM MG132 for 10 h and cell lysates analyzed by WB. ( q ) CoIP showing AR-Hsp90 protein interaction with the absence or presence of LncRNA-SARCC. AR, Hsp90 and LncRNA-SARCC were expressed in 293T cells through transient transfection followed by AR immunoprecipitation. AR and associated Hsp90 protein was detected by immunoblot analysis. Data shown are mean±S.D. ( n =3). * P
    Figure Legend Snippet: LncRNA-SARCC is physically associated with and negatively correlated with AR. ( a ) Immunoblot of AR expression in a series of RCC cell lines or immortalized proximal tubule epithelial cell line from normal adult human kidney (HK2, A498, SW839, 769-P, ACHN, 786-O, OSRC-2, Caki-1 and Caki-2), with prostate cancer cell line C4-2 as positive control. ( b ) A schematic illustration of the procedure used to discover and define LncRNAs binding to AR in RCC tissues. ( c ) RIP assays for the potential LncRNA candidates endogenously associated with AR in SW839 cells. Total RNA was subjected to qRT-PCR assays. ( d ) Primary RCC and adjacent non-cancerous renal tissues were subjected to RNA FISH and analyzed by ultraviolet light excitation using a fluorescence microscope. ( e ) qRT-PCR for LncRNA-SARCC, GAPDH and U1 from RNA extracted from cytoplasmic and nuclear fractions. ( f ) SW839 and OSRC-2 cells were cross-linked with/without 4% paraformaldehyde before RIP assays were carried out. ( g ) RIP assay in 10 nM DHT-treated SW839 cells at the indicated time points. ( h ) RNAs corresponding to different fragments of LncRNA-SARCC were biotinylated and incubated with SW839 cell extracts, targeted with streptavidin beads and washed. Associated AR protein was detected by WB and compared between full length LncRNA-SARCC and its antisense RNA. ( i and j ) qRT-PCR assays (left panels) for the shRNA-SARCC mRNA level in stable SW839 ( i ) and OSRC-2 ( j ) cell clones. AR protein and luciferase level were measured by WB (middle panels) and luciferase reporter assay (right panels). ( k and l ) qRT-PCR assays for the oe-SARCC mRNA levels in stable SW839 ( k ) and OSRC-2 ( l ) cell clones. AR protein and luciferase level were measured by WB (middle panels) and luciferase reporter assay (right panels). ( m ) Immunofluorescence staining of AR. SW839-control and SW839-LncRNA-SARCC cells were hormone-starved for 3 days and the SW839-control cells treated with DMSO or 1 nM R1881, whereas the SW839-LncRNA-SARCC cells were treated with R1881 for 24 h before subjected to immunostaining using an anti-AR antibody. ( n ) SW839 cells expressing control shRNA or LncRNA-SARCC shRNA were treated with 20 mg/ml cycloheximide (CHX) for the indicated time periods and cell lysates analyzed by WB. ( o ) SW839 cells expressing mock or oe-LncRNA-SARCC were treated as in ( n ) and analyzed by WB. ( p ) SW839 cells expressing shRNA-control (−) or shRNA-SARCC (+) were cultured with/without 5 mM MG132 for 10 h and cell lysates analyzed by WB. ( q ) CoIP showing AR-Hsp90 protein interaction with the absence or presence of LncRNA-SARCC. AR, Hsp90 and LncRNA-SARCC were expressed in 293T cells through transient transfection followed by AR immunoprecipitation. AR and associated Hsp90 protein was detected by immunoblot analysis. Data shown are mean±S.D. ( n =3). * P

    Techniques Used: Expressing, Positive Control, Binding Assay, Quantitative RT-PCR, Fluorescence In Situ Hybridization, Fluorescence, Microscopy, Incubation, Western Blot, shRNA, Clone Assay, Luciferase, Reporter Assay, Immunofluorescence, Staining, Immunostaining, Cell Culture, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation

    3) Product Images from "Effects of Inner Nuclear Membrane Proteins SUN1/UNC-84A and SUN2/UNC-84B on the Early Steps of HIV-1 Infection"

    Article Title: Effects of Inner Nuclear Membrane Proteins SUN1/UNC-84A and SUN2/UNC-84B on the Early Steps of HIV-1 Infection

    Journal: Journal of Virology

    doi: 10.1128/JVI.00463-17

    In vitro -synthesized HIV-1 CANC nanotubes capture SUN1 and SUN2 from cell lysates. (A) Cell lysates from 293T cells transfected with HALUC, HACPSF6, HASUN1, or HASUN2 were incubated with in vitro -synthesized CANC nanotubes before centrifugation through a sucrose cushion and analysis of supernatants and pellets by immunoblotting using HA- and CA-specific antibodies. I, input; S, supernatant; P, pellet. (B) In vitro -synthesized CANC complexes from RHPA were mixed with cell lysates and analyzed as described for the results shown in panel A.
    Figure Legend Snippet: In vitro -synthesized HIV-1 CANC nanotubes capture SUN1 and SUN2 from cell lysates. (A) Cell lysates from 293T cells transfected with HALUC, HACPSF6, HASUN1, or HASUN2 were incubated with in vitro -synthesized CANC nanotubes before centrifugation through a sucrose cushion and analysis of supernatants and pellets by immunoblotting using HA- and CA-specific antibodies. I, input; S, supernatant; P, pellet. (B) In vitro -synthesized CANC complexes from RHPA were mixed with cell lysates and analyzed as described for the results shown in panel A.

    Techniques Used: In Vitro, Synthesized, Transfection, Incubation, Centrifugation

    4) Product Images from "The Long Non-coding RNA lnc-DMP1 Regulates Dmp1 Expression Through H3K27Ac Modification"

    Article Title: The Long Non-coding RNA lnc-DMP1 Regulates Dmp1 Expression Through H3K27Ac Modification

    Journal: Frontiers in Genetics

    doi: 10.3389/fgene.2020.00233

    lnc-DMP1 induces the promoter activity by modulating the H3K27Ac enrichment of the Dmp1 promoter region in MC3T3-E1 cells. (A) Luciferase (LUC) reporter assay: the Dmp1 promoter region [2000 bp before transcription start site (TSS)] was cloned upstream of the firefly luciferase coding region. Their luciferase activities were tested in MC3T3-E1 co-transfected with CMV:Lnc-DMP1 (lnc-DMP1 OE), U6:sh-lnc-DMP1 (lnc-DMP1 RNAi) or empty vectors (Ctrl OE and Ctrl RNAi). The empty vector control was set to a value of 1. (B) Schematic of the potential H3K27Ac binding region in the promoter sequence of Dmp1 . The numbers indicated the nucleotide positions relative to their transcription start site (TSS), which was shown as + 1. (C) qRT-PCR detection of the indicated DNAs retrieved by H3K27Ac-specific antibody compared with immunoglobulin G (IgG) in the CHIP assay within MC3T3-E1. (D) DNA pull down experiment with MC3T3-E1 extract. Specific bands were identified by immunoblotting H3K27Ac. (E) The alteration of the enrichment of H3K27Ac in the promoter region of Dmp1 in lnc-DMP1 OE and RNAi cells. ** P
    Figure Legend Snippet: lnc-DMP1 induces the promoter activity by modulating the H3K27Ac enrichment of the Dmp1 promoter region in MC3T3-E1 cells. (A) Luciferase (LUC) reporter assay: the Dmp1 promoter region [2000 bp before transcription start site (TSS)] was cloned upstream of the firefly luciferase coding region. Their luciferase activities were tested in MC3T3-E1 co-transfected with CMV:Lnc-DMP1 (lnc-DMP1 OE), U6:sh-lnc-DMP1 (lnc-DMP1 RNAi) or empty vectors (Ctrl OE and Ctrl RNAi). The empty vector control was set to a value of 1. (B) Schematic of the potential H3K27Ac binding region in the promoter sequence of Dmp1 . The numbers indicated the nucleotide positions relative to their transcription start site (TSS), which was shown as + 1. (C) qRT-PCR detection of the indicated DNAs retrieved by H3K27Ac-specific antibody compared with immunoglobulin G (IgG) in the CHIP assay within MC3T3-E1. (D) DNA pull down experiment with MC3T3-E1 extract. Specific bands were identified by immunoblotting H3K27Ac. (E) The alteration of the enrichment of H3K27Ac in the promoter region of Dmp1 in lnc-DMP1 OE and RNAi cells. ** P

    Techniques Used: Activity Assay, Luciferase, Reporter Assay, Clone Assay, Transfection, Plasmid Preparation, Binding Assay, Sequencing, Quantitative RT-PCR, Chromatin Immunoprecipitation

    5) Product Images from "Mad2 inhibits the mitotic kinesin MKlp2"

    Article Title: Mad2 inhibits the mitotic kinesin MKlp2

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201003095

    MKlp2 is a novel binding partner of Mad2. (A, B, and D) Immunoblot analysis with the indicated antibodies, and 10% of the input is shown as total lysates. The positions of molecular mass markers (kilodaltons) are indicated. (A) Lysates of HEK293 cells (lanes 1–3) expressing the indicated Myc-Mad2 or HeLa (lanes 4–6) and HEK293 (lanes 7–9) cells coexpressing Myc-Mad2 with HA-MKlp2 were subjected to immunoprecipitation (IP) with antibodies to (α-) Myc or HA. (B) Lysates of HEK293 cells expressing HA-MKlp2 or the indicated mutants were subjected to immunoprecipitation with antibodies to HA. (C) Structural motifs of MKlp2 and alignment of the putative Mad2-binding sequences of MKlp2 with those of human Cdc20 and Mad1 in comparison with human MKlp1. The indicted sequences were aligned using ClustalW ( www.ebi.ac.uk/clustalw ). (D) Lysates of HEK293 cells expressing HA-MKlp2 or HA-MKlp2(871A4) were subjected to immunoprecipitation with antibodies to HA. (E) Autoradiography of in vitro–translated HA-MKlp2 proteins (left) and precipitates of GST pull-down analysis performed with the indicated proteins and either GST-Mad2 or GST (right). 10% of the input for total in vitro–translated product is shown.
    Figure Legend Snippet: MKlp2 is a novel binding partner of Mad2. (A, B, and D) Immunoblot analysis with the indicated antibodies, and 10% of the input is shown as total lysates. The positions of molecular mass markers (kilodaltons) are indicated. (A) Lysates of HEK293 cells (lanes 1–3) expressing the indicated Myc-Mad2 or HeLa (lanes 4–6) and HEK293 (lanes 7–9) cells coexpressing Myc-Mad2 with HA-MKlp2 were subjected to immunoprecipitation (IP) with antibodies to (α-) Myc or HA. (B) Lysates of HEK293 cells expressing HA-MKlp2 or the indicated mutants were subjected to immunoprecipitation with antibodies to HA. (C) Structural motifs of MKlp2 and alignment of the putative Mad2-binding sequences of MKlp2 with those of human Cdc20 and Mad1 in comparison with human MKlp1. The indicted sequences were aligned using ClustalW ( www.ebi.ac.uk/clustalw ). (D) Lysates of HEK293 cells expressing HA-MKlp2 or HA-MKlp2(871A4) were subjected to immunoprecipitation with antibodies to HA. (E) Autoradiography of in vitro–translated HA-MKlp2 proteins (left) and precipitates of GST pull-down analysis performed with the indicated proteins and either GST-Mad2 or GST (right). 10% of the input for total in vitro–translated product is shown.

    Techniques Used: Binding Assay, Expressing, Immunoprecipitation, Autoradiography, In Vitro

    Mad2 binding to MKlp2 depends on the mitotic checkpoint. Immunoblot analysis with the indicated antibodies, and 10% of the input is shown as total lysates. The positions of molecular mass markers (kilodaltons) are indicated. (A) Lysates of HEK293 cells expressing GST-MKlp1, GST-MKlp2, or together with Myc-Mad2 were subjected to GST pull-down analysis. (B) HeLa cells growing asynchronously were treated with or without nocodazole for 6 h. Cells were then lysed and subjected to immunoprecipitation (IP) with antibodies to Mad2. (C and D) HeLa cells transfected with control or Mad1 siRNAs or two different BubR1 siRNAs (#1 and #2; to ensure reproducibility) were synchronously released from the G 1 –S boundary. 7 h after release, cells were collected by mitotic shake off. Total cell lysates were subjected to immunoprecipitation analysis with the indicated antibodies. (E) Lysates of HeLa cells harvested at the indicated times after release from nocodazole block were subjected to immunoprecipitation with antibodies to MKlp2. (F and G) 48 h after transfection with control or MKlp2 siRNAs, HeLa cells were treated with nocodazole for 5 h and subjected to immunoprecipitation with antibodies to Mad1 (F), Cdc27, a core component of the APC (G, lanes 4 and 5), or Cdc20 (G, lanes 7 and 8).
    Figure Legend Snippet: Mad2 binding to MKlp2 depends on the mitotic checkpoint. Immunoblot analysis with the indicated antibodies, and 10% of the input is shown as total lysates. The positions of molecular mass markers (kilodaltons) are indicated. (A) Lysates of HEK293 cells expressing GST-MKlp1, GST-MKlp2, or together with Myc-Mad2 were subjected to GST pull-down analysis. (B) HeLa cells growing asynchronously were treated with or without nocodazole for 6 h. Cells were then lysed and subjected to immunoprecipitation (IP) with antibodies to Mad2. (C and D) HeLa cells transfected with control or Mad1 siRNAs or two different BubR1 siRNAs (#1 and #2; to ensure reproducibility) were synchronously released from the G 1 –S boundary. 7 h after release, cells were collected by mitotic shake off. Total cell lysates were subjected to immunoprecipitation analysis with the indicated antibodies. (E) Lysates of HeLa cells harvested at the indicated times after release from nocodazole block were subjected to immunoprecipitation with antibodies to MKlp2. (F and G) 48 h after transfection with control or MKlp2 siRNAs, HeLa cells were treated with nocodazole for 5 h and subjected to immunoprecipitation with antibodies to Mad1 (F), Cdc27, a core component of the APC (G, lanes 4 and 5), or Cdc20 (G, lanes 7 and 8).

    Techniques Used: Binding Assay, Expressing, Immunoprecipitation, Transfection, Blocking Assay

    6) Product Images from "Effects of Inner Nuclear Membrane Proteins SUN1/UNC-84A and SUN2/UNC-84B on the Early Steps of HIV-1 Infection"

    Article Title: Effects of Inner Nuclear Membrane Proteins SUN1/UNC-84A and SUN2/UNC-84B on the Early Steps of HIV-1 Infection

    Journal: Journal of Virology

    doi: 10.1128/JVI.00463-17

    SUN1 inhibits HIV-1 and HIV-2 ROD , but not SIV mac , FIV, EIAV, or MoMLV infection. U87MG CD4/CXCR4 cells expressing HALUC or HASUN1 were infected with serial dilutions of the indicated VSV-G-pseudotyped GFP retroviral vector, and infectious titers (infectious units [i.u.] per milliliter) were determined. Fold changes of infectious titers (HASUN1/HALUC) were calculated for at least eight different doses of two biological replicates for each vector, and mean values with standard deviations are shown. A paired two-tailed t test was performed. *, P
    Figure Legend Snippet: SUN1 inhibits HIV-1 and HIV-2 ROD , but not SIV mac , FIV, EIAV, or MoMLV infection. U87MG CD4/CXCR4 cells expressing HALUC or HASUN1 were infected with serial dilutions of the indicated VSV-G-pseudotyped GFP retroviral vector, and infectious titers (infectious units [i.u.] per milliliter) were determined. Fold changes of infectious titers (HASUN1/HALUC) were calculated for at least eight different doses of two biological replicates for each vector, and mean values with standard deviations are shown. A paired two-tailed t test was performed. *, P

    Techniques Used: Infection, Expressing, Plasmid Preparation, Two Tailed Test

    SUN1 inhibits HIV-1 2-LTR circle formation. (A) U87MG CD4/CXCR4 cells expressing HALUC or HASUN1 were infected in the presence or absence of 5 μM efavirenz (+RTinh) with HIV-1 GFP LV ++ . Total DNA was extracted at the indicated time points, and parallel samples were used to determine percentages of infected cells by flow cytometry 2 days postinfection. Mean infectivities for three samples and standard deviations are shown. (B and C) Early reverse transcription products for GFP (B) or 2-LTR circles (C) were measured by qPCR and normalized to total DNA input. Mean copy numbers per 100 ng total DNA for three independent samples with standard deviations are shown. Statistical analysis was performed using a paired two-tailed t test. ns, not statistically significant; *, P
    Figure Legend Snippet: SUN1 inhibits HIV-1 2-LTR circle formation. (A) U87MG CD4/CXCR4 cells expressing HALUC or HASUN1 were infected in the presence or absence of 5 μM efavirenz (+RTinh) with HIV-1 GFP LV ++ . Total DNA was extracted at the indicated time points, and parallel samples were used to determine percentages of infected cells by flow cytometry 2 days postinfection. Mean infectivities for three samples and standard deviations are shown. (B and C) Early reverse transcription products for GFP (B) or 2-LTR circles (C) were measured by qPCR and normalized to total DNA input. Mean copy numbers per 100 ng total DNA for three independent samples with standard deviations are shown. Statistical analysis was performed using a paired two-tailed t test. ns, not statistically significant; *, P

    Techniques Used: Expressing, Infection, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Two Tailed Test

    Fusion of the amino-terminal SUN1 domain to Fv1 generates a potent HIV-1-inhibiting factor. (A) Cell lysates of U87MG CD4/CXCR4 cells expressing HALUC (negative control), HASUN1 (positive control), HAFv1 n , or a fusion protein of the amino-terminal 130 amino acids of SUN1 with Fv1 n were subjected to immunoblotting using an HA-specific antibody; MAPK served as the loading control. (B) Parallel cells (to those for which results are shown in panel A) were infected with serial dilutions of VSV-G-pseudotyped NL4.3GFP or BREGFP reporter viruses, and infectious titers were determined. Mean infectious titers (in infecious units [i.u.] per milliliter) with standard deviations were calculated from six independent viral doses of two biological replicates. Statistical analysis was performed using an unpaired two-tailed t test. ns, not statistically significant; **, P
    Figure Legend Snippet: Fusion of the amino-terminal SUN1 domain to Fv1 generates a potent HIV-1-inhibiting factor. (A) Cell lysates of U87MG CD4/CXCR4 cells expressing HALUC (negative control), HASUN1 (positive control), HAFv1 n , or a fusion protein of the amino-terminal 130 amino acids of SUN1 with Fv1 n were subjected to immunoblotting using an HA-specific antibody; MAPK served as the loading control. (B) Parallel cells (to those for which results are shown in panel A) were infected with serial dilutions of VSV-G-pseudotyped NL4.3GFP or BREGFP reporter viruses, and infectious titers were determined. Mean infectious titers (in infecious units [i.u.] per milliliter) with standard deviations were calculated from six independent viral doses of two biological replicates. Statistical analysis was performed using an unpaired two-tailed t test. ns, not statistically significant; **, P

    Techniques Used: Expressing, Negative Control, Positive Control, Infection, Two Tailed Test

    In vitro -synthesized HIV-1 CANC nanotubes capture SUN1 and SUN2 from cell lysates. (A) Cell lysates from 293T cells transfected with HALUC, HACPSF6, HASUN1, or HASUN2 were incubated with in vitro -synthesized CANC nanotubes before centrifugation through a sucrose cushion and analysis of supernatants and pellets by immunoblotting using HA- and CA-specific antibodies. I, input; S, supernatant; P, pellet. (B) In vitro -synthesized CANC complexes from RHPA were mixed with cell lysates and analyzed as described for the results shown in panel A.
    Figure Legend Snippet: In vitro -synthesized HIV-1 CANC nanotubes capture SUN1 and SUN2 from cell lysates. (A) Cell lysates from 293T cells transfected with HALUC, HACPSF6, HASUN1, or HASUN2 were incubated with in vitro -synthesized CANC nanotubes before centrifugation through a sucrose cushion and analysis of supernatants and pellets by immunoblotting using HA- and CA-specific antibodies. I, input; S, supernatant; P, pellet. (B) In vitro -synthesized CANC complexes from RHPA were mixed with cell lysates and analyzed as described for the results shown in panel A.

    Techniques Used: In Vitro, Synthesized, Transfection, Incubation, Centrifugation

    HIV-1 strains have differential sensitivities to the SUN1-induced block. (A) VSV-G-pseudotyped NL4.3GFP reporter virus or virus derived by cotransfecting NL4.3 with the GFP LV pCSGW was produced in parallel and titrated on U87MG CD4/CXCR4 cells expressing HALUC or HASUN1. Percentages of infected cells were determined 48 h later by flow cytometry. (B) U87MG CD4/CXCR4 cells expressing HALUC or HASUN1 were infected with the indicated VSV-G-pseudotyped virus produced by cotransfection with the HIV-1 GFP LV pCSGW, and infectious titers (in infectious units [i.u.] per milliliter) were determined from 10 viral doses of two independent biological replicates. (C) Presentation of the same data as shown in panel B, here with the fold changes (HASUN1/HALUC ratios) for mean titers with standard deviations. Statistical analysis was performed using an unpaired two-tailed t test. ns, not statistically significant; *, P
    Figure Legend Snippet: HIV-1 strains have differential sensitivities to the SUN1-induced block. (A) VSV-G-pseudotyped NL4.3GFP reporter virus or virus derived by cotransfecting NL4.3 with the GFP LV pCSGW was produced in parallel and titrated on U87MG CD4/CXCR4 cells expressing HALUC or HASUN1. Percentages of infected cells were determined 48 h later by flow cytometry. (B) U87MG CD4/CXCR4 cells expressing HALUC or HASUN1 were infected with the indicated VSV-G-pseudotyped virus produced by cotransfection with the HIV-1 GFP LV pCSGW, and infectious titers (in infectious units [i.u.] per milliliter) were determined from 10 viral doses of two independent biological replicates. (C) Presentation of the same data as shown in panel B, here with the fold changes (HASUN1/HALUC ratios) for mean titers with standard deviations. Statistical analysis was performed using an unpaired two-tailed t test. ns, not statistically significant; *, P

    Techniques Used: Blocking Assay, Derivative Assay, Produced, Expressing, Infection, Flow Cytometry, Cytometry, Cotransfection, Two Tailed Test

    Overexpression of SUN1 or SUN2 inhibits HIV-1 infection. (A) Nontransduced U87MG CD4/CXCR4 cells (n.t.) or cells expressing the indicated HA-tagged membrane-associated NE proteins or luciferase (LUC; negative control) were infected with serial dilutions of VSV-G-pseudotyped HIV-1 GFP LV, and the infectious units (i.u.) per ml of inoculum were calculated. Fold changes in mean infectious titers relative to HALUC control cells and standard deviations were determined from at least three viral doses from four independent biological repeats. Unpaired two-tailed t tests were performed. ns, not statistically significant; *, P
    Figure Legend Snippet: Overexpression of SUN1 or SUN2 inhibits HIV-1 infection. (A) Nontransduced U87MG CD4/CXCR4 cells (n.t.) or cells expressing the indicated HA-tagged membrane-associated NE proteins or luciferase (LUC; negative control) were infected with serial dilutions of VSV-G-pseudotyped HIV-1 GFP LV, and the infectious units (i.u.) per ml of inoculum were calculated. Fold changes in mean infectious titers relative to HALUC control cells and standard deviations were determined from at least three viral doses from four independent biological repeats. Unpaired two-tailed t tests were performed. ns, not statistically significant; *, P

    Techniques Used: Over Expression, Infection, Expressing, Luciferase, Negative Control, Two Tailed Test

    The amino-terminal domain of SUN1 is required for the block to HIV-1 and contains signatures of positive selection. (A) Schematic of analyzed SUN1 deletion mutants. (B) Immunoblot detection of HA-tagged proteins. Total protein measured by UV activation of the gel served as the loading control. (C) In parallel experiments to those for the samples shown in panel B, U87MG CD4/CXCR4 cells expressing HALUC, HASUN1, or the indicated SUN1 deletion mutants were infected with serial dilutions of VSV-G-pseudotyped NL4.3GFP, and infectious titers were determined. Mean fold changes (relative to untransduced control cells) of at least 10 infectious titers (in infectious units [i.u.] per milliliter) from three independent experiments and standard deviations are shown. Statistical analysis was performed using an unpaired two-tailed t test. ***, P
    Figure Legend Snippet: The amino-terminal domain of SUN1 is required for the block to HIV-1 and contains signatures of positive selection. (A) Schematic of analyzed SUN1 deletion mutants. (B) Immunoblot detection of HA-tagged proteins. Total protein measured by UV activation of the gel served as the loading control. (C) In parallel experiments to those for the samples shown in panel B, U87MG CD4/CXCR4 cells expressing HALUC, HASUN1, or the indicated SUN1 deletion mutants were infected with serial dilutions of VSV-G-pseudotyped NL4.3GFP, and infectious titers were determined. Mean fold changes (relative to untransduced control cells) of at least 10 infectious titers (in infectious units [i.u.] per milliliter) from three independent experiments and standard deviations are shown. Statistical analysis was performed using an unpaired two-tailed t test. ***, P

    Techniques Used: Blocking Assay, Selection, Activation Assay, Expressing, Infection, Two Tailed Test

    The determinant for the sensitivity to SUN1-induced inhibition maps to CA. (A) Schematic of the generation of chimeric viruses between T/F virus RHPA and NL4.3. (B) VSV-G-pseudotyped GFP reporter viruses were produced by cotransfection with the pCSGW vector, and U87MG CD4/CXCR4 cells were infected with serial dilutions. Infectious titers were determined from at least three different viral doses, and mean titers with standard deviations are shown. Representative results of three independent experiments are shown. Statistical analysis was performed using an unpaired two-tailed t test. ns, not statistically significant; **, P
    Figure Legend Snippet: The determinant for the sensitivity to SUN1-induced inhibition maps to CA. (A) Schematic of the generation of chimeric viruses between T/F virus RHPA and NL4.3. (B) VSV-G-pseudotyped GFP reporter viruses were produced by cotransfection with the pCSGW vector, and U87MG CD4/CXCR4 cells were infected with serial dilutions. Infectious titers were determined from at least three different viral doses, and mean titers with standard deviations are shown. Representative results of three independent experiments are shown. Statistical analysis was performed using an unpaired two-tailed t test. ns, not statistically significant; **, P

    Techniques Used: Inhibition, Produced, Cotransfection, Plasmid Preparation, Infection, Two Tailed Test

    Gene disruption of SUN2 but not SUN1 reduces infectivity of HIV-1 in THP-1 cells. (A) THP-1 CRISPR/Cas9 single-cell clones transduced to express specific guide RNAs against SUN1 or SUN2 were generated. Single-cell cloning followed by PCR-based sequencing across the guide RNA target sites identified two clones for each gene in which the open reading frame was disrupted. (B) Disruption of gene expression was verified by immunoblotting using SUN1- or SUN2-specific antibodies; Hsp90 served as the loading control. CRISPR/Cas9 control cells (Cntrl) expressed an unrelated guide RNA. (C) SUN1- or SUN2-depleted cells were infected with serial dilutions of VSV-G-pseudotyped NL4.3GFP or BREGFP reporter viruses, and infectious titers were determined. Shown are the fold changes (FC) in mean titers compared to control cells determined from at least 10 viral doses of four independent experiments, with standard deviations. Statistical analysis was performed using a paired two-tailed t test. ns, not statistically significant; *, P
    Figure Legend Snippet: Gene disruption of SUN2 but not SUN1 reduces infectivity of HIV-1 in THP-1 cells. (A) THP-1 CRISPR/Cas9 single-cell clones transduced to express specific guide RNAs against SUN1 or SUN2 were generated. Single-cell cloning followed by PCR-based sequencing across the guide RNA target sites identified two clones for each gene in which the open reading frame was disrupted. (B) Disruption of gene expression was verified by immunoblotting using SUN1- or SUN2-specific antibodies; Hsp90 served as the loading control. CRISPR/Cas9 control cells (Cntrl) expressed an unrelated guide RNA. (C) SUN1- or SUN2-depleted cells were infected with serial dilutions of VSV-G-pseudotyped NL4.3GFP or BREGFP reporter viruses, and infectious titers were determined. Shown are the fold changes (FC) in mean titers compared to control cells determined from at least 10 viral doses of four independent experiments, with standard deviations. Statistical analysis was performed using a paired two-tailed t test. ns, not statistically significant; *, P

    Techniques Used: Infection, CRISPR, Clone Assay, Generated, Polymerase Chain Reaction, Sequencing, Expressing, Two Tailed Test

    7) Product Images from "Differential phase partition of a PICS complex is required for piRNA processing and chromosome segregation in C. elegans"

    Article Title: Differential phase partition of a PICS complex is required for piRNA processing and chromosome segregation in C. elegans

    Journal: bioRxiv

    doi: 10.1101/463919

    TOFU-6, PID-1 and PICS-1 are required for 21U-RNA maturation. (A) Normalized 21U-RNA counts in N2, pid-1(ust64) and pics-1(tm2417) animals at late young adult stage. pics-1(tm2417) worms were isolated from balancers. Reads were normalized to total RNA reads. (B) Relative coverage of individual bases of 21U-RNA loci by reads from indicated animals. The annotated 5′ ends of 21U-RNA loci are at position 0. (C, D) Images of PID-1::GFP (C) and PICS-1::GFP (D) and P-granule marker mRuby::PGL-1 in germ cells. (E, F) Images of PID-1::GFP (E) and PICS-1::GFP (F) and histone marker mCherry::H2B after RNAi targeting tofu-6.
    Figure Legend Snippet: TOFU-6, PID-1 and PICS-1 are required for 21U-RNA maturation. (A) Normalized 21U-RNA counts in N2, pid-1(ust64) and pics-1(tm2417) animals at late young adult stage. pics-1(tm2417) worms were isolated from balancers. Reads were normalized to total RNA reads. (B) Relative coverage of individual bases of 21U-RNA loci by reads from indicated animals. The annotated 5′ ends of 21U-RNA loci are at position 0. (C, D) Images of PID-1::GFP (C) and PICS-1::GFP (D) and P-granule marker mRuby::PGL-1 in germ cells. (E, F) Images of PID-1::GFP (E) and PICS-1::GFP (F) and histone marker mCherry::H2B after RNAi targeting tofu-6.

    Techniques Used: Isolation, Marker

    TOFU-6, PICS-1, ERH-2, PID-1 and TOST-1 interact with each other both in vitro and in vivo . (A, B) Images of TOST-1::GFP (A) and ERH-2::GFP (B) and P-granule marker mRuby::PGL-1 in germ cells. (C, D, E, F) Western blotting of pulling down samples to assay protein-protein interactions between TOFU-6, PICS-1, ERH-2, PID-1 and TOST-1 in vitro. (G) Summary of the protein-protein interaction of TOFU-6, PICS-1, ERH-2, PID-1 and TOST-1 both in vitro and in vivo . Red lines indicate interactions in vitro . Blue arrows indicate interaction in vivo .
    Figure Legend Snippet: TOFU-6, PICS-1, ERH-2, PID-1 and TOST-1 interact with each other both in vitro and in vivo . (A, B) Images of TOST-1::GFP (A) and ERH-2::GFP (B) and P-granule marker mRuby::PGL-1 in germ cells. (C, D, E, F) Western blotting of pulling down samples to assay protein-protein interactions between TOFU-6, PICS-1, ERH-2, PID-1 and TOST-1 in vitro. (G) Summary of the protein-protein interaction of TOFU-6, PICS-1, ERH-2, PID-1 and TOST-1 both in vitro and in vivo . Red lines indicate interactions in vitro . Blue arrows indicate interaction in vivo .

    Techniques Used: In Vitro, In Vivo, Marker, Western Blot

    TOFU-6, PICS-1, ERH-2 and TOST-1 are required for chromosome segregation and cell division. (A) Images of GFP::H2B at meta-anaphase stage after RNAi targeting indicated genes in embryos. (B) Diagram displaying percentage of abnormal chromosome segregation. (C) Images of GFP::H2B in late embryos after RNAi targeting indicated genes.
    Figure Legend Snippet: TOFU-6, PICS-1, ERH-2 and TOST-1 are required for chromosome segregation and cell division. (A) Images of GFP::H2B at meta-anaphase stage after RNAi targeting indicated genes in embryos. (B) Diagram displaying percentage of abnormal chromosome segregation. (C) Images of GFP::H2B in late embryos after RNAi targeting indicated genes.

    Techniques Used:

    TOFU-6, PICS-1, ERH-2 and TOST-1 accumulate in nucleus during cell division. (A, B) Images of TOFU-6/PICS-1/ERH-2/TOST-1::GFP in early embryos. Diagram displaying percentage of TOFU-6/PICS-1/ERH-2/TOST-1::GFP accumulating in nucleus in two-cell-embryos (A) and four-cell-embryos (B). (C) Images of TOFU-6/PICS-1/ERH-2/TOST-1::GFP in two-cell-embryos after RNAi targeting indicated genes. Diagram displaying percentage of TOFU-6/PICS-1/ERH-2/TOST-1::GFP accumulating in nucleus after RNAi targeting indicated genes.
    Figure Legend Snippet: TOFU-6, PICS-1, ERH-2 and TOST-1 accumulate in nucleus during cell division. (A, B) Images of TOFU-6/PICS-1/ERH-2/TOST-1::GFP in early embryos. Diagram displaying percentage of TOFU-6/PICS-1/ERH-2/TOST-1::GFP accumulating in nucleus in two-cell-embryos (A) and four-cell-embryos (B). (C) Images of TOFU-6/PICS-1/ERH-2/TOST-1::GFP in two-cell-embryos after RNAi targeting indicated genes. Diagram displaying percentage of TOFU-6/PICS-1/ERH-2/TOST-1::GFP accumulating in nucleus after RNAi targeting indicated genes.

    Techniques Used:

    Identification of IFE-3 as an additional factor interacting with TOFU-6. (A) Western blotting of pull down samples to assay protein-protein interactions between IFE-3 and TOFU-6 in vitro. (B) Images of IFE-3 in germline cells. (C) A working model of the PICS complex in germline and embryos.
    Figure Legend Snippet: Identification of IFE-3 as an additional factor interacting with TOFU-6. (A) Western blotting of pull down samples to assay protein-protein interactions between IFE-3 and TOFU-6 in vitro. (B) Images of IFE-3 in germline cells. (C) A working model of the PICS complex in germline and embryos.

    Techniques Used: Western Blot, In Vitro

    Identification of TOFU-6 binding proteins by functional proteomics. (A) Summary of IP-MS followed by feeding RNAi experiments of TOFU-6::GFP. (B) Images of TOFU-6::GFP and P-granule marker mRuby::PGL-1 after RNAi targeting pid-1 and pics-1 . (C) Images of TOFU-6::GFP in indicated animals. (D) Images of TOFU-6::GFP after RNAi targeting tost-1 and erh-2 . (E) Western blotting of TOFU-6::GFP after RNAi targeting tost-1 and erh-2 .
    Figure Legend Snippet: Identification of TOFU-6 binding proteins by functional proteomics. (A) Summary of IP-MS followed by feeding RNAi experiments of TOFU-6::GFP. (B) Images of TOFU-6::GFP and P-granule marker mRuby::PGL-1 after RNAi targeting pid-1 and pics-1 . (C) Images of TOFU-6::GFP in indicated animals. (D) Images of TOFU-6::GFP after RNAi targeting tost-1 and erh-2 . (E) Western blotting of TOFU-6::GFP after RNAi targeting tost-1 and erh-2 .

    Techniques Used: Binding Assay, Functional Assay, Marker, Western Blot

    tofu-6 is required for 21U-RNA biogenesis and chromosome segregation. (A) Images of TOFU-6::GFP and P-granule marker mRuby::PGL-1 in germ cells. (B) Schematic of four alleles of tofu-6 . (C) Normalized 21U-RNA reads in indicated animals at late young adult stage. Indicated mutant animals were isolated from balancers. Reads were normalized to total RNA reads. (D) Bar diagram displaying brood size of indicated worms. Worms were grown at 20. (E) Images of GFP::H2B at meta-anaphase of two-cells stage embryos in indicated worms. (F) Images of GFP::H2B in indicated later stage embryos.
    Figure Legend Snippet: tofu-6 is required for 21U-RNA biogenesis and chromosome segregation. (A) Images of TOFU-6::GFP and P-granule marker mRuby::PGL-1 in germ cells. (B) Schematic of four alleles of tofu-6 . (C) Normalized 21U-RNA reads in indicated animals at late young adult stage. Indicated mutant animals were isolated from balancers. Reads were normalized to total RNA reads. (D) Bar diagram displaying brood size of indicated worms. Worms were grown at 20. (E) Images of GFP::H2B at meta-anaphase of two-cells stage embryos in indicated worms. (F) Images of GFP::H2B in indicated later stage embryos.

    Techniques Used: Marker, Mutagenesis, Isolation

    8) Product Images from "Effects of Inner Nuclear Membrane Proteins SUN1/UNC-84A and SUN2/UNC-84B on the Early Steps of HIV-1 Infection"

    Article Title: Effects of Inner Nuclear Membrane Proteins SUN1/UNC-84A and SUN2/UNC-84B on the Early Steps of HIV-1 Infection

    Journal: Journal of Virology

    doi: 10.1128/JVI.00463-17

    In vitro -synthesized HIV-1 CANC nanotubes capture SUN1 and SUN2 from cell lysates. (A) Cell lysates from 293T cells transfected with HALUC, HACPSF6, HASUN1, or HASUN2 were incubated with in vitro -synthesized CANC nanotubes before centrifugation through a sucrose cushion and analysis of supernatants and pellets by immunoblotting using HA- and CA-specific antibodies. I, input; S, supernatant; P, pellet. (B) In vitro -synthesized CANC complexes from RHPA were mixed with cell lysates and analyzed as described for the results shown in panel A.
    Figure Legend Snippet: In vitro -synthesized HIV-1 CANC nanotubes capture SUN1 and SUN2 from cell lysates. (A) Cell lysates from 293T cells transfected with HALUC, HACPSF6, HASUN1, or HASUN2 were incubated with in vitro -synthesized CANC nanotubes before centrifugation through a sucrose cushion and analysis of supernatants and pellets by immunoblotting using HA- and CA-specific antibodies. I, input; S, supernatant; P, pellet. (B) In vitro -synthesized CANC complexes from RHPA were mixed with cell lysates and analyzed as described for the results shown in panel A.

    Techniques Used: In Vitro, Synthesized, Transfection, Incubation, Centrifugation

    Overexpression of SUN1 or SUN2 inhibits HIV-1 infection. (A) Nontransduced U87MG CD4/CXCR4 cells (n.t.) or cells expressing the indicated HA-tagged membrane-associated NE proteins or luciferase (LUC; negative control) were infected with serial dilutions of VSV-G-pseudotyped HIV-1 GFP LV, and the infectious units (i.u.) per ml of inoculum were calculated. Fold changes in mean infectious titers relative to HALUC control cells and standard deviations were determined from at least three viral doses from four independent biological repeats. Unpaired two-tailed t tests were performed. ns, not statistically significant; *, P
    Figure Legend Snippet: Overexpression of SUN1 or SUN2 inhibits HIV-1 infection. (A) Nontransduced U87MG CD4/CXCR4 cells (n.t.) or cells expressing the indicated HA-tagged membrane-associated NE proteins or luciferase (LUC; negative control) were infected with serial dilutions of VSV-G-pseudotyped HIV-1 GFP LV, and the infectious units (i.u.) per ml of inoculum were calculated. Fold changes in mean infectious titers relative to HALUC control cells and standard deviations were determined from at least three viral doses from four independent biological repeats. Unpaired two-tailed t tests were performed. ns, not statistically significant; *, P

    Techniques Used: Over Expression, Infection, Expressing, Luciferase, Negative Control, Two Tailed Test

    Gene disruption of SUN2 but not SUN1 reduces infectivity of HIV-1 in THP-1 cells. (A) THP-1 CRISPR/Cas9 single-cell clones transduced to express specific guide RNAs against SUN1 or SUN2 were generated. Single-cell cloning followed by PCR-based sequencing across the guide RNA target sites identified two clones for each gene in which the open reading frame was disrupted. (B) Disruption of gene expression was verified by immunoblotting using SUN1- or SUN2-specific antibodies; Hsp90 served as the loading control. CRISPR/Cas9 control cells (Cntrl) expressed an unrelated guide RNA. (C) SUN1- or SUN2-depleted cells were infected with serial dilutions of VSV-G-pseudotyped NL4.3GFP or BREGFP reporter viruses, and infectious titers were determined. Shown are the fold changes (FC) in mean titers compared to control cells determined from at least 10 viral doses of four independent experiments, with standard deviations. Statistical analysis was performed using a paired two-tailed t test. ns, not statistically significant; *, P
    Figure Legend Snippet: Gene disruption of SUN2 but not SUN1 reduces infectivity of HIV-1 in THP-1 cells. (A) THP-1 CRISPR/Cas9 single-cell clones transduced to express specific guide RNAs against SUN1 or SUN2 were generated. Single-cell cloning followed by PCR-based sequencing across the guide RNA target sites identified two clones for each gene in which the open reading frame was disrupted. (B) Disruption of gene expression was verified by immunoblotting using SUN1- or SUN2-specific antibodies; Hsp90 served as the loading control. CRISPR/Cas9 control cells (Cntrl) expressed an unrelated guide RNA. (C) SUN1- or SUN2-depleted cells were infected with serial dilutions of VSV-G-pseudotyped NL4.3GFP or BREGFP reporter viruses, and infectious titers were determined. Shown are the fold changes (FC) in mean titers compared to control cells determined from at least 10 viral doses of four independent experiments, with standard deviations. Statistical analysis was performed using a paired two-tailed t test. ns, not statistically significant; *, P

    Techniques Used: Infection, CRISPR, Clone Assay, Generated, Polymerase Chain Reaction, Sequencing, Expressing, Two Tailed Test

    9) Product Images from "Tandem RNA isolation reveals functional rearrangement of RNA-binding proteins on CDKN1B/p27Kip1 3’UTRs in cisplatin treated cells"

    Article Title: Tandem RNA isolation reveals functional rearrangement of RNA-binding proteins on CDKN1B/p27Kip1 3’UTRs in cisplatin treated cells

    Journal: RNA Biology

    doi: 10.1080/15476286.2019.1662268

    TobTRIP and MS analysis identifies a network of functionally related RBPs bound to p27(3ʹUTR) reporter mRNAs. (a) Schematic representation of tobTRIP for isolation of tagged mRNAs together with bound proteins: In a first step, poly(A) RNA-protein complexes are isolated using oligo(dT) 25 beads. In a second step, tobramycin aptamer-tagged RNAs are captured with tobramycin-coupled magnetic beads. (b) Immunoblot analysis of the indicated proteins across different steps of tobTRIP. 1:600 of the extract (input), and 1:60 of the 1 st and 2 nd step eluates were loaded. A quantification of band intensities across the 2 nd step eluates is indicated for ELAV1 and hnRNPD. (c) Differential binding of the 54 proteins interacting with GFP-T-p27(3ʹUTR) in CP-treated (+CP) versus untreated (-CP) cells. Mean LFQ values are plotted against log 2 -transformed average foldchanges obtained from normalized intensities. (d) GO terms significantly enriched among the 54 selected proteins. Bars indicate the fractions of proteins annotated with the respective GO term, either across proteins pulled-down with the p27(3ʹUTR) reporter (black bars) or across the entire reference proteome (Uniprot; 20,395 proteins; white bars). Absolute numbers of proteins are shown to the right. Adjusted P -values (Benjamini-Hochberg) for enrichments relative to the reference proteome are indicated. (e) PPI network of 28 proteins with multiple and propagating interactions. Each node corresponds to one RBP, and the physical interaction between RBPs is shown as an edge. The thickness of edges is proportional to the STRING confidence score (between 0–1). Node colour corresponds to average log 2 fold-change of RNA binding in treated (+CP) versus untreated (-CP) cells as indicated in the colour bar.
    Figure Legend Snippet: TobTRIP and MS analysis identifies a network of functionally related RBPs bound to p27(3ʹUTR) reporter mRNAs. (a) Schematic representation of tobTRIP for isolation of tagged mRNAs together with bound proteins: In a first step, poly(A) RNA-protein complexes are isolated using oligo(dT) 25 beads. In a second step, tobramycin aptamer-tagged RNAs are captured with tobramycin-coupled magnetic beads. (b) Immunoblot analysis of the indicated proteins across different steps of tobTRIP. 1:600 of the extract (input), and 1:60 of the 1 st and 2 nd step eluates were loaded. A quantification of band intensities across the 2 nd step eluates is indicated for ELAV1 and hnRNPD. (c) Differential binding of the 54 proteins interacting with GFP-T-p27(3ʹUTR) in CP-treated (+CP) versus untreated (-CP) cells. Mean LFQ values are plotted against log 2 -transformed average foldchanges obtained from normalized intensities. (d) GO terms significantly enriched among the 54 selected proteins. Bars indicate the fractions of proteins annotated with the respective GO term, either across proteins pulled-down with the p27(3ʹUTR) reporter (black bars) or across the entire reference proteome (Uniprot; 20,395 proteins; white bars). Absolute numbers of proteins are shown to the right. Adjusted P -values (Benjamini-Hochberg) for enrichments relative to the reference proteome are indicated. (e) PPI network of 28 proteins with multiple and propagating interactions. Each node corresponds to one RBP, and the physical interaction between RBPs is shown as an edge. The thickness of edges is proportional to the STRING confidence score (between 0–1). Node colour corresponds to average log 2 fold-change of RNA binding in treated (+CP) versus untreated (-CP) cells as indicated in the colour bar.

    Techniques Used: Mass Spectrometry, Isolation, Magnetic Beads, Binding Assay, Transformation Assay, RNA Binding Assay

    KHSRP affects p27 mRNA abundance via the 3ʹUTR and modulates CP sensitivity of MCF7 cancer cells. (a) Extracts prepared from HEK293 cells expressing GFP-tagged KHSRP (lane 1) were incubated with biotinylated RNAs comprising a fragment containing AREs of the 3ʹUTR of LDRL mRNAs (lane 2), the 3ʹUTR of p27 mRNA (lane 3), and RASM as a negative control (lane 4). RNA was captured with streptavidin beads and monitored for the presence of GFP-KHSRP, ELAVL1 and actin by immunoblot analysis with GFP, ELAVL1, and actin antibodies, respectively. (b) HEK293 cells expressing GFP-T and GFP-T-p27(3ʹUTR) were transiently transfected with siKHSRP or scr (siRNA control) for 48 h and treated with 20 µM CP for the last 15 h. The level of GFP in CP-treated (+CP) versus untreated cells (-CP) was assessed by RT-qPCR normalized to β-actin . (c) MCF7 cells were transiently transfected with siRNAs targeting KHSRP (siKHSRP) and Scr control oligos. P27 mRNA levels of CP-treated (+CP, 24 h) relative to untreated cells (-CP) was assessed by RT-qPCR normalized to β-actin . An immunoblot showing knock-down of KHSRP is depicted below. (d) Cell proliferation of MCF7 cells was determined by Trypan Blue assay at the indicated time points after CP treatment. Error bars represent SEM, n = 3. * P
    Figure Legend Snippet: KHSRP affects p27 mRNA abundance via the 3ʹUTR and modulates CP sensitivity of MCF7 cancer cells. (a) Extracts prepared from HEK293 cells expressing GFP-tagged KHSRP (lane 1) were incubated with biotinylated RNAs comprising a fragment containing AREs of the 3ʹUTR of LDRL mRNAs (lane 2), the 3ʹUTR of p27 mRNA (lane 3), and RASM as a negative control (lane 4). RNA was captured with streptavidin beads and monitored for the presence of GFP-KHSRP, ELAVL1 and actin by immunoblot analysis with GFP, ELAVL1, and actin antibodies, respectively. (b) HEK293 cells expressing GFP-T and GFP-T-p27(3ʹUTR) were transiently transfected with siKHSRP or scr (siRNA control) for 48 h and treated with 20 µM CP for the last 15 h. The level of GFP in CP-treated (+CP) versus untreated cells (-CP) was assessed by RT-qPCR normalized to β-actin . (c) MCF7 cells were transiently transfected with siRNAs targeting KHSRP (siKHSRP) and Scr control oligos. P27 mRNA levels of CP-treated (+CP, 24 h) relative to untreated cells (-CP) was assessed by RT-qPCR normalized to β-actin . An immunoblot showing knock-down of KHSRP is depicted below. (d) Cell proliferation of MCF7 cells was determined by Trypan Blue assay at the indicated time points after CP treatment. Error bars represent SEM, n = 3. * P

    Techniques Used: Expressing, Incubation, Negative Control, Transfection, Quantitative RT-PCR

    10) Product Images from "Proteolytic Processing of the ?-Subunit Is Associated with the Failure to Form GlcNAc-1-phosphotransferase Complexes and Mannose 6-Phosphate Residues on Lysosomal Enzymes in Human Macrophages *"

    Article Title: Proteolytic Processing of the ?-Subunit Is Associated with the Failure to Form GlcNAc-1-phosphotransferase Complexes and Mannose 6-Phosphate Residues on Lysosomal Enzymes in Human Macrophages *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.129684

    The GlcNAc-1-phosphotransferase γ-subunit is proteolytically cleaved in macrophages and forms irregular oligomers. Lysates of macrophages, non-transfected, and γ-subunit overexpressing COS7 cells were treated with (+) or without (−) PNGase F and separated under reducing ( A , C , D ) or non-reducing ( B ) conditions by SDS-PAGE. In D , the proteins were separated by Tris-Tricine SDS-PAGE. The expression of GlcNAc-1-phosphotransferase subunits were analyzed by Western blotting using anti β-subunit antiserum (1:500, A ) or anti γ-subunit antiserum (1:250, B–D ). The positions of the molecular mass markers in kDa are given.
    Figure Legend Snippet: The GlcNAc-1-phosphotransferase γ-subunit is proteolytically cleaved in macrophages and forms irregular oligomers. Lysates of macrophages, non-transfected, and γ-subunit overexpressing COS7 cells were treated with (+) or without (−) PNGase F and separated under reducing ( A , C , D ) or non-reducing ( B ) conditions by SDS-PAGE. In D , the proteins were separated by Tris-Tricine SDS-PAGE. The expression of GlcNAc-1-phosphotransferase subunits were analyzed by Western blotting using anti β-subunit antiserum (1:500, A ) or anti γ-subunit antiserum (1:250, B–D ). The positions of the molecular mass markers in kDa are given.

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

    11) Product Images from "Targeting Aurora B to the Equatorial Cortex by MKlp2 Is Required for Cytokinesis"

    Article Title: Targeting Aurora B to the Equatorial Cortex by MKlp2 Is Required for Cytokinesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064826

    MKlp2 is a novel binding partner of myosin-II in vivo and in vitro . ( A ) Asynchronously growing HeLa cells were harvested and subjected to immunoprecipitation analysis using antibodies for pre-immune control IgG (lanes 1, 4), myosin-II (lanes 2, 3), and MKlp2 with (lane 5) or without (lane 3) HeLa cell lysates. Note that the 100 kDa band is specific for MKlp2 (lane 2) and not caused by α-Myosin-II antibodies used for immunoprecipitation as it is not detected in lane 3. N.S. indicates non-specific. ( B – D ) Asynchronously growing HeLa cells were transfected with the indicated expression plasmids, and 24 h after transfection, HeLa cell lysates expressing the indicated MKlp2 or myosin-II proteins were subjected to immunoprecipitation with the indicated antibodies. ( E ) Autoradiography of in vitro -translated Myc-Myosin-II precipitates with the indicated GST-MKlp2 proteins using GST-pulldown analysis (bottom, visualized with Coomassie Blue staining). Overall, 10% of the input for total in vitro -translated product is shown. ( F ) F-actin binding assay. Asynchronously growing HeLa cells were transfected with the indicated expression plasmids, and 24 h after transfection, HeLa cell lysates expressing the indicated HA-MKlp2 proteins were supplemented without (lanes 1-6) or with in vitro -polymerized recombinant F-actin (lanes 7-12) and subjected to ultracentrifugation. Supernatant (S) and pellet (P) fractions were subjected to immunoblot analysis.
    Figure Legend Snippet: MKlp2 is a novel binding partner of myosin-II in vivo and in vitro . ( A ) Asynchronously growing HeLa cells were harvested and subjected to immunoprecipitation analysis using antibodies for pre-immune control IgG (lanes 1, 4), myosin-II (lanes 2, 3), and MKlp2 with (lane 5) or without (lane 3) HeLa cell lysates. Note that the 100 kDa band is specific for MKlp2 (lane 2) and not caused by α-Myosin-II antibodies used for immunoprecipitation as it is not detected in lane 3. N.S. indicates non-specific. ( B – D ) Asynchronously growing HeLa cells were transfected with the indicated expression plasmids, and 24 h after transfection, HeLa cell lysates expressing the indicated MKlp2 or myosin-II proteins were subjected to immunoprecipitation with the indicated antibodies. ( E ) Autoradiography of in vitro -translated Myc-Myosin-II precipitates with the indicated GST-MKlp2 proteins using GST-pulldown analysis (bottom, visualized with Coomassie Blue staining). Overall, 10% of the input for total in vitro -translated product is shown. ( F ) F-actin binding assay. Asynchronously growing HeLa cells were transfected with the indicated expression plasmids, and 24 h after transfection, HeLa cell lysates expressing the indicated HA-MKlp2 proteins were supplemented without (lanes 1-6) or with in vitro -polymerized recombinant F-actin (lanes 7-12) and subjected to ultracentrifugation. Supernatant (S) and pellet (P) fractions were subjected to immunoblot analysis.

    Techniques Used: Binding Assay, In Vivo, In Vitro, Immunoprecipitation, Transfection, Expressing, Autoradiography, Staining, Recombinant

    12) Product Images from "Integrated Microfluidics for Protein Modification Discovery *"

    Article Title: Integrated Microfluidics for Protein Modification Discovery *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M115.053512

    Large-scale Tyr phosphorylation analysis. A , An array of 1024 nuclear proteins. Bars represent average quadruplicates of a single protein, as measured by Cy3-coupled anti-Myc antibodies. B and C , HEK293 extracts containing 10 m m SOV ( B ) or buffer ( C ),
    Figure Legend Snippet: Large-scale Tyr phosphorylation analysis. A , An array of 1024 nuclear proteins. Bars represent average quadruplicates of a single protein, as measured by Cy3-coupled anti-Myc antibodies. B and C , HEK293 extracts containing 10 m m SOV ( B ) or buffer ( C ),

    Techniques Used:

    13) Product Images from "Activated microglia/macrophage whey acidic protein (AMWAP) inhibits NFκB signaling and induces a neuroprotective phenotype in microglia"

    Article Title: Activated microglia/macrophage whey acidic protein (AMWAP) inhibits NFκB signaling and induces a neuroprotective phenotype in microglia

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-015-0296-6

    AMWAP is taken up by microglia and inhibits LPS-mediated NFκB activation. (A-C) BV-2 microglia were incubated with fluorescently labeled recombinant AMWAP (AMWAP-Fluo EX, 10 μg/ml) for 6 and 24 h. Microglia gradually incorporated AMWAP-Fluo EX into their cytosol exhibiting a perinuclear localization as shown by anti-Iba1 antibody co-staining. (D-G) AMWAP-Fluo EX pretreatment (for 1 to 24 h) time-dependently inhibited TLR4-mediated nuclear translocation of NFκB p65 after stimulation with 50 ng/ml LPS for 1 h as shown by immunocytochemistry and quantification of nuclear vs. cytosolic fluorescence intensities. PBS served as vehicle control. Data show mean ± SD ( n = 9/group) with * P
    Figure Legend Snippet: AMWAP is taken up by microglia and inhibits LPS-mediated NFκB activation. (A-C) BV-2 microglia were incubated with fluorescently labeled recombinant AMWAP (AMWAP-Fluo EX, 10 μg/ml) for 6 and 24 h. Microglia gradually incorporated AMWAP-Fluo EX into their cytosol exhibiting a perinuclear localization as shown by anti-Iba1 antibody co-staining. (D-G) AMWAP-Fluo EX pretreatment (for 1 to 24 h) time-dependently inhibited TLR4-mediated nuclear translocation of NFκB p65 after stimulation with 50 ng/ml LPS for 1 h as shown by immunocytochemistry and quantification of nuclear vs. cytosolic fluorescence intensities. PBS served as vehicle control. Data show mean ± SD ( n = 9/group) with * P

    Techniques Used: Activation Assay, Incubation, Labeling, Recombinant, Staining, Translocation Assay, Immunocytochemistry, Fluorescence

    AMWAP prevents LPS-induced degradation of IRAK-1 and IκBα. Control and AMWAP-treated BV-2 microglia were incubated with 50 ng/ml LPS for 0, 1, 2, and 3 h, and cytosolic protein extracts were prepared. Immunoblot analysis of IRAK-1 (A) and IκBα (B) was carried out to determine the level of proteolytic degradation. In AMWAP-treated cells, both signaling molecules were protected from LPS-induced proteolysis. Resynthesized IκBα was detected after 2 and 3 h in control cells. Actin served as loading control.
    Figure Legend Snippet: AMWAP prevents LPS-induced degradation of IRAK-1 and IκBα. Control and AMWAP-treated BV-2 microglia were incubated with 50 ng/ml LPS for 0, 1, 2, and 3 h, and cytosolic protein extracts were prepared. Immunoblot analysis of IRAK-1 (A) and IκBα (B) was carried out to determine the level of proteolytic degradation. In AMWAP-treated cells, both signaling molecules were protected from LPS-induced proteolysis. Resynthesized IκBα was detected after 2 and 3 h in control cells. Actin served as loading control.

    Techniques Used: Incubation

    14) Product Images from "Biodistribution Analysis of Oncolytic Adenoviruses in Patient Autopsy Samples Reveals Vascular Transduction of Noninjected Tumors and Tissues"

    Article Title: Biodistribution Analysis of Oncolytic Adenoviruses in Patient Autopsy Samples Reveals Vascular Transduction of Noninjected Tumors and Tissues

    Journal: Molecular Therapy

    doi: 10.1038/mt.2015.125

    Oncolytic adenovirus DNA is recovered from a wide range of injected and noninjected neoplastic and normal tissues . ( a - j ) DNA was extracted from paraffin embedded tissue samples collected in autopsies and qPCR was performed with primers and probes detecting genetic modifications of the used oncolytic viruses (black bars) and wild-type E1A region (open bars) and normalized with the β-actin housekeeping gene. ( k ) All samples containing normal or metastatic brain tissue. (n), histologically normal tissue; Ln, lymph nodes. *Tissue with tumor/metastasis, not injected directly; **injected tumor/metastasis tissue. n = 1–15 samples per tissue, expressed as mean + standard error of mean for tissues with multiple samples.
    Figure Legend Snippet: Oncolytic adenovirus DNA is recovered from a wide range of injected and noninjected neoplastic and normal tissues . ( a - j ) DNA was extracted from paraffin embedded tissue samples collected in autopsies and qPCR was performed with primers and probes detecting genetic modifications of the used oncolytic viruses (black bars) and wild-type E1A region (open bars) and normalized with the β-actin housekeeping gene. ( k ) All samples containing normal or metastatic brain tissue. (n), histologically normal tissue; Ln, lymph nodes. *Tissue with tumor/metastasis, not injected directly; **injected tumor/metastasis tissue. n = 1–15 samples per tissue, expressed as mean + standard error of mean for tissues with multiple samples.

    Techniques Used: Injection, Real-time Polymerase Chain Reaction

    15) Product Images from "Identification and characterization of CMP-NeuAc:GalNAc-IgA1 ?2,6-sialyltransferase in IgA1-producing cells"

    Article Title: Identification and characterization of CMP-NeuAc:GalNAc-IgA1 ?2,6-sialyltransferase in IgA1-producing cells

    Journal:

    doi: 10.1016/j.jmb.2007.03.002

    Sialylation of terminal GalNAc in the hinge region of IgA1 (Mce) by DAKIKI cell extracts
    Figure Legend Snippet: Sialylation of terminal GalNAc in the hinge region of IgA1 (Mce) by DAKIKI cell extracts

    Techniques Used:

    16) Product Images from "Low Expression of lncRNA-GAS5 Is Implicated in Human Primary Varicose Great Saphenous Veins"

    Article Title: Low Expression of lncRNA-GAS5 Is Implicated in Human Primary Varicose Great Saphenous Veins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0120550

    The effects of lncRNA-GAS5 mediated by Annexin A2. A2 = Annexin A2; Mock = control group, only empty plasmid transfected; NC = Negative control group, only control siRNA transfected; A2 siRNA = Annexin A2 knockdown group transfected with silence siRNA; A2 over = Annexin A2 overexpression group transfected with Annexin A2 expression plasmid; GAS5 down = lncRNA-GAS5 knockdown group transfected with silence siRNA. A: Silver stains for protein gels obtained by lncRNA-GAS5 RNA Pulldown. Bio-GAS5 (sense): treatment group; Bio-GAS5 (antisense): control groups; GAS5 (sense): negative control groups. The GAS5- specific-binding protein gels (in Red box) was identified by MALDI-TOF-MS. Results show that Annexin A2 isoform 2 is a direct binding protein to lncRNA-GAS5. N = 2. B: The Annexin A2 expression level was knockdowned in HSVSMCs effectively by siRNA. Values are mean±SE, N = 3; *, P
    Figure Legend Snippet: The effects of lncRNA-GAS5 mediated by Annexin A2. A2 = Annexin A2; Mock = control group, only empty plasmid transfected; NC = Negative control group, only control siRNA transfected; A2 siRNA = Annexin A2 knockdown group transfected with silence siRNA; A2 over = Annexin A2 overexpression group transfected with Annexin A2 expression plasmid; GAS5 down = lncRNA-GAS5 knockdown group transfected with silence siRNA. A: Silver stains for protein gels obtained by lncRNA-GAS5 RNA Pulldown. Bio-GAS5 (sense): treatment group; Bio-GAS5 (antisense): control groups; GAS5 (sense): negative control groups. The GAS5- specific-binding protein gels (in Red box) was identified by MALDI-TOF-MS. Results show that Annexin A2 isoform 2 is a direct binding protein to lncRNA-GAS5. N = 2. B: The Annexin A2 expression level was knockdowned in HSVSMCs effectively by siRNA. Values are mean±SE, N = 3; *, P

    Techniques Used: Plasmid Preparation, Transfection, Negative Control, Over Expression, Expressing, Binding Assay, Mass Spectrometry

    17) Product Images from "Upregulation of the Voltage-Gated Sodium Channel β2 Subunit in Neuropathic Pain Models: Characterization of Expression in Injured and Non-Injured Primary Sensory Neurons"

    Article Title: Upregulation of the Voltage-Gated Sodium Channel β2 Subunit in Neuropathic Pain Models: Characterization of Expression in Injured and Non-Injured Primary Sensory Neurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3066-05.2005

    Peripheral nerve injury induces a rapid and persistent increase in β2 subunit immunoreactivity in the DRG. A , Western blot analyses of the L4/L5 DRGs 1 week after an SNI, total axotomy of the sciatic nerve (Axo), or 48 h and 1 week (1W) after intraplantar CFA induced inflammation (longer exposure). B , D , Representative Western blots from at least four independent experiments at 1, 3, 5, and 7 d after SNI and 1, 2, 3, and 4 weeks after SNI. C , Quantitation of β2 subunit protein levels in the L4/L5 DRGs at the indicated days after SNI. Fold represents comparative increase over control level after normalizing to total ERK2 ( n = 4 for each time point; * p
    Figure Legend Snippet: Peripheral nerve injury induces a rapid and persistent increase in β2 subunit immunoreactivity in the DRG. A , Western blot analyses of the L4/L5 DRGs 1 week after an SNI, total axotomy of the sciatic nerve (Axo), or 48 h and 1 week (1W) after intraplantar CFA induced inflammation (longer exposure). B , D , Representative Western blots from at least four independent experiments at 1, 3, 5, and 7 d after SNI and 1, 2, 3, and 4 weeks after SNI. C , Quantitation of β2 subunit protein levels in the L4/L5 DRGs at the indicated days after SNI. Fold represents comparative increase over control level after normalizing to total ERK2 ( n = 4 for each time point; * p

    Techniques Used: Western Blot, Quantitation Assay

    18) Product Images from "Biochemical characterization of the Yersinia YopT protease: Cleavage site and recognition elements in Rho GTPases"

    Article Title: Biochemical characterization of the Yersinia YopT protease: Cleavage site and recognition elements in Rho GTPases

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

    doi: 10.1073/pnas.252770599

    Determination of the YopT cleavage site in RhoA. ( A ) The prenylated [ 35 S]Cys, but not the [ 14 C]Gly (N-terminal to the prenylated cysteine), in RhoA was cleaved off by YopT. ( Upper ) Scintillation counting of the chloroform-extracted YopT cleavage product of RhoA with either [ 14 C]Gly or [ 35 S]Cys labeling. ( Lower ) Levels of radiolabeled RhoA by autoradiography ( Left ) and total RhoA protein by Western blot ( Right ) present in each cleavage reaction. ( B ) Nanoelectrospray MS/MS analysis of the lipid YopT cleavage product from RhoA. Shown is the fragmentation pattern for the GGCM standard ( Bottom ) and for a single charged parent ion ( m / z 408.7) from the RhoA sample after a cleavage reaction with YopT ( Middle ). ( Top ) Chemical structure of the GGCM standard. The highlighted fragments correspond to the fragment ions observed in the MS/MS spectrum of the standard and the cleavage product from RhoA.
    Figure Legend Snippet: Determination of the YopT cleavage site in RhoA. ( A ) The prenylated [ 35 S]Cys, but not the [ 14 C]Gly (N-terminal to the prenylated cysteine), in RhoA was cleaved off by YopT. ( Upper ) Scintillation counting of the chloroform-extracted YopT cleavage product of RhoA with either [ 14 C]Gly or [ 35 S]Cys labeling. ( Lower ) Levels of radiolabeled RhoA by autoradiography ( Left ) and total RhoA protein by Western blot ( Right ) present in each cleavage reaction. ( B ) Nanoelectrospray MS/MS analysis of the lipid YopT cleavage product from RhoA. Shown is the fragmentation pattern for the GGCM standard ( Bottom ) and for a single charged parent ion ( m / z 408.7) from the RhoA sample after a cleavage reaction with YopT ( Middle ). ( Top ) Chemical structure of the GGCM standard. The highlighted fragments correspond to the fragment ions observed in the MS/MS spectrum of the standard and the cleavage product from RhoA.

    Techniques Used: Labeling, Autoradiography, Western Blot, Mass Spectrometry

    19) Product Images from "Calcineurin promotes APC/C activation at meiotic exit by acting on both XErp1 and Cdc20"

    Article Title: Calcineurin promotes APC/C activation at meiotic exit by acting on both XErp1 and Cdc20

    Journal: EMBO Reports

    doi: 10.15252/embr.201846433

    Characterization of pSer335 XErp1 antibody CSF extract was treated with Myc‐XErp1 IVT carrying the indicated combinations of the mutations DSG − (S33N S38N), DSA − (S284N S288N), ZBR − (C583A) and CaMKII − (T195A). Calcium was added, samples were taken at the indicated time points and as indicated treated with λ‐phosphatase. Samples were immunoblotted for the Myc‐tag and cyclin B2. The cyclin B2 membrane was stripped and reprobed for α‐tubulin. Asterisk indicates unspecific bands. Several lanes were removed at the dashed line. CSF extract was treated with Myc‐XErp1 CaMKII − ZBR − (T195A C583A) IVT at the indicated dilutions. An empty IVT not expressing XErp1 and an untreated condition were used as controls. Samples were taken, treated as indicated with λ‐phosphatase and immunoblotted for XErp1, pSer335 XErp1, and α‐tubulin. The XErp1 membrane was stripped and reprobed for the Myc‐tag. Asterisks indicate unspecific bands. CSF extract was treated with Myc‐XErp1 CaMKII − ZBR − (T195A C583A) IVT that was either wild‐type or mutated to alanine at Ser335. An empty IVT reaction not expressing Myc‐XErp1 served as control. Samples were taken, treated as indicated with λ‐phosphatase and immunoblotted for XErp1, the Myc‐tag, pSer335 XErp1, and α‐tubulin. Asterisks indicate unspecific bands. Source data are available online for this figure.
    Figure Legend Snippet: Characterization of pSer335 XErp1 antibody CSF extract was treated with Myc‐XErp1 IVT carrying the indicated combinations of the mutations DSG − (S33N S38N), DSA − (S284N S288N), ZBR − (C583A) and CaMKII − (T195A). Calcium was added, samples were taken at the indicated time points and as indicated treated with λ‐phosphatase. Samples were immunoblotted for the Myc‐tag and cyclin B2. The cyclin B2 membrane was stripped and reprobed for α‐tubulin. Asterisk indicates unspecific bands. Several lanes were removed at the dashed line. CSF extract was treated with Myc‐XErp1 CaMKII − ZBR − (T195A C583A) IVT at the indicated dilutions. An empty IVT not expressing XErp1 and an untreated condition were used as controls. Samples were taken, treated as indicated with λ‐phosphatase and immunoblotted for XErp1, pSer335 XErp1, and α‐tubulin. The XErp1 membrane was stripped and reprobed for the Myc‐tag. Asterisks indicate unspecific bands. CSF extract was treated with Myc‐XErp1 CaMKII − ZBR − (T195A C583A) IVT that was either wild‐type or mutated to alanine at Ser335. An empty IVT reaction not expressing Myc‐XErp1 served as control. Samples were taken, treated as indicated with λ‐phosphatase and immunoblotted for XErp1, the Myc‐tag, pSer335 XErp1, and α‐tubulin. Asterisks indicate unspecific bands. Source data are available online for this figure.

    Techniques Used: Expressing

    20) Product Images from "The long noncoding RNA HOTAIR activates the Hippo pathway by directly binding to SAV1 in renal cell carcinoma"

    Article Title: The long noncoding RNA HOTAIR activates the Hippo pathway by directly binding to SAV1 in renal cell carcinoma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17414

    HOTAIR directly binds to SAV1 (A–D) Methylation status and rate of SAV1 CpG island in 786O and OSRC2 cells. (E) RNA-pulldown revealed that HOTAIR binds to SAV1.
    Figure Legend Snippet: HOTAIR directly binds to SAV1 (A–D) Methylation status and rate of SAV1 CpG island in 786O and OSRC2 cells. (E) RNA-pulldown revealed that HOTAIR binds to SAV1.

    Techniques Used: Methylation

    21) Product Images from "Dlg1, Sec8, and Mtmr2 Regulate Membrane Homeostasis in Schwann Cell Myelination"

    Article Title: Dlg1, Sec8, and Mtmr2 Regulate Membrane Homeostasis in Schwann Cell Myelination

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1423-09.2009

    Myelin outfoldings are reproduced in Mtmr2 -null Schwann cells/DRG neuron cocultures. Control and Mtmr2 -null Schwann cell/DRG neuron cocultures were treated for 15 d with ascorbic acid to induce myelination. A , A′ inset, C , After MBP labeling, myelinated fibers had a normal shape in control. B , B′ inset, D , Abnormal shape in Mtmr2 -null explants. A , B , Neurofilament (NF) was used to detect axons. E–H , Electron microscopy demonstrated that myelin outfoldings were present in myelin-forming cocultures. The arrowheads in B′ , D , and F–H indicate myelin outfoldings. The myelin outfolding phenotype is fully penetrant in vitro , as at least 40 different Mtmr2 -null embryos were evaluated in this work. Scale bars: (in A ) A , B , 10 μm; A′ , B′ , C , D , 7 μm; (in H ) E–H , 1 μm.
    Figure Legend Snippet: Myelin outfoldings are reproduced in Mtmr2 -null Schwann cells/DRG neuron cocultures. Control and Mtmr2 -null Schwann cell/DRG neuron cocultures were treated for 15 d with ascorbic acid to induce myelination. A , A′ inset, C , After MBP labeling, myelinated fibers had a normal shape in control. B , B′ inset, D , Abnormal shape in Mtmr2 -null explants. A , B , Neurofilament (NF) was used to detect axons. E–H , Electron microscopy demonstrated that myelin outfoldings were present in myelin-forming cocultures. The arrowheads in B′ , D , and F–H indicate myelin outfoldings. The myelin outfolding phenotype is fully penetrant in vitro , as at least 40 different Mtmr2 -null embryos were evaluated in this work. Scale bars: (in A ) A , B , 10 μm; A′ , B′ , C , D , 7 μm; (in H ) E–H , 1 μm.

    Techniques Used: Labeling, Electron Microscopy, In Vitro

    Loss of Mtmr2 and Mtmr2/Dlg1 interaction causes myelin outfoldings in vitro . Myelin outfoldings are rescued by MTMR2 replacement using LV transduction. A , Transduction of Mtmr2 -null Schwann cell/DRG neuron cocultures with a GFP LV. A′ , Inset, MBP-positive fibers with myelin outfoldings are indicated by arrowheads. B , Transduction of Mtmr2 -null Schwann cell/DRG neuron cocultures with FLAG-MTMR2 LV rescues myelin outfoldings. DAPI, 4′,6′-Diamidino-2-phenylindole. B′ , Inset. C , Electron microscopy on FLAG-MTMR2 LV transduced cocultures showing that myelin compaction is preserved on MTMR2 overexpression. At least 30 myelinated fibers were evaluated and compared with cultures infected with GFP alone. D , Quantification of MBP-positive fibers with myelin outfoldings in not transduced (NT) explants (75.5 ± 2.3; n = 6), transduced with GFP alone (76.5 ± 5.0; n = 4), and with FLAG-MTMR2 (10.7 ± 5.3; n = 9) ( p = 8.7 × 10 −6 ) (from at least 2 different experiments). E , Dlg1 coimmunoprecipitates with FLAG-MTMR2 but not with MTMR2 devoid of the PDZ-binding domain at the C terminus, termed FLAG-MTMR2ΔPDZBD. Input is the lysate. F , Western blot analysis showing similar level of expression of both FLAG-MTMR2 and FLAG-MTMR2ΔPDZBD on LV transduction of Mtmr2 -null DRG explants at MOI12.5. G , H , Examples of myelin-forming cocultures transduced with FLAG-MTMR2 or FLAG-MTMR2ΔPDZBD LV at MOI12.5. I , Quantification of MBP-positive fibers with myelin outfoldings on LV transduction of Mtmr2 -null explants with FLAG-MTMR2 (17.99 ± 2.20; n = 9) and FLAG-MTMR2ΔPDZBD (29.97 ± 1.76; n = 9) ( p = 0.00085); FLAG-MTMR2ΔPDZBD (29.97 ± 1.76; n = 9) and GFP (76.5 ± 5.0; n = 4) ( p = 0.00097) (2 different experiments). Error bars indicate SEM. ** p
    Figure Legend Snippet: Loss of Mtmr2 and Mtmr2/Dlg1 interaction causes myelin outfoldings in vitro . Myelin outfoldings are rescued by MTMR2 replacement using LV transduction. A , Transduction of Mtmr2 -null Schwann cell/DRG neuron cocultures with a GFP LV. A′ , Inset, MBP-positive fibers with myelin outfoldings are indicated by arrowheads. B , Transduction of Mtmr2 -null Schwann cell/DRG neuron cocultures with FLAG-MTMR2 LV rescues myelin outfoldings. DAPI, 4′,6′-Diamidino-2-phenylindole. B′ , Inset. C , Electron microscopy on FLAG-MTMR2 LV transduced cocultures showing that myelin compaction is preserved on MTMR2 overexpression. At least 30 myelinated fibers were evaluated and compared with cultures infected with GFP alone. D , Quantification of MBP-positive fibers with myelin outfoldings in not transduced (NT) explants (75.5 ± 2.3; n = 6), transduced with GFP alone (76.5 ± 5.0; n = 4), and with FLAG-MTMR2 (10.7 ± 5.3; n = 9) ( p = 8.7 × 10 −6 ) (from at least 2 different experiments). E , Dlg1 coimmunoprecipitates with FLAG-MTMR2 but not with MTMR2 devoid of the PDZ-binding domain at the C terminus, termed FLAG-MTMR2ΔPDZBD. Input is the lysate. F , Western blot analysis showing similar level of expression of both FLAG-MTMR2 and FLAG-MTMR2ΔPDZBD on LV transduction of Mtmr2 -null DRG explants at MOI12.5. G , H , Examples of myelin-forming cocultures transduced with FLAG-MTMR2 or FLAG-MTMR2ΔPDZBD LV at MOI12.5. I , Quantification of MBP-positive fibers with myelin outfoldings on LV transduction of Mtmr2 -null explants with FLAG-MTMR2 (17.99 ± 2.20; n = 9) and FLAG-MTMR2ΔPDZBD (29.97 ± 1.76; n = 9) ( p = 0.00085); FLAG-MTMR2ΔPDZBD (29.97 ± 1.76; n = 9) and GFP (76.5 ± 5.0; n = 4) ( p = 0.00097) (2 different experiments). Error bars indicate SEM. ** p

    Techniques Used: In Vitro, Transduction, Electron Microscopy, Over Expression, Infection, Binding Assay, Western Blot, Expressing

    Loss of kif13B and Dlg1 in myelin-forming cocultures impairs active myelination. A , kif13B shRNA LV transduction of isolated rat Schwann cells revealed a decrease of kif13B, Dlg1, and Sec8 expression. Three independent experiments were performed. B , kif13B shRNA LV transduction of dissociated Schwann cell/DRG neuron cocultures also revealed a decrease of kif13B, Dlg1, and Sec8 expression. C , Downregulation of Dlg1 expression on Dlg1 shRNA LV transduction of isolated rat Schwann cells. D–F , In dissociated cultures transduced with kif13B shRNA LV, myelination, as the ratio between the number of MBP-positive segments and number of Schwann cell nuclei, was significantly decreased with respect to control shRNA ( kif13B shRNA, 34.80 ± 14.73, n = 12; control shRNA, 100 ± 16.60, n = 16; p = 0.00779; 2 experiments). G–I , The amount of MBP-positive segments is also decreased in Schwann cell/DRG neuron cocultures on Dlg1 shRNA transduction ( Dlg1 shRNA, 19 ± 10.2, n = 7; control shRNA, 100 ± 25.5, n = 7; p = 0.0107). Error bars indicate SEM. * p
    Figure Legend Snippet: Loss of kif13B and Dlg1 in myelin-forming cocultures impairs active myelination. A , kif13B shRNA LV transduction of isolated rat Schwann cells revealed a decrease of kif13B, Dlg1, and Sec8 expression. Three independent experiments were performed. B , kif13B shRNA LV transduction of dissociated Schwann cell/DRG neuron cocultures also revealed a decrease of kif13B, Dlg1, and Sec8 expression. C , Downregulation of Dlg1 expression on Dlg1 shRNA LV transduction of isolated rat Schwann cells. D–F , In dissociated cultures transduced with kif13B shRNA LV, myelination, as the ratio between the number of MBP-positive segments and number of Schwann cell nuclei, was significantly decreased with respect to control shRNA ( kif13B shRNA, 34.80 ± 14.73, n = 12; control shRNA, 100 ± 16.60, n = 16; p = 0.00779; 2 experiments). G–I , The amount of MBP-positive segments is also decreased in Schwann cell/DRG neuron cocultures on Dlg1 shRNA transduction ( Dlg1 shRNA, 19 ± 10.2, n = 7; control shRNA, 100 ± 25.5, n = 7; p = 0.0107). Error bars indicate SEM. * p

    Techniques Used: shRNA, Transduction, Isolation, Expressing

    Sec8 is involved in myelin formation in Schwann cells. A–D , A significant decrease in myelination was observed in dissociated Schwann cell/DRG neuron cocultures on Sec8 shRNA LV transduction ( Sec8 shRNA, 28.01 ± 5.95, n = 6; control shRNA, 100 ± 18.01, n = 10; p = 0.00296). B , The amount of MBP-positive segments was normalized to the Schwann cell number. E–G , Dissociated cocultures were transduced with different amounts of Sec8 shRNA LV. The highest concentration of LV that did not affect myelination was chosen to transduce Mtmr2 -null dissociated cocultures. H–J , Myelin outfoldings were significantly rescued also when myelination is not impaired ( Sec8 shRNA, 51.79 ± 1.67, n = 6; control shRNA, 64.78 ± 3.22, n = 6; p = 0.00716). Error bars indicate SEM. * p
    Figure Legend Snippet: Sec8 is involved in myelin formation in Schwann cells. A–D , A significant decrease in myelination was observed in dissociated Schwann cell/DRG neuron cocultures on Sec8 shRNA LV transduction ( Sec8 shRNA, 28.01 ± 5.95, n = 6; control shRNA, 100 ± 18.01, n = 10; p = 0.00296). B , The amount of MBP-positive segments was normalized to the Schwann cell number. E–G , Dissociated cocultures were transduced with different amounts of Sec8 shRNA LV. The highest concentration of LV that did not affect myelination was chosen to transduce Mtmr2 -null dissociated cocultures. H–J , Myelin outfoldings were significantly rescued also when myelination is not impaired ( Sec8 shRNA, 51.79 ± 1.67, n = 6; control shRNA, 64.78 ± 3.22, n = 6; p = 0.00716). Error bars indicate SEM. * p

    Techniques Used: shRNA, Transduction, Concentration Assay

    Related Articles

    Transfection:

    Article Title: Existence and functions of a kisspeptin neuropeptide signaling system in a non-chordate deuterostome species
    Article Snippet: .. The plasmid constructs were transfected into HEK293 cells using X-tremeGENE HP (Roche), according to the manufacturer's instructions. .. Two days after transfection, stably expressing cells can be selected by the addition of 800 mg/L G418 to avoid impacts from inefficient transfection in experiments.

    Article Title: Stimulation of GCMa Transcriptional Activity by Cyclic AMP/Protein Kinase A Signaling Is Attributed to CBP-Mediated Acetylation of GCMa †
    Article Snippet: .. To study the effect of acetylation on GCMa ubiquitination, 293T cells were transfected with different combinations of pHA-Ub, pGCMa-Myc, lysine-to-arginine mutant pGCMa-Myc, pCBP-Flag, pCBPHAT− -Flag, and pPKAcata, followed by immunoprecipitation with a mouse monoclonal anti-Myc antibody (Myc MAb; Roche) and immunoblotting with HA MAb. .. To map the acetylation domains in GCMa in vitro, 0.2 μg of GST or the indicated GST-GCMa fusion protein was incubated with 0.4 μg of Flag-CBP under the same reaction conditions as described for in vitro acetylation analysis.

    Article Title: The Cdk8/19-cyclin C transcription regulator functions in genome replication through metazoan Sld7
    Article Snippet: .. IP For coimmunoprecipitations of tagged Treslin/TICRR and MTBP from 293T cells, a 10-cm plate of transiently transfected 293T cells were lysed in 5× pellet volume of lysis buffer (20 mM HEPES, 150 mM NaCl, 10% glycerol, complete EDTA-free protease inhibitor cocktail [Roche, 05056489001], 0.1% Triton X-100, 2 mM 2-mercaptoethanol). .. For flag affinity purifications 50% of cell lysates was incubated with 1 μg anti-FLAG mouse monoclonal antibody or 1 μg mouse IgG coupled to 150 μg Protein G Dynabeads (Invitrogen, 100-04D).

    Stable Transfection:

    Article Title: Arginine GlcNAcylation of Rab small GTPases by the pathogen Salmonella Typhimurium
    Article Snippet: .. To purify Flag-Rab1 for total molecular MS, Rab1 stably expressed 293T cells were collected in lysis buffer B containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 20 mM n -octyl-β-d -glucopyranoside (INALCO), and 5% glycerol, supplemented with an EDTA-free protease inhibitor mixture (Roche Molecular Biochemicals). .. Cells were lysed by ultrasonication and precleared lysates were incubated with Flag M2 beads.

    Immunoprecipitation:

    Article Title: Stimulation of GCMa Transcriptional Activity by Cyclic AMP/Protein Kinase A Signaling Is Attributed to CBP-Mediated Acetylation of GCMa †
    Article Snippet: .. To study the effect of acetylation on GCMa ubiquitination, 293T cells were transfected with different combinations of pHA-Ub, pGCMa-Myc, lysine-to-arginine mutant pGCMa-Myc, pCBP-Flag, pCBPHAT− -Flag, and pPKAcata, followed by immunoprecipitation with a mouse monoclonal anti-Myc antibody (Myc MAb; Roche) and immunoblotting with HA MAb. .. To map the acetylation domains in GCMa in vitro, 0.2 μg of GST or the indicated GST-GCMa fusion protein was incubated with 0.4 μg of Flag-CBP under the same reaction conditions as described for in vitro acetylation analysis.

    Mutagenesis:

    Article Title: Stimulation of GCMa Transcriptional Activity by Cyclic AMP/Protein Kinase A Signaling Is Attributed to CBP-Mediated Acetylation of GCMa †
    Article Snippet: .. To study the effect of acetylation on GCMa ubiquitination, 293T cells were transfected with different combinations of pHA-Ub, pGCMa-Myc, lysine-to-arginine mutant pGCMa-Myc, pCBP-Flag, pCBPHAT− -Flag, and pPKAcata, followed by immunoprecipitation with a mouse monoclonal anti-Myc antibody (Myc MAb; Roche) and immunoblotting with HA MAb. .. To map the acetylation domains in GCMa in vitro, 0.2 μg of GST or the indicated GST-GCMa fusion protein was incubated with 0.4 μg of Flag-CBP under the same reaction conditions as described for in vitro acetylation analysis.

    Protease Inhibitor:

    Article Title: Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signaling networks using SILAC-based phosphoproteomics
    Article Snippet: .. For co-immunoprecipitation (IP) experiments, cells were lysed in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% NP-40, 1 mM DTT, 2 mM MgCl2 , and benzonase supplemented with a protease inhibitor (Roche) cocktail tablet. .. After 30 min incubation on ice, cell lysates were clarified by centrifugation (15,000 xg , 4 °C, 20 min).

    Article Title: The Cdk8/19-cyclin C transcription regulator functions in genome replication through metazoan Sld7
    Article Snippet: .. IP For coimmunoprecipitations of tagged Treslin/TICRR and MTBP from 293T cells, a 10-cm plate of transiently transfected 293T cells were lysed in 5× pellet volume of lysis buffer (20 mM HEPES, 150 mM NaCl, 10% glycerol, complete EDTA-free protease inhibitor cocktail [Roche, 05056489001], 0.1% Triton X-100, 2 mM 2-mercaptoethanol). .. For flag affinity purifications 50% of cell lysates was incubated with 1 μg anti-FLAG mouse monoclonal antibody or 1 μg mouse IgG coupled to 150 μg Protein G Dynabeads (Invitrogen, 100-04D).

    Article Title: Arginine GlcNAcylation of Rab small GTPases by the pathogen Salmonella Typhimurium
    Article Snippet: .. To purify Flag-Rab1 for total molecular MS, Rab1 stably expressed 293T cells were collected in lysis buffer B containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 20 mM n -octyl-β-d -glucopyranoside (INALCO), and 5% glycerol, supplemented with an EDTA-free protease inhibitor mixture (Roche Molecular Biochemicals). .. Cells were lysed by ultrasonication and precleared lysates were incubated with Flag M2 beads.

    Article Title: Identification of the viral RNA promoter stem loop A (SLA)-binding site on Zika virus polymerase NS5
    Article Snippet: .. For protein purification, the cells were resuspended in lysis buffer containing 50 mM Tris (pH 8.0), 500 mM NaCl, 2 mM β-mercaptoethanol, and 1 tablet of EDTA-free protease inhibitor (Roche Applied Science, Penzberg, Germany), and lysed by sonication. .. After centrifugation to clear the insoluble debris, the protein in the soluble fraction was purified by Talon metal affinity chromatography (Clontech, Mountain View, CA).

    Northern Blot:

    Article Title: Discovery and Characterization of Two Novel Salt-Tolerance Genes in Puccinellia tenuiflora
    Article Snippet: .. Northern Blot Analysis Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) from various organs of P. tenuiflora and cultured P. tenuiflora cells treated with 200 mM NaCl for 0, 6, 12, 24 and 48 h. For northern blot analysis, hybridization signals were detected using anti-DIG antibody conjugated with alkaline phosphatase (Roche, Indianapolis, IN, USA) and CDP-Star (Roche), as described by Sambrook et al. [ ]. .. Signals were detected using a luminescent image analyzer (Fujifilm, LAS-4000 mini, Tokyo, Japan).

    Construct:

    Article Title: Existence and functions of a kisspeptin neuropeptide signaling system in a non-chordate deuterostome species
    Article Snippet: .. The plasmid constructs were transfected into HEK293 cells using X-tremeGENE HP (Roche), according to the manufacturer's instructions. .. Two days after transfection, stably expressing cells can be selected by the addition of 800 mg/L G418 to avoid impacts from inefficient transfection in experiments.

    Protein Purification:

    Article Title: Identification of the viral RNA promoter stem loop A (SLA)-binding site on Zika virus polymerase NS5
    Article Snippet: .. For protein purification, the cells were resuspended in lysis buffer containing 50 mM Tris (pH 8.0), 500 mM NaCl, 2 mM β-mercaptoethanol, and 1 tablet of EDTA-free protease inhibitor (Roche Applied Science, Penzberg, Germany), and lysed by sonication. .. After centrifugation to clear the insoluble debris, the protein in the soluble fraction was purified by Talon metal affinity chromatography (Clontech, Mountain View, CA).

    Concentration Assay:

    Article Title: UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II
    Article Snippet: .. Pellets were resuspended in an equal volume of ice-cold lysis buffer with additives consisting of 1 mM DTT, 4 mM ATP, 2 mM PMSF, complete EDTA-free protease inhibitors (Roche), and diisopropyl fluorophosphate at a final concentration of 0.5 mM. ..

    Cell Culture:

    Article Title: Discovery and Characterization of Two Novel Salt-Tolerance Genes in Puccinellia tenuiflora
    Article Snippet: .. Northern Blot Analysis Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) from various organs of P. tenuiflora and cultured P. tenuiflora cells treated with 200 mM NaCl for 0, 6, 12, 24 and 48 h. For northern blot analysis, hybridization signals were detected using anti-DIG antibody conjugated with alkaline phosphatase (Roche, Indianapolis, IN, USA) and CDP-Star (Roche), as described by Sambrook et al. [ ]. .. Signals were detected using a luminescent image analyzer (Fujifilm, LAS-4000 mini, Tokyo, Japan).

    Lysis:

    Article Title: UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II
    Article Snippet: .. Pellets were resuspended in an equal volume of ice-cold lysis buffer with additives consisting of 1 mM DTT, 4 mM ATP, 2 mM PMSF, complete EDTA-free protease inhibitors (Roche), and diisopropyl fluorophosphate at a final concentration of 0.5 mM. ..

    Article Title: The Cdk8/19-cyclin C transcription regulator functions in genome replication through metazoan Sld7
    Article Snippet: .. IP For coimmunoprecipitations of tagged Treslin/TICRR and MTBP from 293T cells, a 10-cm plate of transiently transfected 293T cells were lysed in 5× pellet volume of lysis buffer (20 mM HEPES, 150 mM NaCl, 10% glycerol, complete EDTA-free protease inhibitor cocktail [Roche, 05056489001], 0.1% Triton X-100, 2 mM 2-mercaptoethanol). .. For flag affinity purifications 50% of cell lysates was incubated with 1 μg anti-FLAG mouse monoclonal antibody or 1 μg mouse IgG coupled to 150 μg Protein G Dynabeads (Invitrogen, 100-04D).

    Article Title: Arginine GlcNAcylation of Rab small GTPases by the pathogen Salmonella Typhimurium
    Article Snippet: .. To purify Flag-Rab1 for total molecular MS, Rab1 stably expressed 293T cells were collected in lysis buffer B containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 20 mM n -octyl-β-d -glucopyranoside (INALCO), and 5% glycerol, supplemented with an EDTA-free protease inhibitor mixture (Roche Molecular Biochemicals). .. Cells were lysed by ultrasonication and precleared lysates were incubated with Flag M2 beads.

    Article Title: Identification of the viral RNA promoter stem loop A (SLA)-binding site on Zika virus polymerase NS5
    Article Snippet: .. For protein purification, the cells were resuspended in lysis buffer containing 50 mM Tris (pH 8.0), 500 mM NaCl, 2 mM β-mercaptoethanol, and 1 tablet of EDTA-free protease inhibitor (Roche Applied Science, Penzberg, Germany), and lysed by sonication. .. After centrifugation to clear the insoluble debris, the protein in the soluble fraction was purified by Talon metal affinity chromatography (Clontech, Mountain View, CA).

    Sonication:

    Article Title: Identification of the viral RNA promoter stem loop A (SLA)-binding site on Zika virus polymerase NS5
    Article Snippet: .. For protein purification, the cells were resuspended in lysis buffer containing 50 mM Tris (pH 8.0), 500 mM NaCl, 2 mM β-mercaptoethanol, and 1 tablet of EDTA-free protease inhibitor (Roche Applied Science, Penzberg, Germany), and lysed by sonication. .. After centrifugation to clear the insoluble debris, the protein in the soluble fraction was purified by Talon metal affinity chromatography (Clontech, Mountain View, CA).

    Plasmid Preparation:

    Article Title: Existence and functions of a kisspeptin neuropeptide signaling system in a non-chordate deuterostome species
    Article Snippet: .. The plasmid constructs were transfected into HEK293 cells using X-tremeGENE HP (Roche), according to the manufacturer's instructions. .. Two days after transfection, stably expressing cells can be selected by the addition of 800 mg/L G418 to avoid impacts from inefficient transfection in experiments.

    Hybridization:

    Article Title: Discovery and Characterization of Two Novel Salt-Tolerance Genes in Puccinellia tenuiflora
    Article Snippet: .. Northern Blot Analysis Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) from various organs of P. tenuiflora and cultured P. tenuiflora cells treated with 200 mM NaCl for 0, 6, 12, 24 and 48 h. For northern blot analysis, hybridization signals were detected using anti-DIG antibody conjugated with alkaline phosphatase (Roche, Indianapolis, IN, USA) and CDP-Star (Roche), as described by Sambrook et al. [ ]. .. Signals were detected using a luminescent image analyzer (Fujifilm, LAS-4000 mini, Tokyo, Japan).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93
    Roche pronase
    Clofoctol can specifically bind UNR and regulate KLF13, thus inducing GSC inhibition. ( A ) Western blot showing protection of the UNR protein from <t>pronase</t> digestion upon clofoctol binding in the DARTS assay. Samples of more than 3 individual experiments were assayed. ( B . ( C ) Knockdown of UNR could decrease the expression of KLF13 at the RNA and protein levels. ( D ) Limiting dilution tumorsphere assay in control or UNR-knockdown GSC2 cells. Ten wells were analyzed for each group. ( E ) Percent cell viability inhibition of GSC2 cells transiently transfected with UNR-si3 and UNR-si4 treated with clofoctol for 48 hours. ( F ) RIP assay analysis of the binding between UNR and KLF13 mRNA. PABPC1 is the positive control. ( G ) Clofoctol treatment (3 μM) increased the binding between UNR and KLF13 mRNA. ( H and I ) KLF13 mRNA stability assay in GSC2 cells without ( H ) or with ( I ) UNR knockdown. ( J and K ) Analysis of the expression of KLF13 mRNA ( J ) and the expression of UNR and KLF13 at the protein level ( K ) under clofoctol treatment with or without UNR knockdown. Data are presented as the mean ± SEM. For C , E – G , and J , 2-tailed Student’s t test was used. * P
    Pronase, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pronase/product/Roche
    Average 93 stars, based on 33 article reviews
    Price from $9.99 to $1999.99
    pronase - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    88
    Roche caspase lysis buffer
    Nutlin prevents <t>caspase</t> activation and γH2AX accumulation in response to Wee1 inhibitor and/or gemcitabine A . U2OS cells were treated with 8μM Nutlin for 24 hrs, followed by treatment with 1μM Wee1 inhibitor, 300nM gemcitabine, and/or 8μM Nutlin in the absence and presence of 50μM ZVAD-FMK for another 24 hrs. Cells were harvested and immunoblot analysis was performed to detect poly-ADP ribose polymerase (PARP) and γH2AX. B ., C . U2OS cells were treated as in (A). The cells were then fixed and stained for γH2AX by immunofluorescence. Detection and analysis was performed using automated immunofluorescence microscopy (BD Pathway). Figure panel (B) shows images of γH2AX staining for each treatment condition. Quantitation of γH2AX intensities was done using the BD pathway analysis tool and depicted in figure panel (C). Error bars represent the SD, n=3. D . U2OS cells were treated with 8μM Nutlin for 24 hrs, followed by treatment with 1μM Wee1 inhibitor, 300nM gemcitabine, 8μM Nutlin in the absence and presence ( Supplementary Figure 1 ) of 50μM ZVAD-FMK for another 24 hrs. The cells were harvested and lysed for caspase activity assay. Fluorescent intensity measurements were obtained for each treatment. The activity (arbitrary units of fluorescence/min) was calculated for each treatment at the linear part of the curve (cf. Supplementary Figure 1 ). Error bars represent the S.D, n=3.
    Caspase Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/caspase lysis buffer/product/Roche
    Average 88 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    caspase lysis buffer - by Bioz Stars, 2020-09
    88/100 stars
      Buy from Supplier

    85
    Roche ns5a lysis buffer
    Mechanistic model illustrating formation of the HCV replication complex, with a focus on the role of tyrosine phosphorylation events. A, ). B, ) between LCS II or D2 of <t>NS5A</t> and the SH3 domain may also contribute ( black dotted arrows ). NS5B is complexed through c-Src–SH3 ( gray dotted arrow ) and NS5A ( black broken arrows for a discussion of steric restraints). DAAs like daclatasvir can bind to NS5A lacking phosphorylated Tyr-93 but are ineffective in the presence of pTyr-93. Regulatory tyrosine phosphorylation in c-Src is highlighted in yellow ; NS5A–D1 phosphorylation at Tyr-93 is marked in red (abbreviations used are: AH, amphipathic helix; TD, transmembrane domain).
    Ns5a Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ns5a lysis buffer/product/Roche
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ns5a lysis buffer - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    84
    Roche mix staged transgenic worms expressing tofu 6
    <t>TOFU-6,</t> PID-1 and PICS-1 are required for 21U-RNA maturation. (A) Normalized 21U-RNA counts in N2, pid-1(ust64) and pics-1(tm2417) animals at late young adult stage. pics-1(tm2417) worms were isolated from balancers. Reads were normalized to total RNA reads. (B) Relative coverage of individual bases of 21U-RNA loci by reads from indicated animals. The annotated 5′ ends of 21U-RNA loci are at position 0. (C, D) Images of PID-1::GFP (C) and PICS-1::GFP (D) and P-granule marker mRuby::PGL-1 in germ cells. (E, F) Images of PID-1::GFP (E) and PICS-1::GFP (F) and histone marker mCherry::H2B after RNAi targeting tofu-6.
    Mix Staged Transgenic Worms Expressing Tofu 6, supplied by Roche, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mix staged transgenic worms expressing tofu 6/product/Roche
    Average 84 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mix staged transgenic worms expressing tofu 6 - by Bioz Stars, 2020-09
    84/100 stars
      Buy from Supplier

    Image Search Results


    Clofoctol can specifically bind UNR and regulate KLF13, thus inducing GSC inhibition. ( A ) Western blot showing protection of the UNR protein from pronase digestion upon clofoctol binding in the DARTS assay. Samples of more than 3 individual experiments were assayed. ( B . ( C ) Knockdown of UNR could decrease the expression of KLF13 at the RNA and protein levels. ( D ) Limiting dilution tumorsphere assay in control or UNR-knockdown GSC2 cells. Ten wells were analyzed for each group. ( E ) Percent cell viability inhibition of GSC2 cells transiently transfected with UNR-si3 and UNR-si4 treated with clofoctol for 48 hours. ( F ) RIP assay analysis of the binding between UNR and KLF13 mRNA. PABPC1 is the positive control. ( G ) Clofoctol treatment (3 μM) increased the binding between UNR and KLF13 mRNA. ( H and I ) KLF13 mRNA stability assay in GSC2 cells without ( H ) or with ( I ) UNR knockdown. ( J and K ) Analysis of the expression of KLF13 mRNA ( J ) and the expression of UNR and KLF13 at the protein level ( K ) under clofoctol treatment with or without UNR knockdown. Data are presented as the mean ± SEM. For C , E – G , and J , 2-tailed Student’s t test was used. * P

    Journal: The Journal of Clinical Investigation

    Article Title: The antibiotic clofoctol suppresses glioma stem cell proliferation by activating KLF13

    doi: 10.1172/JCI124979

    Figure Lengend Snippet: Clofoctol can specifically bind UNR and regulate KLF13, thus inducing GSC inhibition. ( A ) Western blot showing protection of the UNR protein from pronase digestion upon clofoctol binding in the DARTS assay. Samples of more than 3 individual experiments were assayed. ( B . ( C ) Knockdown of UNR could decrease the expression of KLF13 at the RNA and protein levels. ( D ) Limiting dilution tumorsphere assay in control or UNR-knockdown GSC2 cells. Ten wells were analyzed for each group. ( E ) Percent cell viability inhibition of GSC2 cells transiently transfected with UNR-si3 and UNR-si4 treated with clofoctol for 48 hours. ( F ) RIP assay analysis of the binding between UNR and KLF13 mRNA. PABPC1 is the positive control. ( G ) Clofoctol treatment (3 μM) increased the binding between UNR and KLF13 mRNA. ( H and I ) KLF13 mRNA stability assay in GSC2 cells without ( H ) or with ( I ) UNR knockdown. ( J and K ) Analysis of the expression of KLF13 mRNA ( J ) and the expression of UNR and KLF13 at the protein level ( K ) under clofoctol treatment with or without UNR knockdown. Data are presented as the mean ± SEM. For C , E – G , and J , 2-tailed Student’s t test was used. * P

    Article Snippet: After incubation, the mixture was digested using pronase (catalog 10165921001, Roche) at room temperature for 30 minutes.

    Techniques: Inhibition, Western Blot, Binding Assay, Expressing, Transfection, Positive Control, Stability Assay

    Nutlin prevents caspase activation and γH2AX accumulation in response to Wee1 inhibitor and/or gemcitabine A . U2OS cells were treated with 8μM Nutlin for 24 hrs, followed by treatment with 1μM Wee1 inhibitor, 300nM gemcitabine, and/or 8μM Nutlin in the absence and presence of 50μM ZVAD-FMK for another 24 hrs. Cells were harvested and immunoblot analysis was performed to detect poly-ADP ribose polymerase (PARP) and γH2AX. B ., C . U2OS cells were treated as in (A). The cells were then fixed and stained for γH2AX by immunofluorescence. Detection and analysis was performed using automated immunofluorescence microscopy (BD Pathway). Figure panel (B) shows images of γH2AX staining for each treatment condition. Quantitation of γH2AX intensities was done using the BD pathway analysis tool and depicted in figure panel (C). Error bars represent the SD, n=3. D . U2OS cells were treated with 8μM Nutlin for 24 hrs, followed by treatment with 1μM Wee1 inhibitor, 300nM gemcitabine, 8μM Nutlin in the absence and presence ( Supplementary Figure 1 ) of 50μM ZVAD-FMK for another 24 hrs. The cells were harvested and lysed for caspase activity assay. Fluorescent intensity measurements were obtained for each treatment. The activity (arbitrary units of fluorescence/min) was calculated for each treatment at the linear part of the curve (cf. Supplementary Figure 1 ). Error bars represent the S.D, n=3.

    Journal: Oncotarget

    Article Title: Mdm2 inhibition confers protection of p53-proficient cells from the cytotoxic effects of Wee1 inhibitors

    doi:

    Figure Lengend Snippet: Nutlin prevents caspase activation and γH2AX accumulation in response to Wee1 inhibitor and/or gemcitabine A . U2OS cells were treated with 8μM Nutlin for 24 hrs, followed by treatment with 1μM Wee1 inhibitor, 300nM gemcitabine, and/or 8μM Nutlin in the absence and presence of 50μM ZVAD-FMK for another 24 hrs. Cells were harvested and immunoblot analysis was performed to detect poly-ADP ribose polymerase (PARP) and γH2AX. B ., C . U2OS cells were treated as in (A). The cells were then fixed and stained for γH2AX by immunofluorescence. Detection and analysis was performed using automated immunofluorescence microscopy (BD Pathway). Figure panel (B) shows images of γH2AX staining for each treatment condition. Quantitation of γH2AX intensities was done using the BD pathway analysis tool and depicted in figure panel (C). Error bars represent the SD, n=3. D . U2OS cells were treated with 8μM Nutlin for 24 hrs, followed by treatment with 1μM Wee1 inhibitor, 300nM gemcitabine, 8μM Nutlin in the absence and presence ( Supplementary Figure 1 ) of 50μM ZVAD-FMK for another 24 hrs. The cells were harvested and lysed for caspase activity assay. Fluorescent intensity measurements were obtained for each treatment. The activity (arbitrary units of fluorescence/min) was calculated for each treatment at the linear part of the curve (cf. Supplementary Figure 1 ). Error bars represent the S.D, n=3.

    Article Snippet: The pelleted cells were resuspended in 250μl caspase lysis buffer (1M Tris-HCl, 2mM MgCl2 , 150mM NaCl, 10mM DTT, Roche complete mini protease-inhibitor mix).

    Techniques: Activation Assay, Staining, Immunofluorescence, Microscopy, Quantitation Assay, Caspase Activity Assay, Activity Assay, Fluorescence

    Mechanistic model illustrating formation of the HCV replication complex, with a focus on the role of tyrosine phosphorylation events. A, ). B, ) between LCS II or D2 of NS5A and the SH3 domain may also contribute ( black dotted arrows ). NS5B is complexed through c-Src–SH3 ( gray dotted arrow ) and NS5A ( black broken arrows for a discussion of steric restraints). DAAs like daclatasvir can bind to NS5A lacking phosphorylated Tyr-93 but are ineffective in the presence of pTyr-93. Regulatory tyrosine phosphorylation in c-Src is highlighted in yellow ; NS5A–D1 phosphorylation at Tyr-93 is marked in red (abbreviations used are: AH, amphipathic helix; TD, transmembrane domain).

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphorylated tyrosine 93 of hepatitis C virus nonstructural protein 5A is essential for interaction with host c-Src and efficient viral replication

    doi: 10.1074/jbc.RA119.007656

    Figure Lengend Snippet: Mechanistic model illustrating formation of the HCV replication complex, with a focus on the role of tyrosine phosphorylation events. A, ). B, ) between LCS II or D2 of NS5A and the SH3 domain may also contribute ( black dotted arrows ). NS5B is complexed through c-Src–SH3 ( gray dotted arrow ) and NS5A ( black broken arrows for a discussion of steric restraints). DAAs like daclatasvir can bind to NS5A lacking phosphorylated Tyr-93 but are ineffective in the presence of pTyr-93. Regulatory tyrosine phosphorylation in c-Src is highlighted in yellow ; NS5A–D1 phosphorylation at Tyr-93 is marked in red (abbreviations used are: AH, amphipathic helix; TD, transmembrane domain).

    Article Snippet: For protein purification, cells were resuspended in NS5A-lysis buffer (100 m m Tris-HCl, 100 m m NaCl, 5 m m imidazole, protease inhibitor mixture (Roche Applied Science), 1% (v/v) Triton X-100, DNase, 0.2 m m OV (for ELK tyrosine–phosphorylated proteins), pH 8.0) and homogenized in a cell disruptor at 2000 bar, and after centrifugation at 50,000 × g for 1 h, the supernatant was applied to a nickel-nitrilotriacetic acid–affinity column (Macherey-Nagel, Düren, Germany) connected to an Äkta prime system (GE Healthcare) and equilibrated in NS5A affinity buffer (100 m m Tris-HCl, 300 m m NaCl, 5 m m imidazole, pH 8.0).

    Techniques:

    Schematic representation of Tyr-containing peptides derived from NS5A ( A ) and screening of NS5A tyrosine residues for binding to c-Src–SH2 ( B ). Binding of the GST–c-Src constructs to the various immobilized biotinylated peptides was measured colorimetrically. Binding was normalized to the best binder (pTyr-93), which was arbitrarily set to 100. Mean and S.D. of n = 3 experiments are shown.

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphorylated tyrosine 93 of hepatitis C virus nonstructural protein 5A is essential for interaction with host c-Src and efficient viral replication

    doi: 10.1074/jbc.RA119.007656

    Figure Lengend Snippet: Schematic representation of Tyr-containing peptides derived from NS5A ( A ) and screening of NS5A tyrosine residues for binding to c-Src–SH2 ( B ). Binding of the GST–c-Src constructs to the various immobilized biotinylated peptides was measured colorimetrically. Binding was normalized to the best binder (pTyr-93), which was arbitrarily set to 100. Mean and S.D. of n = 3 experiments are shown.

    Article Snippet: For protein purification, cells were resuspended in NS5A-lysis buffer (100 m m Tris-HCl, 100 m m NaCl, 5 m m imidazole, protease inhibitor mixture (Roche Applied Science), 1% (v/v) Triton X-100, DNase, 0.2 m m OV (for ELK tyrosine–phosphorylated proteins), pH 8.0) and homogenized in a cell disruptor at 2000 bar, and after centrifugation at 50,000 × g for 1 h, the supernatant was applied to a nickel-nitrilotriacetic acid–affinity column (Macherey-Nagel, Düren, Germany) connected to an Äkta prime system (GE Healthcare) and equilibrated in NS5A affinity buffer (100 m m Tris-HCl, 300 m m NaCl, 5 m m imidazole, pH 8.0).

    Techniques: Derivative Assay, Binding Assay, Construct

    Binding affinities of NS5A-derived peptides to c-Src–derived constructs determined by fluorescence polarization. Dissociation constants ( K d ) of different NS5A peptides toward c-Src–ΔSH1 ( A ), c-Src–SH2 ( B ), and c-Src–SH3SH2 ( C .

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphorylated tyrosine 93 of hepatitis C virus nonstructural protein 5A is essential for interaction with host c-Src and efficient viral replication

    doi: 10.1074/jbc.RA119.007656

    Figure Lengend Snippet: Binding affinities of NS5A-derived peptides to c-Src–derived constructs determined by fluorescence polarization. Dissociation constants ( K d ) of different NS5A peptides toward c-Src–ΔSH1 ( A ), c-Src–SH2 ( B ), and c-Src–SH3SH2 ( C .

    Article Snippet: For protein purification, cells were resuspended in NS5A-lysis buffer (100 m m Tris-HCl, 100 m m NaCl, 5 m m imidazole, protease inhibitor mixture (Roche Applied Science), 1% (v/v) Triton X-100, DNase, 0.2 m m OV (for ELK tyrosine–phosphorylated proteins), pH 8.0) and homogenized in a cell disruptor at 2000 bar, and after centrifugation at 50,000 × g for 1 h, the supernatant was applied to a nickel-nitrilotriacetic acid–affinity column (Macherey-Nagel, Düren, Germany) connected to an Äkta prime system (GE Healthcare) and equilibrated in NS5A affinity buffer (100 m m Tris-HCl, 300 m m NaCl, 5 m m imidazole, pH 8.0).

    Techniques: Binding Assay, Derivative Assay, Construct, Fluorescence

    Substitution of Tyr-93 with phenylalanine in NS5A severely impairs HCV replication and virus production in human hepatoma cells. A, Huh-7 cells were transfected with either WT HCV replicon plasmid pFK-I377/NS3-NS3′ ( Rep ) or the mutated plasmid pFK-I377/NS3-NS3′-Y93F ( Rep Y93F ). B and C, Huh-7.5 cells were transfected with either WT HCV cc JC1 plasmid pFK-JFH1J6C-846_dg (JC1) or the mutated plasmid pFK-JFH1J6C-846_dg-Y93F (JC1 Y93F). A and C, total mRNA was determined by semi-quantitative rtPCR, and results were calculated using the ΔΔ CT method and SDHA as the control gene. Data are provided in relation to the respective control cells (Rep or JC1, set as 100) and are depicted as means ± S.D. of at least three independent experiments. The respective differences were significant ( p = 0.037 and p = 0.032, respectively) as indicated by the one-tailed Mann-Whitney test. B, virus was collected from the supernatants, concentrated by PEG precipitation and infective titers determined by the TCID 50 assay. Significant differences ( p = 0.029) were confirmed using the one-tailed Mann-Whitney test.

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphorylated tyrosine 93 of hepatitis C virus nonstructural protein 5A is essential for interaction with host c-Src and efficient viral replication

    doi: 10.1074/jbc.RA119.007656

    Figure Lengend Snippet: Substitution of Tyr-93 with phenylalanine in NS5A severely impairs HCV replication and virus production in human hepatoma cells. A, Huh-7 cells were transfected with either WT HCV replicon plasmid pFK-I377/NS3-NS3′ ( Rep ) or the mutated plasmid pFK-I377/NS3-NS3′-Y93F ( Rep Y93F ). B and C, Huh-7.5 cells were transfected with either WT HCV cc JC1 plasmid pFK-JFH1J6C-846_dg (JC1) or the mutated plasmid pFK-JFH1J6C-846_dg-Y93F (JC1 Y93F). A and C, total mRNA was determined by semi-quantitative rtPCR, and results were calculated using the ΔΔ CT method and SDHA as the control gene. Data are provided in relation to the respective control cells (Rep or JC1, set as 100) and are depicted as means ± S.D. of at least three independent experiments. The respective differences were significant ( p = 0.037 and p = 0.032, respectively) as indicated by the one-tailed Mann-Whitney test. B, virus was collected from the supernatants, concentrated by PEG precipitation and infective titers determined by the TCID 50 assay. Significant differences ( p = 0.029) were confirmed using the one-tailed Mann-Whitney test.

    Article Snippet: For protein purification, cells were resuspended in NS5A-lysis buffer (100 m m Tris-HCl, 100 m m NaCl, 5 m m imidazole, protease inhibitor mixture (Roche Applied Science), 1% (v/v) Triton X-100, DNase, 0.2 m m OV (for ELK tyrosine–phosphorylated proteins), pH 8.0) and homogenized in a cell disruptor at 2000 bar, and after centrifugation at 50,000 × g for 1 h, the supernatant was applied to a nickel-nitrilotriacetic acid–affinity column (Macherey-Nagel, Düren, Germany) connected to an Äkta prime system (GE Healthcare) and equilibrated in NS5A affinity buffer (100 m m Tris-HCl, 300 m m NaCl, 5 m m imidazole, pH 8.0).

    Techniques: Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, One-tailed Test, MANN-WHITNEY

    NS5A tyrosine screening confirms the presence of a canonical SH2-binding motif in NS5A

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphorylated tyrosine 93 of hepatitis C virus nonstructural protein 5A is essential for interaction with host c-Src and efficient viral replication

    doi: 10.1074/jbc.RA119.007656

    Figure Lengend Snippet: NS5A tyrosine screening confirms the presence of a canonical SH2-binding motif in NS5A

    Article Snippet: For protein purification, cells were resuspended in NS5A-lysis buffer (100 m m Tris-HCl, 100 m m NaCl, 5 m m imidazole, protease inhibitor mixture (Roche Applied Science), 1% (v/v) Triton X-100, DNase, 0.2 m m OV (for ELK tyrosine–phosphorylated proteins), pH 8.0) and homogenized in a cell disruptor at 2000 bar, and after centrifugation at 50,000 × g for 1 h, the supernatant was applied to a nickel-nitrilotriacetic acid–affinity column (Macherey-Nagel, Düren, Germany) connected to an Äkta prime system (GE Healthcare) and equilibrated in NS5A affinity buffer (100 m m Tris-HCl, 300 m m NaCl, 5 m m imidazole, pH 8.0).

    Techniques: Binding Assay

    TOFU-6, PID-1 and PICS-1 are required for 21U-RNA maturation. (A) Normalized 21U-RNA counts in N2, pid-1(ust64) and pics-1(tm2417) animals at late young adult stage. pics-1(tm2417) worms were isolated from balancers. Reads were normalized to total RNA reads. (B) Relative coverage of individual bases of 21U-RNA loci by reads from indicated animals. The annotated 5′ ends of 21U-RNA loci are at position 0. (C, D) Images of PID-1::GFP (C) and PICS-1::GFP (D) and P-granule marker mRuby::PGL-1 in germ cells. (E, F) Images of PID-1::GFP (E) and PICS-1::GFP (F) and histone marker mCherry::H2B after RNAi targeting tofu-6.

    Journal: bioRxiv

    Article Title: Differential phase partition of a PICS complex is required for piRNA processing and chromosome segregation in C. elegans

    doi: 10.1101/463919

    Figure Lengend Snippet: TOFU-6, PID-1 and PICS-1 are required for 21U-RNA maturation. (A) Normalized 21U-RNA counts in N2, pid-1(ust64) and pics-1(tm2417) animals at late young adult stage. pics-1(tm2417) worms were isolated from balancers. Reads were normalized to total RNA reads. (B) Relative coverage of individual bases of 21U-RNA loci by reads from indicated animals. The annotated 5′ ends of 21U-RNA loci are at position 0. (C, D) Images of PID-1::GFP (C) and PICS-1::GFP (D) and P-granule marker mRuby::PGL-1 in germ cells. (E, F) Images of PID-1::GFP (E) and PICS-1::GFP (F) and histone marker mCherry::H2B after RNAi targeting tofu-6.

    Article Snippet: Immunoprecipitation followed by mass spectrometry analysis The mix-staged transgenic worms expressing TOFU-6::GFP, TOST-1::GFP, PICS-1::GFP, and ERH-2::GFP were resuspended in the same volume of 2x lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1% Triton-X100, Roche®cOmplete™ EDTA-free Protease Inhibitor Cocktail, 10 mM NaF, 2 mM Na3 VO4) and lysed in a FastPrep-24™ 5G Homogenizer.

    Techniques: Isolation, Marker

    TOFU-6, PICS-1, ERH-2, PID-1 and TOST-1 interact with each other both in vitro and in vivo . (A, B) Images of TOST-1::GFP (A) and ERH-2::GFP (B) and P-granule marker mRuby::PGL-1 in germ cells. (C, D, E, F) Western blotting of pulling down samples to assay protein-protein interactions between TOFU-6, PICS-1, ERH-2, PID-1 and TOST-1 in vitro. (G) Summary of the protein-protein interaction of TOFU-6, PICS-1, ERH-2, PID-1 and TOST-1 both in vitro and in vivo . Red lines indicate interactions in vitro . Blue arrows indicate interaction in vivo .

    Journal: bioRxiv

    Article Title: Differential phase partition of a PICS complex is required for piRNA processing and chromosome segregation in C. elegans

    doi: 10.1101/463919

    Figure Lengend Snippet: TOFU-6, PICS-1, ERH-2, PID-1 and TOST-1 interact with each other both in vitro and in vivo . (A, B) Images of TOST-1::GFP (A) and ERH-2::GFP (B) and P-granule marker mRuby::PGL-1 in germ cells. (C, D, E, F) Western blotting of pulling down samples to assay protein-protein interactions between TOFU-6, PICS-1, ERH-2, PID-1 and TOST-1 in vitro. (G) Summary of the protein-protein interaction of TOFU-6, PICS-1, ERH-2, PID-1 and TOST-1 both in vitro and in vivo . Red lines indicate interactions in vitro . Blue arrows indicate interaction in vivo .

    Article Snippet: Immunoprecipitation followed by mass spectrometry analysis The mix-staged transgenic worms expressing TOFU-6::GFP, TOST-1::GFP, PICS-1::GFP, and ERH-2::GFP were resuspended in the same volume of 2x lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1% Triton-X100, Roche®cOmplete™ EDTA-free Protease Inhibitor Cocktail, 10 mM NaF, 2 mM Na3 VO4) and lysed in a FastPrep-24™ 5G Homogenizer.

    Techniques: In Vitro, In Vivo, Marker, Western Blot

    TOFU-6, PICS-1, ERH-2 and TOST-1 are required for chromosome segregation and cell division. (A) Images of GFP::H2B at meta-anaphase stage after RNAi targeting indicated genes in embryos. (B) Diagram displaying percentage of abnormal chromosome segregation. (C) Images of GFP::H2B in late embryos after RNAi targeting indicated genes.

    Journal: bioRxiv

    Article Title: Differential phase partition of a PICS complex is required for piRNA processing and chromosome segregation in C. elegans

    doi: 10.1101/463919

    Figure Lengend Snippet: TOFU-6, PICS-1, ERH-2 and TOST-1 are required for chromosome segregation and cell division. (A) Images of GFP::H2B at meta-anaphase stage after RNAi targeting indicated genes in embryos. (B) Diagram displaying percentage of abnormal chromosome segregation. (C) Images of GFP::H2B in late embryos after RNAi targeting indicated genes.

    Article Snippet: Immunoprecipitation followed by mass spectrometry analysis The mix-staged transgenic worms expressing TOFU-6::GFP, TOST-1::GFP, PICS-1::GFP, and ERH-2::GFP were resuspended in the same volume of 2x lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1% Triton-X100, Roche®cOmplete™ EDTA-free Protease Inhibitor Cocktail, 10 mM NaF, 2 mM Na3 VO4) and lysed in a FastPrep-24™ 5G Homogenizer.

    Techniques:

    TOFU-6, PICS-1, ERH-2 and TOST-1 accumulate in nucleus during cell division. (A, B) Images of TOFU-6/PICS-1/ERH-2/TOST-1::GFP in early embryos. Diagram displaying percentage of TOFU-6/PICS-1/ERH-2/TOST-1::GFP accumulating in nucleus in two-cell-embryos (A) and four-cell-embryos (B). (C) Images of TOFU-6/PICS-1/ERH-2/TOST-1::GFP in two-cell-embryos after RNAi targeting indicated genes. Diagram displaying percentage of TOFU-6/PICS-1/ERH-2/TOST-1::GFP accumulating in nucleus after RNAi targeting indicated genes.

    Journal: bioRxiv

    Article Title: Differential phase partition of a PICS complex is required for piRNA processing and chromosome segregation in C. elegans

    doi: 10.1101/463919

    Figure Lengend Snippet: TOFU-6, PICS-1, ERH-2 and TOST-1 accumulate in nucleus during cell division. (A, B) Images of TOFU-6/PICS-1/ERH-2/TOST-1::GFP in early embryos. Diagram displaying percentage of TOFU-6/PICS-1/ERH-2/TOST-1::GFP accumulating in nucleus in two-cell-embryos (A) and four-cell-embryos (B). (C) Images of TOFU-6/PICS-1/ERH-2/TOST-1::GFP in two-cell-embryos after RNAi targeting indicated genes. Diagram displaying percentage of TOFU-6/PICS-1/ERH-2/TOST-1::GFP accumulating in nucleus after RNAi targeting indicated genes.

    Article Snippet: Immunoprecipitation followed by mass spectrometry analysis The mix-staged transgenic worms expressing TOFU-6::GFP, TOST-1::GFP, PICS-1::GFP, and ERH-2::GFP were resuspended in the same volume of 2x lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1% Triton-X100, Roche®cOmplete™ EDTA-free Protease Inhibitor Cocktail, 10 mM NaF, 2 mM Na3 VO4) and lysed in a FastPrep-24™ 5G Homogenizer.

    Techniques:

    Identification of IFE-3 as an additional factor interacting with TOFU-6. (A) Western blotting of pull down samples to assay protein-protein interactions between IFE-3 and TOFU-6 in vitro. (B) Images of IFE-3 in germline cells. (C) A working model of the PICS complex in germline and embryos.

    Journal: bioRxiv

    Article Title: Differential phase partition of a PICS complex is required for piRNA processing and chromosome segregation in C. elegans

    doi: 10.1101/463919

    Figure Lengend Snippet: Identification of IFE-3 as an additional factor interacting with TOFU-6. (A) Western blotting of pull down samples to assay protein-protein interactions between IFE-3 and TOFU-6 in vitro. (B) Images of IFE-3 in germline cells. (C) A working model of the PICS complex in germline and embryos.

    Article Snippet: Immunoprecipitation followed by mass spectrometry analysis The mix-staged transgenic worms expressing TOFU-6::GFP, TOST-1::GFP, PICS-1::GFP, and ERH-2::GFP were resuspended in the same volume of 2x lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1% Triton-X100, Roche®cOmplete™ EDTA-free Protease Inhibitor Cocktail, 10 mM NaF, 2 mM Na3 VO4) and lysed in a FastPrep-24™ 5G Homogenizer.

    Techniques: Western Blot, In Vitro

    Identification of TOFU-6 binding proteins by functional proteomics. (A) Summary of IP-MS followed by feeding RNAi experiments of TOFU-6::GFP. (B) Images of TOFU-6::GFP and P-granule marker mRuby::PGL-1 after RNAi targeting pid-1 and pics-1 . (C) Images of TOFU-6::GFP in indicated animals. (D) Images of TOFU-6::GFP after RNAi targeting tost-1 and erh-2 . (E) Western blotting of TOFU-6::GFP after RNAi targeting tost-1 and erh-2 .

    Journal: bioRxiv

    Article Title: Differential phase partition of a PICS complex is required for piRNA processing and chromosome segregation in C. elegans

    doi: 10.1101/463919

    Figure Lengend Snippet: Identification of TOFU-6 binding proteins by functional proteomics. (A) Summary of IP-MS followed by feeding RNAi experiments of TOFU-6::GFP. (B) Images of TOFU-6::GFP and P-granule marker mRuby::PGL-1 after RNAi targeting pid-1 and pics-1 . (C) Images of TOFU-6::GFP in indicated animals. (D) Images of TOFU-6::GFP after RNAi targeting tost-1 and erh-2 . (E) Western blotting of TOFU-6::GFP after RNAi targeting tost-1 and erh-2 .

    Article Snippet: Immunoprecipitation followed by mass spectrometry analysis The mix-staged transgenic worms expressing TOFU-6::GFP, TOST-1::GFP, PICS-1::GFP, and ERH-2::GFP were resuspended in the same volume of 2x lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1% Triton-X100, Roche®cOmplete™ EDTA-free Protease Inhibitor Cocktail, 10 mM NaF, 2 mM Na3 VO4) and lysed in a FastPrep-24™ 5G Homogenizer.

    Techniques: Binding Assay, Functional Assay, Marker, Western Blot

    tofu-6 is required for 21U-RNA biogenesis and chromosome segregation. (A) Images of TOFU-6::GFP and P-granule marker mRuby::PGL-1 in germ cells. (B) Schematic of four alleles of tofu-6 . (C) Normalized 21U-RNA reads in indicated animals at late young adult stage. Indicated mutant animals were isolated from balancers. Reads were normalized to total RNA reads. (D) Bar diagram displaying brood size of indicated worms. Worms were grown at 20. (E) Images of GFP::H2B at meta-anaphase of two-cells stage embryos in indicated worms. (F) Images of GFP::H2B in indicated later stage embryos.

    Journal: bioRxiv

    Article Title: Differential phase partition of a PICS complex is required for piRNA processing and chromosome segregation in C. elegans

    doi: 10.1101/463919

    Figure Lengend Snippet: tofu-6 is required for 21U-RNA biogenesis and chromosome segregation. (A) Images of TOFU-6::GFP and P-granule marker mRuby::PGL-1 in germ cells. (B) Schematic of four alleles of tofu-6 . (C) Normalized 21U-RNA reads in indicated animals at late young adult stage. Indicated mutant animals were isolated from balancers. Reads were normalized to total RNA reads. (D) Bar diagram displaying brood size of indicated worms. Worms were grown at 20. (E) Images of GFP::H2B at meta-anaphase of two-cells stage embryos in indicated worms. (F) Images of GFP::H2B in indicated later stage embryos.

    Article Snippet: Immunoprecipitation followed by mass spectrometry analysis The mix-staged transgenic worms expressing TOFU-6::GFP, TOST-1::GFP, PICS-1::GFP, and ERH-2::GFP were resuspended in the same volume of 2x lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1% Triton-X100, Roche®cOmplete™ EDTA-free Protease Inhibitor Cocktail, 10 mM NaF, 2 mM Na3 VO4) and lysed in a FastPrep-24™ 5G Homogenizer.

    Techniques: Marker, Mutagenesis, Isolation