Journal: bioRxiv

Article Title: Synthetic essentiality of metabolic regulator PDHK1 in PTEN-deficient cells and cancers

doi: 10.1101/441295

Figure Lengend Snippet: (A) (Left) Workflow of the unbiased, global phospho-proteomic profiling used in PTEN-deficient cancer cell lines stably expressing PTEN WT or PTEN G129E or PTEN Y138L or GFP to identify the phospho-peptides (and corresponding proteins) affected specifically by the protein or lipid phosphatase (PP or LP) activity of PTEN. (Right) Venn-diagram showing the number of proteins with phospho-sites specifically affected by the protein phosphatase activity of PTEN in H1650 (n = 169) and A2058 (n = 248) cells, with the phospho-proteins (including NFκB activating protein, NKAP highlighted in white) affected in both cell lines shown in the overlap (n = 42). (B) Schematic representation of NKAP phospho-sites at serines 9 and 149 affected by the protein phosphatase activity of PTEN. (C) Phosphate released (μM) from a phospho-S9-NKAP or phospho-S149-NKAP or phospho-S72-Rab7 (control) peptide after incubation without or with recombinant WT PTEN or mutant Y138L PTEN enzyme in an in-vitro Malachite green based colorimetric assay. Data are shown as mean ± SD (n=2 replicates). * p < 0.05; ** p < 0.01; **** p < 0.0001 compared to ‘no PTEN’ control by two-tailed unpaired t test with Welch’s correction. (D) Detection of phospho-NKAP and its de-phosphorylated species (indicated by the red dotted inset) in PTEN-deficient cancer cell lines stably expressing either PTEN WT or PTEN G129E or PTEN Y138L or GFP by Phos-tag PAGE and immunoblotting. De-phosphorylation score indicates the extent to which expression of each PTEN mutant in PTEN-deficient cells suppresses NKAP de-phosphorylation relative to WT PTEN (set at 1). A lower de-phosphorylation score indicates less de-phosphorylation of NKAP. (E) Co-immunoprecipitation of NKAP (indicated by the red dotted inset) with PTEN upon over-expression of both NKAP-V5 and FLAG-PTEN and not NKAP-V5 over-expression alone or no over-expression in 293T cells followed by IP-FLAG is shown. Arrowhead denotes NKAP-V5; while the dark band below is background due to secondary antibody cross-reactivity to the immunoglobulin heavy chain (IgH) used in IP. See also .

Article Snippet: HEK 293T cells were transfected with pCMV-2xFLAG PTEN WT and pLX-TRC304-NKAP-V5 expression plasmids using TransIT-LT1 transfection reagent (Mirus Bio) according to manufacturer’s instructions.

Techniques: Stable Transfection, Expressing, Activity Assay, Incubation, Recombinant, Mutagenesis, In Vitro, Colorimetric Assay, Two Tailed Test, Western Blot, De-Phosphorylation Assay, Immunoprecipitation, Over Expression

Journal: bioRxiv

Article Title: Synthetic essentiality of metabolic regulator PDHK1 in PTEN-deficient cells and cancers

doi: 10.1101/441295

Figure Lengend Snippet: (A) Phospho-proteins (n=42, listed alphabetically), including NKAP (highlighted in red box), regulated specifically by the PTEN protein-phosphatase in both H1650 and A2058 PTEN-deficient cancer cell lines stably expressing either PTEN WT or PTEN G129E or PTEN Y138L or GFP identified by an unbiased global phospho-proteomic profiling analysis (Methods) are shown. Venn diagram representation of the phospho-proteins regulated specifically by the PTEN protein phosphatase in either H1650 (n = 169) or A2058 (n = 248) or both (n = 42) cell lines. (B) Co-immunoprecipitation of NHERF2 with PTEN upon over-expression of both GST-NHERF2 and FLAG-PTEN in 293T cells followed by IP-FLAG is shown.

Article Snippet: HEK 293T cells were transfected with pCMV-2xFLAG PTEN WT and pLX-TRC304-NKAP-V5 expression plasmids using TransIT-LT1 transfection reagent (Mirus Bio) according to manufacturer’s instructions.

Techniques: Stable Transfection, Expressing, Immunoprecipitation, Over Expression

Journal: Cell

Article Title: TMEM41B Is a Pan-flavivirus Host Factor

doi: 10.1016/j.cell.2020.12.005

Figure Lengend Snippet:

Article Snippet: Human: HEK293T/17 (embryonic kidney epithelial) , ATCC , Cat.#CRL-11268 ; RRID: CVCL_1926.

Techniques: Virus, Subcloning, Western Blot, Recombinant, Infection, Transfection, Protease Inhibitor, Staining, Bicinchoninic Acid Protein Assay, Immunoprecipitation, SYBR Green Assay, cDNA Synthesis, Sequencing, Derivative Assay, Plasmid Preparation, Software

Journal: eLife

Article Title: Piezo1/2 mediate mechanotransduction essential for bone formation through concerted activation of NFAT-YAP1-ß-catenin

doi: 10.7554/eLife.52779

Figure Lengend Snippet: ( a ) Luciferase reporter assays of NFATc1 (pGL3-NFAT), YAP1 (8XGTIIC) and CTNNB1 (SUPERTOPFLASH) transcription activities in HEK293T cells. N = 3, data are shown as means ± SD. *p<0.05, **p<0.01, ***p<0.001, two-tailed unpaired Student’s t -test and one-way ANOVA followed by Tukey’s multiple comparisons tests when ANOVA was significant . Expression one of NFATc1, YAP1 and CTNNB1 led to activation of the other two. The only exception is that YAP1 overexpression reducd NFATc1 activties. ( b, d ) Immunoprecipitation (IP) assays of HEK293T cell lysates with the indicated expression constructs. ( b ) Yoda1 treatment (40 μM, 4 hr) promoted complex formation of NFATc1 and YAP1, which was inhibited by CsA or Gd 3+ treatment. ( c ) Western blotting assay of HEK 293 T cells transfected with HA-tagged YAP1 and S-tagged PPP3CA as indicated. DMSO (vehicle) or Yoda1 (40 μM) treatment was performed 24 hr after transfection for 4 hr. Arrow indicates endogenous PPP3CA. ( d ) Expression of NFATc1, YAP1 and CTNNB1 together promoted their complex formation. ( e ) Western blotting analyses of Piezo1/2 deficient BMSCs treated with Yoda1. Yoda1 treatment failed to reduce phosphorylation of Ctnnb1 and Yap1. Ppp3ca: Catalytic subunit of CaN. ( f ) Western blotting analyses of wild-type primary BMSCs treated by Gd 3+ . Increased Nfatc1 phosphorylation (blue arrow), as well as increased phosphorylation of Yap1 and Ctnnb1 was observed . Figure 9—figure supplement 1—source data 1. Original Western blots.

Article Snippet: Cell line ( Homo sapiens ) , HEK 293T , ATCC , Cat# ACS-4500, RRID: CVCL_4V93 , .

Techniques: Luciferase, Two Tailed Test, Expressing, Activation Assay, Over Expression, Immunoprecipitation, Construct, Western Blot, Transfection, Phospho-proteomics

Journal: eLife

Article Title: Piezo1/2 mediate mechanotransduction essential for bone formation through concerted activation of NFAT-YAP1-ß-catenin

doi: 10.7554/eLife.52779

Figure Lengend Snippet:

Article Snippet: Cell line ( Homo sapiens ) , HEK 293T , ATCC , Cat# ACS-4500, RRID: CVCL_4V93 , .

Techniques: Transfection, Construct, Negative Control, Isolation, Recombinant, Plasmid Preparation, Luciferase, Sequencing, Gel Extraction, RNAscope, Gene Expression, Reporter Assay, Enzyme-linked Immunosorbent Assay, Staining, TUNEL Assay, SYBR Green Assay, Reverse Transcription, Labeling, Blocking Assay, Software

Journal: Oncogene

Article Title: USP4 inhibits p53 and NF-κB through deubiquitinating and stabilizing HDAC2

doi: 10.1038/onc.2015.349

Figure Lengend Snippet: USP4 interacts with HDAC2. ( a ) The 293T cells were co-transfected with the indicated plasmids, and cell lysates were immunoprecipitated with the indicated antibodies and immunoblotted with anti-Flag or anti-Myc antibody. ( b ) Immunoprecipitation of HDAC2 from H1299 cells, followed by western blotting of the precipitated proteins with anti-HDAC2 and anti-USP4 antibodies. ( c ) In vitro binding assay. Purified His-USP4 (2 μg) from E. coli BL21 was incubated with GST or GST-HDAC2 bound to glutathione–sepharose beads. Proteins retained on the beads were then blotted with the indicated antibodies. ( d ) Mapping of the HDAC2 domains responsible for binding to USP4. The 293T cells were co-transfected with Myc-USP4 and the indicated truncated Flag-HDAC2 mutants, and cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with anti-Myc antibody. ( e ) Schematic representation of HAC2 and HDAC2 deletion mutants used in the experiment in ( d ). +, interaction; –, no interaction. ( f ) Mapping of the USP4 domains responsible for binding to HDAC2. The 293T cells were co-transfected with Flag-HDAC2 and the truncated Myc-USP4 mutants as indicated, and cell lysates were immunoprecipitated with anti- Myc antibody and then immunoblotted with anti-Flag antibody. ( g ) Schematic representation of USP4 and USP4 deletion mutants used in the experiment in ( f ). +, interaction; –, no interaction. ( h ) Immunofluorescent detection of Flag-HDAC2 and Myc-USP4 in H1299 cells. Nuclei are counterstained by DAPI.

Article Snippet: Human H1299, U2OS, HEK293 and 293T cells were from ATCC (Manassas, VA, USA).

Techniques: Transfection, Immunoprecipitation, Western Blot, In Vitro, Binding Assay, Purification, Incubation

Journal: Oncogene

Article Title: USP4 inhibits p53 and NF-κB through deubiquitinating and stabilizing HDAC2

doi: 10.1038/onc.2015.349

Figure Lengend Snippet: USP4 deubiquitinates HDAC2. ( a , b ) The 293T cells were transfected with the indicated combinations of expression vectors. Cells, 24 h after transfection, were treated with MG132 (5 μ M ) for 3 h and then lysed. Cell lysates were immunoprecipitated with anti-Flag antibody (against Flag-HDAC2) and immunoblotted with anti-HA antibody (against HA-ubiquitin). ( c ) The H1299-control or H1299-USP4-RNAi cells were transfected with the indicated combinations of expression vectors. Cells, 24 h after transfection, were treated with MG132 (5 μ M ) for 3 h and then lysed. Cell lysates were immunoprecipitated with anti-HDAC2 antibody and immunoblotted with anti-ubiquitin antibody. ( d ) Recombinant USP4 deubiquitinates HDAC2 in vitro . HEK-293T cells were transfected with expression vectors encoding Flag-HDAC2 and HA-ubiquitin. Cells were lysed in the lysis buffer only with phenylmethylsulfonyl fluoride (PMSF) as a protease inhibitor. Flag-HDAC2 proteins in the cell lysates were immunoprecipitated with anti- Flag antibodies and coincubated with purified recombinant His-USP4-WT or -C311A mutant for 2 h in the deubiquitination buffer before being analyzed by immunoblotting with the anti-HA antibodies.

Article Snippet: Human H1299, U2OS, HEK293 and 293T cells were from ATCC (Manassas, VA, USA).

Techniques: Transfection, Expressing, Immunoprecipitation, Ubiquitin Proteomics, Control, Recombinant, In Vitro, Lysis, Protease Inhibitor, Purification, Mutagenesis, Western Blot

Journal: Virologica Sinica

Article Title: Classical swine fever virus NS5A protein antagonizes innate immune response by inhibiting the NF-κB signaling.

doi: 10.1016/j.virs.2023.09.002

Figure Lengend Snippet: Fig. 1. The CSFV NS5A interacts with NEMO. A, B HEK293T cells in 6-well plates were transfected with p3FLAG-NS5A or p3FLAG-CMV-10 (2.5 μg/well). At 48 h post-transfection, the cells were lysed for co-immunoprecipitation (co-IP) and Western blotting with indicated antibodies. C PK15 cells were infected with CSFV Shimen strain at an MOI of 1. At 48 hpi, the cells were lysed for co-IP and Western blotting with indicated antibodies.

Article Snippet: Briefly, DynabeadsTM (Thermo Fisher) with bound His-NS5A or His tag were incubated with the cell lysates from HEK293T cells expressing Myc-NEMOΔN1, Myc-NEMOΔN2, Myc-NEMOΔN3 or Myc-NEMOΔN1 for 10 min on a roller at 37 C. Proteins co-purified with His-NS5A or His-tagged protein were detected by western blotting with rabbit anti-Myc polyclonal antibody (Proteintech, China) for probing NEMO mutants and mouse anti-His tag mAb (Proteintech, China) for probing NS5A.

Techniques: Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Infection

Journal: Virologica Sinica

Article Title: Classical swine fever virus NS5A protein antagonizes innate immune response by inhibiting the NF-κB signaling.

doi: 10.1016/j.virs.2023.09.002

Figure Lengend Snippet: Fig. 2. Determination of the domain of NEMO required for the association with NS5A. A Schematic representation of NEMO mutant constructs. B HEK293T cells in 6- well plates were transfected with NEMO mutants (2.5 μg/well). After 48 h of transfection, the cells were lysed and incubated with Dynabeads™coupled with His-NS5A or His tag. The proteins co-purified with fusion protein His-NS5A or His tag and cell lysates were subjected to Western blotting analysis using the indicated antibodies.

Article Snippet: Briefly, DynabeadsTM (Thermo Fisher) with bound His-NS5A or His tag were incubated with the cell lysates from HEK293T cells expressing Myc-NEMOΔN1, Myc-NEMOΔN2, Myc-NEMOΔN3 or Myc-NEMOΔN1 for 10 min on a roller at 37 C. Proteins co-purified with His-NS5A or His-tagged protein were detected by western blotting with rabbit anti-Myc polyclonal antibody (Proteintech, China) for probing NEMO mutants and mouse anti-His tag mAb (Proteintech, China) for probing NS5A.

Techniques: Mutagenesis, Construct, Transfection, Incubation, Western Blot

Journal: Virologica Sinica

Article Title: Classical swine fever virus NS5A protein antagonizes innate immune response by inhibiting the NF-κB signaling.

doi: 10.1016/j.virs.2023.09.002

Figure Lengend Snippet: Fig. 3. Determination of the region of NS5A required for NS5A-NEMO interaction. A Schematic representation of NS5A mutant constructs. B HEK293T cells in 24-well plates were transfected with full-length NS5A or NS5A truncated mutant (1 μg/well). FLAG tag was used as control. After 48 h of transfection, the cells were lysed for co-immunoprecipitation and Western blotting assay with the indicated antibodies.

Article Snippet: Briefly, DynabeadsTM (Thermo Fisher) with bound His-NS5A or His tag were incubated with the cell lysates from HEK293T cells expressing Myc-NEMOΔN1, Myc-NEMOΔN2, Myc-NEMOΔN3 or Myc-NEMOΔN1 for 10 min on a roller at 37 C. Proteins co-purified with His-NS5A or His-tagged protein were detected by western blotting with rabbit anti-Myc polyclonal antibody (Proteintech, China) for probing NEMO mutants and mouse anti-His tag mAb (Proteintech, China) for probing NS5A.

Techniques: Mutagenesis, Construct, Transfection, FLAG-tag, Control, Immunoprecipitation, Western Blot

Journal: Virologica Sinica

Article Title: Classical swine fever virus NS5A protein antagonizes innate immune response by inhibiting the NF-κB signaling.

doi: 10.1016/j.virs.2023.09.002

Figure Lengend Snippet: Fig. 4. Determination of the region of NS5A required for inhibition of the NF-κB signaling. A–C HEK293T cells in 6-well plate were transfected with full-length NS5A or NS5A truncated mutant (2.5 μg/well). After 48 h, the cells were treated with or without TNF-α for 15 min and then harvested for detection of IKKα/β phos- phorylation (A), IκBα degradation (B), quantification of IFN-α mRNA (C). The expressing level of phosphorylated IKKα/β and IκBα was analyzed by Western blotting. IFN-α mRNA was detected by RT-PCR using the One-Step SYBR Prime Script RT-PCR kit. D HEK293T cells in 24-well plate were transfected with pNF-κB-Luc reporter (1 μg/well), PRL-TK internal control (0.5 μg/well). After 48 h, the cells were treated with or without TNF-α for 15 min and the NF-κB transcriptional activity was analyzed using the Dual-Lumi Luciferase Reporter Gene Assay Kit. E The expression level of NS5A and its truncated mutants in above experiments was detected by Western blotting. Data presented as means SD. Statistical analyses were performed using GraphPad Prism version 6.01 software (GraphPad Software, La Jolla, CA, USA).**, P < 0.01; ***, P < 0.001; NS, not significant.

Article Snippet: Briefly, DynabeadsTM (Thermo Fisher) with bound His-NS5A or His tag were incubated with the cell lysates from HEK293T cells expressing Myc-NEMOΔN1, Myc-NEMOΔN2, Myc-NEMOΔN3 or Myc-NEMOΔN1 for 10 min on a roller at 37 C. Proteins co-purified with His-NS5A or His-tagged protein were detected by western blotting with rabbit anti-Myc polyclonal antibody (Proteintech, China) for probing NEMO mutants and mouse anti-His tag mAb (Proteintech, China) for probing NS5A.

Techniques: Inhibition, Transfection, Mutagenesis, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control, Activity Assay, Luciferase, Reporter Gene Assay, Software

Journal: Virologica Sinica

Article Title: Classical swine fever virus NS5A protein antagonizes innate immune response by inhibiting the NF-κB signaling.

doi: 10.1016/j.virs.2023.09.002

Figure Lengend Snippet: Fig. 5. Reduction of NEMO induced by CSFV or NS5A. PK-15 cells (A) or PAM cells (B) were infected with CSFV Shimen strain at an MOI of 3 or 5. At 48 hpi, the cells were lysed for Western blotting analysis with the indicated antibodies. C, D HEK293T cells cultured in 6-well plates were transfected with the indicated vector (pcDNA-NEMO, 2.5 μg/well). At 48 h post-transfection, the cells were lysed for Western blotting analysis with the indicated antibodies. Data presented as means SD. Statistical analyses were performed using GraphPad Prism version 6.01 software (GraphPad Software, La Jolla, CA, USA).*, P < 0.05; **, P < 0.01.

Article Snippet: Briefly, DynabeadsTM (Thermo Fisher) with bound His-NS5A or His tag were incubated with the cell lysates from HEK293T cells expressing Myc-NEMOΔN1, Myc-NEMOΔN2, Myc-NEMOΔN3 or Myc-NEMOΔN1 for 10 min on a roller at 37 C. Proteins co-purified with His-NS5A or His-tagged protein were detected by western blotting with rabbit anti-Myc polyclonal antibody (Proteintech, China) for probing NEMO mutants and mouse anti-His tag mAb (Proteintech, China) for probing NS5A.

Techniques: Infection, Western Blot, Cell Culture, Transfection, Plasmid Preparation, Software

Journal: Virologica Sinica

Article Title: Classical swine fever virus NS5A protein antagonizes innate immune response by inhibiting the NF-κB signaling.

doi: 10.1016/j.virs.2023.09.002

Figure Lengend Snippet: Fig. 6. NS5A mediates proteasomal degradation of NEMO. HEK293T cells cultured in 6-well plates were co-transfected with pcDNA-NEMO and p3FLAG-NS5A vectors (2.5 μg/well) (A) or transfected with p3FLAG-NS5A vector alone (2.5 μg/well) (B). At 24 h post-transfection, the cells were incubated with MG132 for 24 h, then the cells were lysed for Western blotting analysis with the indicated antibodies. C Cell viability after MG132 treatment was evaluated by MTT assay. Data presented as means SD. Statistical analyses were performed using GraphPad Prism version 6.01 software (GraphPad Software, La Jolla, CA, USA). *, P < 0.05; **, P < 0.01; NS, not significant.

Article Snippet: Briefly, DynabeadsTM (Thermo Fisher) with bound His-NS5A or His tag were incubated with the cell lysates from HEK293T cells expressing Myc-NEMOΔN1, Myc-NEMOΔN2, Myc-NEMOΔN3 or Myc-NEMOΔN1 for 10 min on a roller at 37 C. Proteins co-purified with His-NS5A or His-tagged protein were detected by western blotting with rabbit anti-Myc polyclonal antibody (Proteintech, China) for probing NEMO mutants and mouse anti-His tag mAb (Proteintech, China) for probing NS5A.

Techniques: Cell Culture, Transfection, Plasmid Preparation, Incubation, Western Blot, MTT Assay, Software

Journal: Virologica Sinica

Article Title: Classical swine fever virus NS5A protein antagonizes innate immune response by inhibiting the NF-κB signaling.

doi: 10.1016/j.virs.2023.09.002

Figure Lengend Snippet: Fig. 7. NS5A mediates NEMO ubiquitination. PK-15 and HEK293T cells in 6-well plates were transfected with p3FLAG-NS5A or p3FLAG-CMV-10 (2.5 μg/well). At 24 h post-transfection, the cells were incubated with MG132 (10 μmol/L) for 24 h, and then the cells were lysed for immunoprecipitation with rabbit anti-NEMO mAb or nonspecific IgG. Western blotting analysis was performed by using mouse anti-Ub mAb, mouse anti-NEMO mAb and mouse anti-FLAG mAb.

Article Snippet: Briefly, DynabeadsTM (Thermo Fisher) with bound His-NS5A or His tag were incubated with the cell lysates from HEK293T cells expressing Myc-NEMOΔN1, Myc-NEMOΔN2, Myc-NEMOΔN3 or Myc-NEMOΔN1 for 10 min on a roller at 37 C. Proteins co-purified with His-NS5A or His-tagged protein were detected by western blotting with rabbit anti-Myc polyclonal antibody (Proteintech, China) for probing NEMO mutants and mouse anti-His tag mAb (Proteintech, China) for probing NS5A.

Techniques: Ubiquitin Proteomics, Transfection, Incubation, Immunoprecipitation, Western Blot

Journal: Virologica Sinica

Article Title: Classical swine fever virus NS5A protein antagonizes innate immune response by inhibiting the NF-κB signaling.

doi: 10.1016/j.virs.2023.09.002

Figure Lengend Snippet: Fig. 8. NS5A does not mediate K48-linked polyubiquitination of NEMO. PK-15 and HEK293T cells in 6-well plates were transfected with p3FLAG-NS5A or p3FLAG-CMV-10 (2.5 μg/well). At 24 h post-transfection, the cells were incubated with MG132 (10 μmol/L) for 24 h. Then the cells were lysed for immunopre- cipitation with rabbit anti-NEMO mAb, and Western blotting analysis was performed by using K48-linkage specific polyubiquitin (D9D5) rabbit mAb, mouse anti- NEMO mAb and mouse anti-FLAG mAb.

Article Snippet: Briefly, DynabeadsTM (Thermo Fisher) with bound His-NS5A or His tag were incubated with the cell lysates from HEK293T cells expressing Myc-NEMOΔN1, Myc-NEMOΔN2, Myc-NEMOΔN3 or Myc-NEMOΔN1 for 10 min on a roller at 37 C. Proteins co-purified with His-NS5A or His-tagged protein were detected by western blotting with rabbit anti-Myc polyclonal antibody (Proteintech, China) for probing NEMO mutants and mouse anti-His tag mAb (Proteintech, China) for probing NS5A.

Techniques: Transfection, Incubation, Western Blot

Journal: Virologica Sinica

Article Title: Classical swine fever virus NS5A protein antagonizes innate immune response by inhibiting the NF-κB signaling.

doi: 10.1016/j.virs.2023.09.002

Figure Lengend Snippet: Fig. 9. NS5A induces K27-linked polyubiquitination of NEMO. PK-15 (A and B) and HEK293T (C and D) cells in 6-well plates were transfected with p3FLAG-NS5A or p3FLAG-CMV-10 (2.5 μg/well). At 24 h post-transfection, the cells were incubated with MG132 (10 μmol/L) for 24 h. Then the cells were harvested for immunoprecipitation with rabbit anti-NEMO mAb and ubiquitination assay of NEMO with mouse anti-HA mAb.

Article Snippet: Briefly, DynabeadsTM (Thermo Fisher) with bound His-NS5A or His tag were incubated with the cell lysates from HEK293T cells expressing Myc-NEMOΔN1, Myc-NEMOΔN2, Myc-NEMOΔN3 or Myc-NEMOΔN1 for 10 min on a roller at 37 C. Proteins co-purified with His-NS5A or His-tagged protein were detected by western blotting with rabbit anti-Myc polyclonal antibody (Proteintech, China) for probing NEMO mutants and mouse anti-His tag mAb (Proteintech, China) for probing NS5A.

Techniques: Transfection, Incubation, Immunoprecipitation, Ubiquitin Proteomics

Journal: Virologica Sinica

Article Title: Classical swine fever virus NS5A protein antagonizes innate immune response by inhibiting the NF-κB signaling.

doi: 10.1016/j.virs.2023.09.002

Figure Lengend Snippet: Fig. 10. NS5A inhibits K63-linked polyubiquitination of NEMO. PK-15 (A) and HEK293T (B) cells in 6-well plates were transfected with or without p3FLAG-NS5A or p3FLAG-CMV-10 vector (2.5 μg/well). Following incubation with MG132 (10 μmol/L) for 24 h and infection with SeV (500 HAU/well) for 12 h, the cells were lysed for immunoprecipitation with rabbit anti-NEMO mAb or nonspecific IgG, and Western blotting analysis was performed by using K63-linkage specific polyubiquitin (D7A11) rabbit mAb, and mouse anti-NEMO mAb or mouse anti-FLAG mAb.

Article Snippet: Briefly, DynabeadsTM (Thermo Fisher) with bound His-NS5A or His tag were incubated with the cell lysates from HEK293T cells expressing Myc-NEMOΔN1, Myc-NEMOΔN2, Myc-NEMOΔN3 or Myc-NEMOΔN1 for 10 min on a roller at 37 C. Proteins co-purified with His-NS5A or His-tagged protein were detected by western blotting with rabbit anti-Myc polyclonal antibody (Proteintech, China) for probing NEMO mutants and mouse anti-His tag mAb (Proteintech, China) for probing NS5A.

Techniques: Transfection, Plasmid Preparation, Incubation, Infection, Immunoprecipitation, Western Blot

Journal:

Article Title: K-bZIP of Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 (KSHV/HHV-8) Binds KSHV/HHV-8 Rta and Represses Rta-Mediated Transactivation

doi: 10.1128/JVI.77.6.3809-3815.2003

Figure Lengend Snippet: K-bZIP represses Rta-mediated transactivation. (A) Transactivation of RtaRE220-Luc by Rta is repressed by K-bZIP. A DNA fragment containing a 220-bp nucleotide sequence upstream of the ORF K8 translational start site was inserted upstream of the promoterless firefly luciferase gene in a reporter plasmid, pA3Pluc, to generate RtaRE220-Luc. RtaRE220-Luc was then used as a reporter for Rta-mediated transactivation. The cDNA for KSHV/HHV-8 Rta was amplified from a lambda phage-based cDNA library of a PEL cell line, BCBL1, using the primers 5′ AGATCT(BglII)CCATGG CGC AAG ATG ACAAG and 5′ GAATTC(EcoRI)TCA GTC TCG GAA GTA ATT ACG. The PCR product was cloned into the PCR2.1 TA-cloning vector, digested with BglII and EcoRI, and ligated into the BamHI and EcoRI sites of pcDNA3.1(+) vector to obtain CMV-Rta. The K-bZIP expression vector, pCMV-HA-K-bZIP, has been described (17). Approximately 106 HEK 293 cells/well in a six-well plate were cotransfected with the RtaRE220 (0.5 μg) in the presence (lanes 1 to 4) or absence (lane 5) of the Rta expression plasmid, CMV-Rta (1 μg), together with 0, 0.5, 1 and 2 μg of pCMV-HA-K-bZIP (lanes 1-4, respectively) using Lipofectamine (Invitrogen Corp.) as prescribed by the manufacturer. The total amount of transfected DNA (3 μg) in each well was kept constant by the addition of the empty vector plasmid pcDNA3.1(+). Cell lysates were prepared 48 h after transfection by dissolving the DNA-transfected cells from each well in 200 μl of the reporter lysis buffer (Promega Corp.). Twenty microliters of the lysate was then placed in each well of a 96-well plate. After injection of 100 μl of luciferase substrate buffer, the luciferase activity was measured immediately in an MLX microtiter plate luminometer (Dynex Technologies). Transfections and luciferase assays for each experiment were performed in triplicate with the averages and standard deviations of luciferase activities shown. (B) Expression of Rta driven by the CMV immediate early enhancer-promoter is not affected by K-bZIP. HEK 293 cells were transfected as described for panel A. Twenty micrograms of total cell proteins from each transfection was resolved in an SDS-12% polyacrylamide gel and immunoblotted with a rabbit polyclonal antibody against a peptide containing amino acid residues 527 to 539 (KKRKALTVPEADT) of Rta and an anti-HA mouse monoclonal antibody (Santa Cruz Biotechnology, Inc.). (C) Repression of Rta-mediated transactivation by K-bZIP at different Rta-to-K-bZIP ratios. DNA transfection was carried out as described for panel A, with increasing amounts of CMV-Rta and a fixed amount of K-bZIP. (D) Detection of Rta and K-bZIP in cell lysates from panel C. Immunoblots were carried out as for panel B using antibodies against Rta and HA epitopes. Each lane contains 10 μl of cell lysates (∼20 μg of total protein). (E) Repression of Rta transactivation by K-bZIP in NC37 cells. Two million NC37 B-cells in each well of a six-well plate were cotransfected with 2 μg of CMV-Rta and 0.1, 0.5, 1, or 2 μg of pCMV-HA-K-bZIP, using the GENEporter 2 transfection reagent (Gene Therapy System) as per the manufacturer's protocol. Forty-eight hours after transfection, cells were collected and luciferase assays were performed as described for panel A. A TK-promoter driven Renilla luciferase vector, pTK-RL (Promega Corp.), was used to normalize the transfection efficiency. The total amount of plasmid DNA in each well was kept constant by adding pcDNA3.1(+) empty vector.

Article Snippet: HEK 293 cells were transfected as described for panel A. Twenty micrograms of total cell proteins from each transfection was resolved in an SDS-12% polyacrylamide gel and immunoblotted with a rabbit polyclonal antibody against a peptide containing amino acid residues 527 to 539 (KKRKALTVPEADT) of Rta and an anti-HA mouse monoclonal antibody (Santa Cruz Biotechnology, Inc.). (C) Repression of Rta-mediated transactivation by K-bZIP at different Rta-to-K-bZIP ratios.

Techniques: Sequencing, Luciferase, Plasmid Preparation, Amplification, cDNA Library Assay, Clone Assay, TA Cloning, Expressing, Transfection, Lysis, Injection, Activity Assay, Western Blot

Journal:

Article Title: K-bZIP of Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 (KSHV/HHV-8) Binds KSHV/HHV-8 Rta and Represses Rta-Mediated Transactivation

doi: 10.1128/JVI.77.6.3809-3815.2003

Figure Lengend Snippet: K-bZIP interacts with Rta in vivo and in vitro. (A) K-bZIP and Rta coimmunoprecipitate. Approximately 106 HEK 293 cells in each well of a six-well plate were transiently transfected with 1 μg of CMV-Rta alone (lanes 1) or together with 1 (lanes 2) or (lanes 3) 2 μg of pCMV-HA-K-bZIP or 1 μg of the empty vector pcDNA3.1(+) (lanes 4). Forty-eight hours after transfection, 500 μg of whole-cell lysates prepared in 250 μl of radioimmunoprecipitation assay buffer (9.1 mM dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS) containing 1× protease inhibitor cocktail (Roche Diagnostics) was immunoprecipitated with 10 μl of a rabbit polyclonal Rta antibody or 10 μl of an HA monoclonal antibody together with 20 μl of protein A-Sepharose beads (Invitrogen) overnight at 4°C. After the beads had been washed with radioimmunoprecipitation assay buffer four times, the bead-bound proteins were heated in the loading buffer and resolved on an SDS-12% polyacrylamide gel, transferred to a nitrocellulose membrane, and immunoblotted with anti-HA antibody (IP: Rta) or anti-Rta antibody (IP: HA). Panels labeled “lysate” show HA-K-bZIP (top) and Rta (bottom) expression in total cell lysates. (B) K-bZIP directly binds Rta. The complete coding sequence of K-bZIP was PCR amplified from pCMV-HA-K-bZIP by using the primers 5′-GCCGAATTC(EcoRI)ATGCCCAGAATGA-3′ and 5′-CGGGATCC(BamHI)TCAACATGGTGGGA-3′, digested with EcoRI and BamHI, and inserted in frame into the EcoRI and BamHI site of pMAL Cx2 (New England Biolabs, Inc.) to produce pMBP-K-bZIP. The MBP-K-bZIP fusion protein was expressed and purified according to the manufacturer's specifications. To derive Rta protein, the complete coding sequence of Rta was amplified by PCR from CMV-Rta and inserted into a bacterial expression vector, pTrc2His2-TOPO (Invitrogen Corp.), to produce pTrc2His2-TOPO-Rta, where Rta was tagged with a c-Myc epitope and a hexahistidine extension at the COOH terminus. Expression and purification of hexahistidine-tagged Rta protein by using Ni2+-nitrilotriacetic acid-Sepharose (Qiagen Inc.) were as described previously (34). Five micrograms of MBP or MBP-K-bZIP was incubated with 2 μg of Rta or glutathione S-transferase-IKKγ together with 30 μl of amylose resin (New England Biolabs, Inc.). The resin was then washed with 1 ml of a buffer containing 20 mM Tris-HCl (pH 7.5) and 100 mM NaCl four times. Protein-bound resin was boiled in SDS-PAGE loading buffer and the proteins were resolved in a SDS-12% polyacrylamide gel, transferred to a nitrocellulose membrane, and immunoblotted with Rta or IKKγ antibody.

Article Snippet: HEK 293 cells were transfected as described for panel A. Twenty micrograms of total cell proteins from each transfection was resolved in an SDS-12% polyacrylamide gel and immunoblotted with a rabbit polyclonal antibody against a peptide containing amino acid residues 527 to 539 (KKRKALTVPEADT) of Rta and an anti-HA mouse monoclonal antibody (Santa Cruz Biotechnology, Inc.). (C) Repression of Rta-mediated transactivation by K-bZIP at different Rta-to-K-bZIP ratios.

Techniques: In Vivo, In Vitro, Transfection, Plasmid Preparation, Radio Immunoprecipitation, Protease Inhibitor, Immunoprecipitation, Labeling, Expressing, Sequencing, Amplification, Purification, Incubation, SDS Page

Journal:

Article Title: K-bZIP of Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 (KSHV/HHV-8) Binds KSHV/HHV-8 Rta and Represses Rta-Mediated Transactivation

doi: 10.1128/JVI.77.6.3809-3815.2003

Figure Lengend Snippet: K-bZIP-Rta interaction requires the bZIP domain of K-bZIP. (A) Domain organization of K-bZIP. Regions of K-bZIP included in the deletion mutants are indicated. aa, amino acids. (B) The ZIP domain of K-bZIP binds Rta. The coding sequences of K-bZIP(1-189), K-bZIP(121-237), and K-bZIP(189-237) were derived by PCR using the respective forward primers 5′-GCCGAATTC(EcoRI)ATGCCCAGAATGA-3′, 5′-GCCGAATTC (EcoRI)ATGCAGCTTCCAACT-3′, and 5′-GCCGAATTC(EcoRI) ATGCAGGCATTAGA-3′ and the reverse primers 5′-CGGGATCC(BamHI)TCAACATGGTGGGA-3′ [K-bZIP(121-237) and K-bZIP(189-237)] and 5′-CGGGATCC(BamHI)TCACTGCTGCAGCT-3′ [K-bZIP(1-189)] and fused to the MBP sequence as described for Fig. ​Fig.3.3. MBP and MBP fusion proteins containing full-length K-bZIP and the three deletions were expressed and purified (lanes 7 to 11, stained with Coomassie brilliant blue), and 5 μg of each was used together with 2 μg of purified Rta protein in pull-down experiments (lanes 1 to 6, Rta immunoblot) as described for Fig. ​Fig.3B.3B. (C) K-bZIP lacking the ZIP domain failed to suppress Rta transactivation. Transient transfection of HEK 293 cells and luciferase assays were performed as described for Fig. ​Fig.11 except that 1 μg each of pCMV-HA-K-bZIP, pCMV-HA-K-bZIP (encoding residues 1 to 189), pCMV-HA-K-bZIP (residues 121 to 237), and pCMV-HA-K-bZIP (residues 189 to 237) were used, respectively, together with 1 μg of CMV-Rta. The expression plasmids for the deletion mutants of HA-K-bZIP were constructed by fusing their respective coding sequences (PCR derived) with the coding region of the HA epitope in pCMV-HA (Invitrogen Corp.) via EcoRI and BamHI site. The forward primers used here were 5′-GCCGAATTC(EcoRI)TGCCCAGAATGA-3′ [K-bZIP(1-189)], 5′-GCCGAATTC(EcoRI)TGCAGCTTCCAACT-3′ [K-bZIP(121-237)], 5′-GCCGAATTC(EcoRI)TGCAGGCATTAGA-3′ [K-bZIP(189-237)]. The reverse primers were as described for the MBP fusion constructs in Fig. ​Fig.3.3. Twenty micrograms of total cell proteins from each transfection was resolved in an SDS-12% polyacrylamide gel and immunoblotted (IB) with an anti-HA mouse monoclonal antibody and a rabbit polyclonal antibody against Rta. Luciferase assays were carried out as described for Fig. ​Fig.1A1A.

Article Snippet: HEK 293 cells were transfected as described for panel A. Twenty micrograms of total cell proteins from each transfection was resolved in an SDS-12% polyacrylamide gel and immunoblotted with a rabbit polyclonal antibody against a peptide containing amino acid residues 527 to 539 (KKRKALTVPEADT) of Rta and an anti-HA mouse monoclonal antibody (Santa Cruz Biotechnology, Inc.). (C) Repression of Rta-mediated transactivation by K-bZIP at different Rta-to-K-bZIP ratios.

Techniques: Derivative Assay, Sequencing, Purification, Staining, Western Blot, Transfection, Luciferase, Expressing, Construct

Journal:

Article Title: K-bZIP of Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 (KSHV/HHV-8) Binds KSHV/HHV-8 Rta and Represses Rta-Mediated Transactivation

doi: 10.1128/JVI.77.6.3809-3815.2003

Figure Lengend Snippet: Overexpression of p300 or P/CAF cannot relieve the repression by K-bZIP. (A) Activities of luciferase reporters. Approximately 107 HEK 293 cells in 100-mm dishes were cotransfected with reporter plasmids RtaRE220 (0.5 μg), pTK-RL (0.1 μg), and the indicated amounts of CMV-Rta and pCMV-HA-K-bZIP together with CMV-HA-p300, CMV-P/CAF, or CMV-p300 plus CMV-P/CAF as for Fig. ​Fig.4.4. The total amount of transfected DNA (7.6 μg) in each dish was kept constant by the addition of the empty vector plasmid pcDNA3.1(+). Preparation of cell lysates and luciferase assays were performed as described for Fig. ​Fig.1A.1A. (B) Immunoblot analyses of cell lysates from panel A. Immunoblots were carried out as for Fig. ​Fig.1B1B by using antibodies against Rta, HA, and FLAG epitopes. Each lane contains 10 μl of cell lysates (∼20 μg of total protein). MW, molecular weight (in thousands).

Article Snippet: HEK 293 cells were transfected as described for panel A. Twenty micrograms of total cell proteins from each transfection was resolved in an SDS-12% polyacrylamide gel and immunoblotted with a rabbit polyclonal antibody against a peptide containing amino acid residues 527 to 539 (KKRKALTVPEADT) of Rta and an anti-HA mouse monoclonal antibody (Santa Cruz Biotechnology, Inc.). (C) Repression of Rta-mediated transactivation by K-bZIP at different Rta-to-K-bZIP ratios.

Techniques: Over Expression, Luciferase, Transfection, Plasmid Preparation, Western Blot, Molecular Weight

Journal: Molecular Neurobiology

Article Title: PKCγ-mediated Phosphorylation of Mtss1 Regulates the Dendritic Outgrowth and Spine Development of Cerebellar Purkinje Cells

doi: 10.1007/s12035-025-05526-9

Figure Lengend Snippet: Mtss1 expression is regulated by PKCγ activity. ( A ) Proteomic analysis of cerebellar protein samples from P21 PKCγ-A24E +/+ and PKCγ-A24E wt/wt mice was performed to profile protein differences between genotypes. ( B ) Volcano plot of proteomics analysis of PKCγ-A24E +/+ versus PKCγ-A24E wt/wt mice (n = 3) at P21. Differentially enriched proteins are shown in the volcano plot; downregulated (left, blue) and upregulated (right, green). Mtss1 is among the proteins with a significant upregulation, pointed in green to differentiate it. X axis is log2 fold change and Y axis is p-value. ( C ) IPA showed that many differentially enriched proteins are involved in branching of neurons, including Mtss1 (pointed with a green arrow). Blue proteins represent downregulation and green proteins represent upregulation. ( D ) Western blot staining of Mtss1 and corresponding graph of Mtss1 relative expression normalized to Actin show Mtss1 protein upregulation in HEK293T cells co-transfected with PKCγ-WT and Mtss1-WT and treated with PMA for 6 h, and in HEK293T cells co-transfected with PKCγ-A24E construct and Mtss1-WT (Control = 1 ± 0,07; PMA = 2,74 ± 0,33; A24E = 2,12 ± 0,13). ( E ) Western blot analysis of whole cerebellar lysates shows Mtss1 protein upregulation in PKCγ-A24E +/+ (hom) and PKCγ-A24E wt/+ (het) mice compared to PKCγ-A24E wt/wt (wt). Relative protein quantification of Mtss1 was normalized to Actin. P14, n = 4: wt = 1 ± 0,16; het = 1,30 ± 0,17; hom = 1,31 ± 0,16. ( F ) Representative images of sagittal cerebellar sections of lobules V-VI comparing PKCγ-A24E wt/wt , in PKCγ-A24E wt/+ , and in PKCγ-A24E +/+ at P14. Protein distribution of Mtss1 and Calbindin displayed in green and yellow, respectively. ( G ) Representative images of WT sagittal cerebellar sections of lobule VIb at P7, P14, P21, and P35. Protein distribution of Mtss1 (green) and Calbindin (yellow) is shown. ( H ) Western blot analysis of WT cerebellar lysates over time shows the highest Mtss1 expression at P14. Mtss1 protein quantification was normalized to Actin. P7 = 1 ± 0,21; P14 = 2,87 ± 0,68; P21 = 2,09 ± 0,39; P35 = 0,75 ± 0,15; P266 = 0,54 ± 0,10; n = 4. Scale bar = 50 μm. Ordinary one-way ANOVA with post-hoc Sidak comparisons test; * = p-value < 0,05; ** = p-value < 0,01; *** = P-value < 0,001; **** = p-value < 0,0001. Error bars indicate SD

Article Snippet: Human embryonic kidney cells (HEK293T) (RRID:CVCL_0063; ATCC; Molsheim Cedex, France) were grown under standard conditions (37 ̊C, 5% CO 2 ).

Techniques: Expressing, Activity Assay, Western Blot, Staining, Transfection, Construct, Control

Journal: Molecular Neurobiology

Article Title: PKCγ-mediated Phosphorylation of Mtss1 Regulates the Dendritic Outgrowth and Spine Development of Cerebellar Purkinje Cells

doi: 10.1007/s12035-025-05526-9

Figure Lengend Snippet: Phosphorylation of Mtss1 is increased in models with PKCγ overactivation. ( A ) To assess phosphorylation differences between PKCγ-A24E +/+ mutant and PKCγ-A24E wt/wt mice, phospho-proteomic analysis was performed on cerebellar protein samples from P21 pups. ( B ) Volcano plot of phospho-proteomics analysis of PKCγ-A24E +/+ versus PKCγ-A24E wt/wt mice (n = 3) at P21. Peptides differentially phosphorylated are shown in the volcano plot; decreased phosphorylation (left, blue) and increased phosphorylation (right, green). Mtss1 is among the proteins with a significant increased phosphorylation, pointed in dark green to differentiate it. X axis is log2 fold change and Y axis is p-value. ( C ) IPA showed that many proteins with an altered phosphorylation are involved in the morphology of the cytoskeleton, including Mtss1 (pointed with a green arrow). Blue proteins represent a decrease in phosphorylation whereas green proteins represent an increase. ( D ) Immunoprecipitation demonstrates that Mtss1 is associated with PKCγ and is detected as a phospho-serine (pSer) PKC substrate. ( E ) Immunoblot of pSer PKC-substrates overlapping with Mtss1 expression in lysates of HEK293T cells co-transfected with PKCγ-WT and Mtss1-WT, and treated for 1 h with 300 nM PMA, 10 μM Gö6983, or 0,1% DMSO (Control). ( F ) Corresponding quantification showing a significant increase or decrease in pSer-Mtss1 relative protein expression, normalized to Actin and to Mtss1, in cells treated with PMA or Gö6983 compared to DMSO controls, respectively (DMSO = 1 ± 0,10; PMA = 1,79 ± 0,07; Gö6983 = 0,57 ± 0,20). ( G ) Western blot of HEK293T cells co-transfected with Mtss1-WT and either mock (empty vector), PKCγ-WT, or PKCγ-A24E constructs. ( H ) Corresponding quantification showing a significant increase in pSer-Mtss1 relative expression, normalized to Actin and to Mtss1, in cells co-transfected with Mtss1-WT and PKCγ-A24E compared to controls (mock and PKCγ-WT) (mock = 1 ± 0,28; PKCγ-WT = 1,73 ± 0,10; PKCγ-A24E = 2,27 ± 0,12). Red squares in ( E ) and ( G ) indicate the pSer PKC substrate signal corresponding to Mtss1 (~ 110 KDa). One-way ANOVA; * = p-value < 0,05; ** = p-value < 0,01; *** = P-value < 0,001; **** = P-value < 0,0001. Error bars indicate SD

Article Snippet: Human embryonic kidney cells (HEK293T) (RRID:CVCL_0063; ATCC; Molsheim Cedex, France) were grown under standard conditions (37 ̊C, 5% CO 2 ).

Techniques: Phospho-proteomics, Mutagenesis, Immunoprecipitation, Western Blot, Expressing, Transfection, Control, Plasmid Preparation, Construct

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: The Adhesion-GPCR BAI1 Regulates Synaptogenesis by Controlling the Recruitment of the Par3/Tiam1 Polarity Complex to Synaptic Sites

doi: 10.1523/JNEUROSCI.3978-12.2013

Figure Lengend Snippet: BAI1 interacts with cell polarity machinery. A, Shown are results of a two-hybrid screen using an embryonic rat spinal cord library and the Tiam1 PDZ as bait. B, BAI1 and vector, flag-Tiam1, or flag-Tiam1-PDZ were expressed in 293T cells and immunoprecipitated using antiflag antibodies. Shown are blots of lysates and immunoprecipitates probed for BAI1. Images are representative of three independent experiments. C, Rat hippocampal neurons in culture were lysed on the days indicated and immunoprecipitated with nonimmune serum (NI) or antibodies against Tiam1. Shown are blots of immunoprecipitates and lysates representative of eight experiments. D, The indicated immunoprecipitations were performed on postnuclear brain lysates collected from adult rat brain. A longer exposure was required to visualize the Tiam1 band in the lysates than the other bands (indicated by the line on the blot). Data are representative of three independent experiments. E, The 21 DIV rat hippocampal neurons were stained for Tiam1 and BAI1. Shown are images representative of 50 neurons from five preparations, as well as an image showing the areas of significant colocalization. F, Rat hippocampal neurons in culture were lysed on the days indicated and immunoprecipitated with antibodies against Tiam1, BAI1, or Par3 or NI serum. Lysates and immunoprecipitates shown are representative of eight experiments. Bands at the top and bottom of each panel each are known isoforms of Par3. G, BAI1, BAI1· ΔTEV, and flag-Tiam1 were expressed in the indicated combinations and the cells lysed and immunoprecipitated with antiflag antibodies. Shown are blots of immunoprecipitates and lysates representative of seven experiments. H, The intensity of BAI1 staining in the flag immunoprecipitates is quantified (n=7). I,J, myc-Par3 and BAI1 or BAI1· ΔTEV were expressed in HEK 293T cells and the cells processed for immunocytochemistry. Pearson’s correlation coefficients were calculated for overlap between the two proteins. I, The line at 0.3 represents the value above which colocalization is greater than chance. J, Representative images (n=18, N=3). Data are mean ± SEM. *0.05 > p > 0.01. ***p < 0.001.

Article Snippet: HEK 293T cells were transfected using calcium phosphate, and NIH 3T3 cells were transfected using Lipofectamine 2000 (Invitrogen).

Techniques: Two Hybrid Screening, Plasmid Preparation, Immunoprecipitation, Staining, Immunocytochemistry

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: The Adhesion-GPCR BAI1 Regulates Synaptogenesis by Controlling the Recruitment of the Par3/Tiam1 Polarity Complex to Synaptic Sites

doi: 10.1523/JNEUROSCI.3978-12.2013

Figure Lengend Snippet: BAI1 controls the localization of Par3 and Tiam1 in neurons. A, HEK 293T cells were transfected as indicated and Rac1 activation measured indirectly by blotting for phospho-PAK, which autophosphorylates in response to its activation by Rac1 · GTP. PAK and β-actin serve as loading controls in this experiment. Data are representative of five independent experiments. B, Hippocampal neurons transfected with the high-efficiency Neon system were probed for Tiam1 and Par3 as indicated. The blots shown are for the same preparation as that shown in Figure 2B; BAI1 was knocked down by >50%. Blot is representative of four experiments. C, EGFP and BAI1 were visualized at 21 DIV in rat neurons with or without Par3 knockdown. BAI1 was overexpressed at low levels in this experiment. Scale bar, 5 μm. D, The BAI1 gradient between spines and dendrites for these experiments was quantified (n = 30 neurons, N = 3). EGFP and endogenous Par3 (E) (n = 40; N = 4), exogenous Par3 (F) (n = 30; N = 3), Tiam1 (G) (n = 50; N = 5), PKCζ (K) (n = 40, N = 4), or N-cadherin (L) (n = 40, N = 4) were visualized at 21 DIV in control rat hippocampal neurons or in BAI1 knockdown neurons. J, Gradients of N-cadherin between spines and dendrites (black) and spines and somata (gray) were measured in control and BAI1 knockdown neurons. Gradients of Par3, Tiam1, and PKCζ from spines to dendrites (H) and spines to somata (I) are also given. Dataset sizes are given above. Data are shown ± SEM. ***p < 0.001.

Article Snippet: HEK 293T cells were transfected using calcium phosphate, and NIH 3T3 cells were transfected using Lipofectamine 2000 (Invitrogen).

Techniques: Transfection, Activation Assay, Knockdown, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MNAT1 is overexpressed in colorectal cancer and mediates p53 ubiquitin-degradation to promote colorectal cancer malignance

doi: 10.1186/s13046-018-0956-3

Figure Lengend Snippet: MNAT1 decreases p53 expression. A, HCT116 cells were transfected with Flag-MNAT1 (a), and DLD1 cells were transfected with pSIN-MNAT1 (b), p53 expressions the tranfected cells were detected using Western-blotting. B, PT53 mRNA expressions in the transfected cells were detected using Real-time PCR. C, HEK293T cells were cotransfected with HA-p53 and Flag-MNAT1 at various concentrations, Flag and HA expressions were detected (a). Abundances of Flag and HA were analyzed (b). D, LoVo cells were transfected with shsramble, shMNAT1#1 and #2, and then p53 and MNAT1 expressions in the transfected cells were detected using Western-blotting. E. LoVo cells were treated with COX at the indicated concentrations, and then MNAT1 and p53 expressions were detected. F, HEK293T cells were transfected with pSIN-MNAT1 or blank vector, and then treated with DOX. MNAT1 and p53 expressions were detected using Western-blotting. G, HEK293T cells transfected with pSIN-MNAT1 were treated with DOX and/or MG132, and then MNAT1 and p53 were detected. DOX, Doxorubicin; *, p < 0.05

Article Snippet: CRC cell lines, SW480, HT-29, SW620, DLD1, HCT116, loVo, RK0, HCT116 p53 +/+ , HCT116 p53 −/− , and HEK293T (an embryonic kidney cell line 293 T) were obtained from American Type Culture Collection (Maryland).

Techniques: Expressing, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Plasmid Preparation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MNAT1 is overexpressed in colorectal cancer and mediates p53 ubiquitin-degradation to promote colorectal cancer malignance

doi: 10.1186/s13046-018-0956-3

Figure Lengend Snippet: MNAT1 interacts with p53. A, the lysates of LoVo cells were immunoprecipitated using anti-IgG, anti-MNAT1 or anti-p53 antibodies, and immunoprecipitation products were analyzed by Western-blotting with the indicated antibody. B, HEK293T cells were cotransfected with Flag-MNAT1 and HA-p53. Flag or HA was immunoprecipitated in the transfected cells, and the immunocomplexes were detected using Western-blotting with HA- or Flag-antibody. C, The various construct plasmids of TP53 were constructed using PCR (a) . HEK293T cells were cotransfected with Flag-MNAT1 and the indicated plasmids of TP53 . Immunoprecipitation was performed using anti-HA antibody, and the immunoprecipitated products were detected using Western-blotting with anti-Flag antibody. D, The plasmids containing various MNAT1 gene segments were constructed (a). HEK293T cells were cotransfected with HA- TP53 and the indicated plasmids of Flag-MNAT1. Immunoprecipitation was performed using anti-Flag antibody, and the immunoprecipitated products were detected using Western-blotting with anti-HA antibody. FL, full length p53; IP, immunoprecipitation; WCL, whole cell lysate

Article Snippet: CRC cell lines, SW480, HT-29, SW620, DLD1, HCT116, loVo, RK0, HCT116 p53 +/+ , HCT116 p53 −/− , and HEK293T (an embryonic kidney cell line 293 T) were obtained from American Type Culture Collection (Maryland).

Techniques: Immunoprecipitation, Western Blot, Transfection, Construct

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MNAT1 is overexpressed in colorectal cancer and mediates p53 ubiquitin-degradation to promote colorectal cancer malignance

doi: 10.1186/s13046-018-0956-3

Figure Lengend Snippet: MNAT1 promotes p53 ubiquitin-mediated degradation. A, HEK293T cells were cotransfected with HA-Ub, Flag-p53, and v5-MNAT1. Flag was immunoprecipitated in the transfected cells, and p53 was detected using Western-blotting (a), and p53 life-time was calculated (b). B, LoVo cells were cotransfected with HA-Ub, Flag-p53 and pLVX-shMNAT1 # 1 or pLVX-shMNAT1 # 2, respectively. The transfected cells were immunoprecipitated with Flag antibody, and p53 in the immunocomplexes were detected. C, HCT116 cells were cotransfected with pLVX-shMNAT1 # 1 or pLVX-shMNAT1 # 2, respectively. The transfected cells were immunoprecipitated with p53 antibody, and then ubiquitin in the immunocomplexes was detected. D, HEK293 cells were transfected with pSIN-MNAT1, and p53 in the transfected cells was immunoprecipitated. Ubiquitin in the immunocomplexes was detected. E, HCT116 cells were cotransfected with pLVX-shMNAT1 # 1 and # 2. p53 in the transfeccted cells was immunoprecipitated, and its ubiquitination was detected. IP, immunoprecipitation; Ub, ubiquitin; WCL, whole cell lysate

Article Snippet: CRC cell lines, SW480, HT-29, SW620, DLD1, HCT116, loVo, RK0, HCT116 p53 +/+ , HCT116 p53 −/− , and HEK293T (an embryonic kidney cell line 293 T) were obtained from American Type Culture Collection (Maryland).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Transfection, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MNAT1 is overexpressed in colorectal cancer and mediates p53 ubiquitin-degradation to promote colorectal cancer malignance

doi: 10.1186/s13046-018-0956-3

Figure Lengend Snippet: MNAT1 regulates p53 down-stream molecules. A, HEK293T cells were transfected with pSIN or pSIN-MANT1, and then MNAT1, p53, p21, cleaved caspase3, caspase3, cleaved PARP, PARP, BAX, RAD51, and Bcl2 were detected with Western-blotting. pSIN served as a control. B, HCT116 p53 +/+ and HCT116 p53 −/− cells were transfected with pLVX-shscramble, pLVX-shMNAT1 # 1 or pLVX-shMNAT1 # 2, respectively. pLVX-shscramble served as a control. MANT1, p53, p21, BAX, PARP, and RAD51 were detected. C, LoVo cells were transfected with pLVX-shMNAT1 # 1, and HEK293T cells were transfected with pSIN-MANT1. And then the transfected cells were treated with DOX. The treated cells were evaluated MNAT1, PARP, p53 expression. GAPDH served as a loading control. D, LoVo cells were infected with pLVX-shMNAT1 # 1 or pLVX-shMNAT1 # 2, and HEK293T cells were transfected with pSIN-MANT1, and then treated with 5-Fu. The treated cells were evaluated MNAT1, p53, and Fas expression. E, The apoptosis of the treated cells was analyzed using flow cytometry, and apoptosis rates were conducted. a, shscramble plus 5-FU; b, shMNAT1 # 1 plus 5-FU; c, shMNAT1 # 2 plus 5-FU; d, blank vector plus 5-FU; e, pSIN-MNAT1 plus 5-FU; f, apoptosis rates of the above treated cell. DOX, Doxorubicin; 5-Fu, 5-fluorouracil; *, p < 0.05

Article Snippet: CRC cell lines, SW480, HT-29, SW620, DLD1, HCT116, loVo, RK0, HCT116 p53 +/+ , HCT116 p53 −/− , and HEK293T (an embryonic kidney cell line 293 T) were obtained from American Type Culture Collection (Maryland).

Techniques: Transfection, Western Blot, Control, Expressing, Infection, Flow Cytometry, Plasmid Preparation