Journal: The Journal of biological chemistry

Article Title: Chimeric erythropoietin-interferon gamma receptors reveal differences in functional architecture of intracellular domains for signal transduction.

doi: 10.1074/jbc.272.8.4993

Figure Lengend Snippet: FIG. 7. Phosphorylation of Jak1 and Jak2 kinases. Untreated cells or cells treated with Hu-IFN-g or Epo were lysed and immunoprecipi- tated (I.P.) with monoclonal anti-phosphotyrosine antibodies (anti-P-Tyr panels), polyclonal anti-Jak1 antibodies (anti-Jak1 panels) or anti-Jak2 antibodies (anti-Jak2 panels) as described under “Materials and Methods.” The cell lines are indicated on the figure and are defined in the legend to Fig. 1. Immunoprecipitates were resolved on SDS-PAGE, transferred to PVDF membranes and the blots probed with anti-Jak1 or anti-Jak2 antibodies as noted under the respective horizontal panels.

Article Snippet: Rabbit anti-Jak2 antibody (catalogue no. SC-294) and rabbit anti-Stat5 antibody (catalogue no. SC-835) were from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, SDS Page

Journal: The Journal of biological chemistry

Article Title: Chimeric erythropoietin-interferon gamma receptors reveal differences in functional architecture of intracellular domains for signal transduction.

doi: 10.1074/jbc.272.8.4993

Figure Lengend Snippet: FIG. 8. Schematic representation of receptor complexes. A represents the IFN-gR1 homodimer bound to IFN-g. The cytoplasmic domains of the two chains are too far apart to permit transactivation of the two Jak1 kinases. B represents the active heteromeric IFN-g receptor com- plex with two IFN-gR1 and two IFN-gR2 subunits per complex. The IFN-g ho- modimer binds to two IFN-gR1 chains, followed by its interaction with two IFN- gR2 chains. The associated Jak2 and Jak1 kinases activate one another by transphosphorylation, with subsequent phosphorylation and dimerization of Stat1a. C depicts the EpoR/gR1 ho- modimer, which, unlike the IFN-gR1 ho- modimer, permits transactivation of the two Jak1 molecules. D illustrates the structure of the heterodimer of EpoR/gR1 and EpoR/gR2, which is the putative ac- tive receptor complex.

Article Snippet: Rabbit anti-Jak2 antibody (catalogue no. SC-294) and rabbit anti-Stat5 antibody (catalogue no. SC-835) were from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 4. Effects of estradiol on cyclin protein expression. The experimental design was described in Fig. 2. Representative Western blots are shown for G1 phase cyclins (D1, D3, E) (A) and S/G2M phase cyclins (A, B1) (B). Controls for cyclin E are as described in Fig. 2.

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 5. Estrogen induction of cyclin D1 and cyclin D3 mRNA expression. MCF-7 cells were pretreated with 10 nM ICI 182780 for 48 h and then treated with 100 nM E2 or vehicle (control) at time 0. At intervals thereafter, total cellular RNA was harvested. A, representative North- ern blots are shown for cyclin D1 and D3 mRNA from estradiol-treated and control cells. Arrows indicate the 4.5- and 1.5-kb cyclin D1 transcripts. B, graphical pres- entation of temporal changes in mRNA (open symbols) and protein (solid symbols, mean of two experiments) for cyclin D1 (left) and cyclin D3 (right).

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Expressing, Control

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 8. Activation of Cdk4 and Cdk2 following estrogen treatment. MCF-7 cells were pretreated with 10 nM ICI 182780 for 48 h and then treated with 100 nM E2 or vehicle (control) at time 0. At intervals thereafter, whole cell lysates were prepared and immunoprecipitated with antibodies to either Cdk4, cyclin E, or Cdk2, and then the kinase activity of the immunoprecipitates was determined by phosphorylation of GST-pRB773–923

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Activation Assay, Control, Immunoprecipitation, Activity Assay, Phospho-proteomics

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 9. Composition of cyclin D1-as- sociated and cyclin E-associated complexes. MCF-7 cells were pretreated with 10 nM ICI 182780 for 48 h and then treated with 100 nM E2 or vehicle (control) at time 0. At intervals thereafter, whole cell lysates were prepared and immuno- precipitated with anti-cyclin D1 anti- serum or anti-cyclin E antibodies, and then these immunoprecipitates were sep- arated by SDS-PAGE and transferred to nitrocellulose membranes. A, cyclin D1 antiserum immunoprecipitates. The same filter was sequentially Western blotted for cyclin D1, Cdk4, p21, and p27. A rep- resentative blot is shown for each. B, rel- ative levels of cyclin D1 (E), Cdk4 (G), p21 (M), and p27 (f) were determined by den- sitometry and are expressed relative to the vehicle treated controls. Points repre- sent the mean of two separate experi- ments. C, cyclin E immunoprecipitates. The same filter was sequentially Western blotted for cyclin E, Cdk2, p21, and p27. A representative blot is shown for each. The asterisk marks the more mobile form of Cdk2 that is phosphorylated on Thr-160.

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Control, SDS Page, Western Blot

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 10. Estrogen decreases inhibi- tory activity toward cyclin E-Cdk2. A, lysates were prepared from cells that were pretreated with antiestrogen and then treated with E2 (1) or vehicle (2). Active cyclin E-Cdk2 complexes that had been prepared from baculovirus-infected Sf9 cells were incubated with either lysis buffer only (labeled input) or with cell lysates. Lysates were also immunode- pleted with either anti-p27 antibodies, anti-p21 antibodies, or both and then in- cubated with recombinant cyclin E-Cdk2 complexes. Recombinant cyclin E-Cdk2 complexes were then recovered and as- sayed for histone (H1) kinase activity. The percentage of the input activity, de- fined as 100%, is also shown numerically below the autoradiograph. The same sam- ples were electrophoresed on a duplicate SDS-PAGE gel and then Western blotted for p21 and p27 to assess the binding of these proteins to recombinant cyclin E- Cdk2. B, the experiment described for panel A was repeated using either boiled lysis buffer (labeled input) or boiled ly- sates from cells treated with estradiol (1) or vehicle (2). C, MCF-7 cells were pre- treated with 10 nM ICI 182780 for 48 h and then treated with 100 nM estradiol or vehicle (control) at time 0. At intervals thereafter, whole cell lysates were pre- pared and assayed for cyclin E-Cdk2 in- hibitory activity as described for panel A.

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Activity Assay, Infection, Incubation, Lysis, Labeling, Recombinant, Autoradiography, SDS Page, Western Blot, Binding Assay, Control

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 11. Cyclin E-Cdk2 activation is accompanied by loss of CDK inhibitor association and Cdk2 Thr-160 phosphorylation. MCF-7 cells were pretreated with 10 nM ICI 182780 for 48 h and then treated with 100 nM E2 or vehicle (control) at time 0. Whole cell lysates were prepared either 8 h after estrogen (8 h E2) or from control cells (ICI 182780). Lysates were then fractionated on a Superose 12 gel filtration column. A, fractions were precipitated with acetone and Western blot- ted for cyclin E (top panels) or assayed for cyclin E-Cdk2 histone (H1) kinase activity (bottom panel). The relative levels of cyclin E protein (E, G) and cyclin E-Cdk2 activity (M, f) in lysates from antiestrogen- pretreated cells and cells treated with estradiol for 8 h were determined by densitometry and are represented graphically. The elution of mark- ers of known molecular weight (ferritin, 440 kDa; catalase, 232 kDa; aldolase, 158 kDa) are indicated at the top of the graph. B, fractions 19 and 24 from the 8 h E2 lysate were immunoprecipitated with an anti- cyclin E antibody, and the immunoprecipitates were electrophoresed on a SDS-PAGE gel and transferred to a nitrocellulose filter. The same filter was then sequentially Western blotted for cyclin E, Cdk2, p21, and p27. A representative blot is shown for each. The asterisk marks the more mobile form of Cdk2 that is phosphorylated on Thr-160.

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Activation Assay, Phospho-proteomics, Control, Filtration, Western Blot, Activity Assay, Molecular Weight, Immunoprecipitation, SDS Page

Journal: bioRxiv

Article Title: Arginine-GlcNAcylation of death domain and NleB/SseK proteins is crucial for bacteria pathogenesis by regulating host cell death

doi: 10.1101/746883

Figure Lengend Snippet: Multiple sequence alignment of 12 death domains (DDs) from human proteins containing death domain. The red asterisk indicates the conserved arginine site among different DDs, which could be GlcNAcylated by NleB/SseKs. (B) Site distribution of amino acids in 12 death receptor domains containing conserved arginine by Weblogo. (C) Phylogenetic tree of the 12 DDs constructed on the basis of sequence similarity. (D-G) Identification of the physiological substrates of arginine GlcNAc transferase NleB/SseKs during bacterial infection of mammalian cells. 293T cells transfected with pCS2-1Flag-TNFR1 DD, pCS2-1Flag-TRADD DD, pCS2-1Flag-FADD, and pCS2-1Flag-RIPK1 DD were infected with the indicated modified EPEC strains or Salmonella strains. After infection, cells were lysed, and proteins were immunoprecipitated with FLAG M2 beads. Samples were loaded onto SDS-PAGE gels and were immunoblotted with anti-Flag, anti-Arg-GlcNAc, and a loading control anti-tubulin. Data in Fig (D - G) are from at least three independent experiments.

Article Snippet: The anti-GlcNAc arginine antibody (ab195033, Abcam) was described previously( ).

Techniques: Sequencing, Construct, Infection, Transfection, Modification, Immunoprecipitation, SDS Page

Journal: bioRxiv

Article Title: Arginine-GlcNAcylation of death domain and NleB/SseK proteins is crucial for bacteria pathogenesis by regulating host cell death

doi: 10.1101/746883

Figure Lengend Snippet: ( A ) Multiple sequence alignment of 37 death domains (DDs) from human death domain-containing proteins. ( B ) Identification of the physiological substrates of arginine GlcNAc transferase NleB/SseKs during EPEC infection of mammalian cells. 293T cells transfected with pCS2-1Flag-TRADD DD, pCS2-1Flag-FADD DD, and pCS2-1Flag-RIPK1 DD were infected with the indicated modified EPEC strains. After infection, cells were lysed, and proteins were immunoprecipitated with FLAG M2 beads. Samples were loaded onto SDS-PAGE gels and were immunoblotted with anti-Flag, anti-Arg-GlcNAc, and a loading control anti-tubulin. Blot data were derived from at least three independent experiments.

Article Snippet: The anti-GlcNAc arginine antibody (ab195033, Abcam) was described previously( ).

Techniques: Sequencing, Infection, Transfection, Modification, Immunoprecipitation, SDS Page, Derivative Assay

Journal: bioRxiv

Article Title: Arginine-GlcNAcylation of death domain and NleB/SseK proteins is crucial for bacteria pathogenesis by regulating host cell death

doi: 10.1101/746883

Figure Lengend Snippet: An arginine point mutation screen of hTRADD to investigate its ability to be GlcNAcylated by SseK1. 293T cells were transfected with the indicated plasmid combinations. The samples of anti-Flag immunoprecipitates (Flag IP) and total cell lysates (Input) were immunoblotted with corresponding antibodies. Anti-tubulin was used as a loading control. (B) Electrospray ionization mass spectrometry (ESI-MS) determination of the total mass of the site-directed TRADD mutants purified from bacteria. GST-TRADD DD, GST-TRADD DD (R235A), GST-TRADD DD (R245A), and GST-TRADD DD (R235A/R245A) were expressed alone (upper panel) or co-expressed with His-SseK1 (lower panel) in E. coli BL21 (DE3) strain. The resulting mass spectra were shown. The resulting mass spectra were shown. The black bar and red bar denote unmodified and GlcNAcylated TRADD DD, respectively. (C) The percentage of site-directed TRADD mutants GlcNAcylated by SseK1. (D) HCD analysis of the peptides of TRADD DD R245 GlcNAcylated by SseK1 in bacteria. The fragmentation patterns of the generated b and y ions were shown along the peptide sequence on the top of the spectrum. (E) Modification of TRADD and TRADD variants by SseK1 upon S. Typhimurine infection. 293T cells was transfected with plasmids carrying TRADD and the site-directed TRADD mutants, and then infected with the indicated Salmonella strains. After 15-hour infection, cells were lysed and proteins were immunoprecipitated with FLAG M2 beads. Samples were loaded onto SDS-PAGE gels, followed by immunoblots with anti-Flag and anti-Arg-GlcNAc antibodies. (F) Identification of the site of TNFR1 GlcNAcylated by SseK3 in bacteria. (G) Summary of ESI-MS determination of the total mass of TNFR1 and its point mutants co-expressed with SseK3 in bacteria. Data in Fig (A, E, and F) are from at least three independent experiments.

Article Snippet: The anti-GlcNAc arginine antibody (ab195033, Abcam) was described previously( ).

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Mass Spectrometry, Purification, Generated, Sequencing, Modification, Infection, Immunoprecipitation, SDS Page, Western Blot

Journal: bioRxiv

Article Title: Arginine-GlcNAcylation of death domain and NleB/SseK proteins is crucial for bacteria pathogenesis by regulating host cell death

doi: 10.1101/746883

Figure Lengend Snippet: (A) Ectopic expression of NleB/SseKs effectors showed the related subcellular localization and modification pattern in transfected HeLa cells. GFP-NleB, GFP-SseK1, GFP-SseK2, and GFP-SseK3 were expressed ectopically in HeLa cells. In HeLa cells, green indicated immunofluorescence staining of GFP and arginine-GlcNAcylated proteins. Blue indicated DAPI staining of nuclei, and red indicated GM130 staining of the Golgi structure. (B) Analysis of NleB/SseKs auto-arginine-GlcNAcylation by Western blot. Recombinant purified NleB/SseKs and their enzymatic mutants were analyzed on SDS-PAGE gels, followed by immunoblotting with anti-Arg-GlcNAc. (C) ESI-MS analysis determination of the total mass of the NleB/SseKs purified from bacteria. The black bar denotes unmodified protein. For NleB and SseK3, the red bar denotes GlcNAcylated form with 203-Da increase, while for SseK1, the red bar denotes GlcNAcylated (203 Da) and acetylated (42 Da) form with 245-Da increase. (D) Arginine-GlcNAcylation percentage of NleB/SseKs. (E) Summary of ESI-MS determination of the total mass of NleB/SseKs. Data in (A and B) are representative from at least three independent experiments.

Article Snippet: The anti-GlcNAc arginine antibody (ab195033, Abcam) was described previously( ).

Techniques: Expressing, Modification, Transfection, Immunofluorescence, Staining, Western Blot, Recombinant, Purification, SDS Page

Journal: bioRxiv

Article Title: Arginine-GlcNAcylation of death domain and NleB/SseK proteins is crucial for bacteria pathogenesis by regulating host cell death

doi: 10.1101/746883

Figure Lengend Snippet: Effects of the auto-arginine-GlcNAcylation of NleB on enzyme activity towards death domain protein. The coupled anti-Arg-GlcNAc beads were incubated with 50 μg of purified NleB for enrichment with auto-arginine-glycosylated NleB. Beads enriched with auto-arginine-glycosylated proteins were used in vitro glycosylation assay. Samples were loaded onto SDS-PAGE gels and were immunoblotted with anti-Flag and anti-Arg-GlcNAc. (B) Mass spectrometry analysis of 203-Da increase in the total molecular weight of the auto-arginine-GlcNAcylation site-directed mutant proteins. (C) Effects of the modification site mutation of NleB/SseKs. 293T cells were transfected with the indicated plasmids. After 24-hour transfection, cells were lysed, and proteins were immunoprecipitated with α-FLAG conjugated beads. Samples were loaded onto SDS-PAGE gels, followed by immunoblot with anti-Flag, anti-Arg-GlcNAc, and a loading control anti-tubulin. (D-F) Effects of the auto-arginine-GlcNAcylation on cell death inhibition of NleB and SseKs. HeLa cells infected with the indicated EPEC strains and Salmonella strains were stimulated with TNF-α and TRAIL. Cell viability was determined by measuring ATP levels. Black bars or white bars denoted unstimulated or stimulated, respectively. Data in (A) and (C-F) are representative from at least three independent experiments.

Article Snippet: The anti-GlcNAc arginine antibody (ab195033, Abcam) was described previously( ).

Techniques: Activity Assay, Incubation, Purification, In Vitro, SDS Page, Mass Spectrometry, Molecular Weight, Mutagenesis, Modification, Transfection, Immunoprecipitation, Western Blot, Inhibition, Infection

Journal: Circulation research

Article Title: E1A can provoke G1 exit that is refractory to p21 and independent of activating cdk2.

doi: 10.1161/01.res.85.4.319

Figure Lengend Snippet: Figure 3. A, Western blot analysis, demonstrating that p21 does not prevent induction of cyclins E and A by E2F-1. Cardiac myocytes infected with the viruses shown were collected for analysis at the indicated times after infection. B, Immune com- plex kinase assays, confirming the induction of endogenous Cdk2 activity by E2F-1 and demonstrating the block to Cdk2 function by p21. Cdk4-dependent phosphorylation of Rb is shown for comparison. Cardiac myocytes infected with the viruses shown were analyzed 24 hours after infection.

Article Snippet: Murine monoclonal antibody to E1A (M73) and rabbit antibodies against E2F-1 (C-20), p21 (C-19), p16 (C-20), cyclin D3 (C-16), cyclin E (M-20), cyclin A (C-19), cdk2 (M2), and cdk4 (C-22) were from Santa Cruz Biotechnology.

Techniques: Western Blot, Infection, Activity Assay, Blocking Assay, Phospho-proteomics, Comparison