gade2 pmel13 cas9 10969 kanr snr52 tef1 guide rna ade2 spcas9 endonuclease additional plasmids origin 13 pmel13 cas9 10969 kanr ref  (Addgene inc)

Journal: iScience

Article Title: Revisiting phosphoregulation of Cdc25C during M-phase induction

doi: 10.1016/j.isci.2024.111603

Figure Lengend Snippet: Characterization of MEE-catalyzed phosphorylation of GST-Cdc25C by mass spectrometry (A) An experimental flowchart for analyzing MEE-phosphorylated GST-Cdc25Cˆ by mass spectrometry. (B) Phosphorylation of GST-Cdc25Cˆ with MEE for 6 h, followed by SDS-PAGE and Coomassie blue staining. Double asterisks indicate sliced bands for mass spectrometry. (C) MEE-phosphorylated GST-Cdc25Cˆ was analyzed by LC-MS/MS involving both HCD and EDT fragmentations. From both output PSM datasets, PSMs with high or medium significance were selected and combined for data analysis by a macro of EXCEL edited with the programming language of Visual Basic, which generated Table S1 . Table S1 was used to calculate numbers and percentages of total, S/T-containing (S/T+) and phosphorylated S/T-containing (pS/T+) PSMs from mass spectrometry of MEE-phosphorylated GST-Cdc25Cˆ. (D) Protein recovery and phosphorylation site identification in the GST, Cdc25C 9-368 and Cdc25C 369-550ˆ regions of GST-Cdc25Cˆ, with non-recovered regions indicated by strikethroughs. Indicated above each of the S/T residues are phosphorylated over total identified in relevant PSMs, which were obtained from the data analysis described for (C). Red and blue types indicate phosphorylated and non-phosphorylated S/T residues, respectively. Red types with + sign indicate phosphorylated Y residues. Shaded red types indicate the S/T/Y residues that were phosphorylated at <5% identification frequencies. (E) Phosphorylation identification frequencies for individual S/T residues in the recovered regions of GST-Cdc25Cˆ were calculated by dividing the total number of PSMs that contained this S/T residue by the total number of PSMs that were phosphorylated at this residue. Both spectra accounts were obtained from the data analysis described for (C). Asterisks mark out the S/T residues in the context of S/TP motifs.

Article Snippet: The expression constructs for GST-tagged Cdc25C proteins were transformed into competent BL21 E. coli (Thermo Scientific, EC0114) by heat shock at 42°C.

Techniques: Mass Spectrometry, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Generated, Residue

Journal: iScience

Article Title: Revisiting phosphoregulation of Cdc25C during M-phase induction

doi: 10.1016/j.isci.2024.111603

Figure Lengend Snippet: Phosphorylation of multiple conserved S/TP motifs in the regulatory domain of Cdc25C is a prerequisite for the supershift-producing phosphorylation (A) Schematic illustration of localization of five conserved S/TP motifs in five relatively conserved regions in the regulatory domain of Cdc25C. (B) Wild type (WT) and indicated single, double or triple T-to-V mutant forms of myc-Cdc25Cˆ were phosphorylated with MEE for indicated hour, and products were immunoblotted with anti-myc tag antibodies. (C) WT and indicated single or double S-to-A mutant forms of myc-Cdc25Cˆ with or without preexisting 48V or 67V single mutation (left panel) or with or without preexisting 48V-T67V-138V triple mutation (right panel) were processed as described for (B). (D) WT and indicated single or double T-to-V (upper panel) or T-to-E (lower panel) mutant forms of myc-Cdc25Cˆ were processed as described for (B). (E) WT and indicated triple T-to-V or T-to-E mutant form of myc-Cdc25Cˆ were processed as described for (B). (F) WT and indicated triple TP-to-EP or TP-to-EE mutant form of myc-Cdc25Cˆ were processed as described for (B). (G) WT and indicated triple TP-to-EP, TP-to-EE or TP-VP mutant form of myc-Cdc25C were phosphorylated with undiluted ME (left panel) or 1:4-diluted ME (right panel) prepared from HEK293T cells for indicated hr, and products were immunoblotted with anti-myc tag antibodies.

Article Snippet: The expression constructs for GST-tagged Cdc25C proteins were transformed into competent BL21 E. coli (Thermo Scientific, EC0114) by heat shock at 42°C.

Techniques: Mutagenesis

Journal: iScience

Article Title: Revisiting phosphoregulation of Cdc25C during M-phase induction

doi: 10.1016/j.isci.2024.111603

Figure Lengend Snippet: Phosphorylation of the three conserved TP motifs positively regulates the density of the site-comprehensive phosphorylation of the regulatory domain of Cdc25C (A) Parallel phosphorylation of immobilized GST, GST-Cdc25Cˆ and GST-Cdc25Cˆ T3V with MEE for 3 h in the presence of [γ- 32 P]-ATP, followed by ponceau S staining and autoradiography of gel-separated proteins on the same blot. (B) Immobilized GST-Cdc25Cˆ was phosphorylated with MEE for 3 or 6 h, and proteins eluted from washed beads were subject to SDS-PAGE and Coomassie blue staining. (C) GST-Cdc25Cˆ T3V was processed as described for (B). (D) Top bands from MEE-phosphorylated GST-Cdc25Cˆ in (B) were analyzed by LC-MS/MS involving HCD fragmentation. From combined output PSM datasets, PSMs with high or medium significance were selected and combined for data analysis by a macro of EXCEL edited with the programming language of Visual Basic to generate total and phosphorylated spectral counts for individual S/T residues. Phosphorylation identification frequencies for individual S/T residues in recovered Cdc25Cˆ regions were then calculated by dividing total spectral count with phosphorylated spectral count for each of the identified S/T residues. Asterisk symbols mark out the S/T residues in the context of S/TP motifs. (E) Top bands from MEE phosphorylated GST-Cdc25Cˆ T3V in (C) were processed as described for (D). Red underlines indicate the phosphorylation sites that were not phosphorylated in Cdc25Cˆ T3V, and black underlines indicate the phosphorylation sites that had ≥2-fold lower phosphorylation identification frequencies than the same site in Cdc25Cˆ. Asterisk symbols mark out the S/T residues in the context of S/TP motifs.

Article Snippet: The expression constructs for GST-tagged Cdc25C proteins were transformed into competent BL21 E. coli (Thermo Scientific, EC0114) by heat shock at 42°C.

Techniques: Staining, Autoradiography, SDS Page, Liquid Chromatography with Mass Spectroscopy

Journal: iScience

Article Title: Revisiting phosphoregulation of Cdc25C during M-phase induction

doi: 10.1016/j.isci.2024.111603

Figure Lengend Snippet: Characterization of the roles of the four major mitotic kinases in MEE-induced supershifts of Cdc25C (A) Summary of the major Cdk1, MAPK, Plx1 and RSK2 phosphorylation sites previously identified in Cdc25C. (B and C) Phosphorylation of myc-Cdc25C with the four major mitotic kinases individually (B) or collectively (C) in parallel with equivalent strengths of MEE for indicated hr, followed by myc-tag immunoblotting. (D) Phosphorylation of myc-Cdc25C with MEE or p9-Cdk1 for indicated hr, followed by myc-tag immunoblotting. (E) Phosphorylation of myc-Cdc25C with MEE in the presence of drug vehicle DMSO, the 4 specific kinase inhibitors combined (RS + VX + SL + BI) or a pan kinase inhibitor (ST) for indicated min, followed by myc-tag immunoblotting. (F) Phosphorylation of myc-Cdc25C with control IgG-absorbed or 4 kinase-depleted MEE for indicated min, followed by myc-tag immunoblotting. The ≈ symbols indicate equivalent timepoint matching.

Article Snippet: The expression constructs for GST-tagged Cdc25C proteins were transformed into competent BL21 E. coli (Thermo Scientific, EC0114) by heat shock at 42°C.

Techniques: Western Blot, Control

Journal: iScience

Article Title: Revisiting phosphoregulation of Cdc25C during M-phase induction

doi: 10.1016/j.isci.2024.111603

Figure Lengend Snippet: The abrupt kinetics of the MEE-induced supershift of Cdc25C stems from intrinsic properties of the site-comprehensive phosphorylation process (A and B) Phosphorylation of myc-Cdc25Cˆ with indicated dilutions of MEE for 1 h (A) or 2 h (B), followed by myc-tag immunoblotting. FL indicates full-length substrate protein. (C and D) Phosphorylation of myc-Cdc25Cˆ (C) or myc-Cdc25C 9-375 (D) with indicated serial dilutions of MEE for indicated min, followed by myc-tag immunoblotting. (E) Myc-Cdc25C 9-375 was either phosphorylated with 1:64-diluted MEE for indicated min (left panel) or first phosphorylated with MEE for 2 min and then phosphorylated with 1:64-diluted MEE for indicated min (right panel). Products were immunoblotted with anti-myc antibodies. (F and G) Immunoprecipitated myc-Cdc25C 9-375 was first phosphorylated with MEE (F) or an exceptionally high activity preparation of p9-Cdk1 (G) for 2 min and then chased in phosphorylation buffer for indicated min after 2× wash with EB. Proteins eluted from start and treated immunocomplexes were immunoblotted with anti-myc antibodies. Phosphorylation with MEE (F) or the high activity preparation of p9-Cdk1 (G) for 160 min served as a positive control.

Article Snippet: The expression constructs for GST-tagged Cdc25C proteins were transformed into competent BL21 E. coli (Thermo Scientific, EC0114) by heat shock at 42°C.

Techniques: Western Blot, Immunoprecipitation, Activity Assay, Positive Control

Journal: iScience

Article Title: Revisiting phosphoregulation of Cdc25C during M-phase induction

doi: 10.1016/j.isci.2024.111603

Figure Lengend Snippet: The site-comprehensive phosphorylation of the regulatory domain of Cdc25C is required for robust activation of G2-state Cdc25C (A) IOE-Cdc25C was mock-treated with EB or phosphorylated with either individual TNT kinase samples or MEE for 2 h. Washed immunocomplexes were assayed for Cdc25C shifts by myc-tag immunoblotting (left) and for Cdc25C activity by measuring Cdk1 dephosphorylation for indicated hr (right). (B) Same as described for (A) except that 1:4-diluted TNT kinase samples or MEE were used to phosphorylate IOE-Cdc25C. (C) IOE-Cdc25C was mock-treated or phosphorylated with either 4 TNT kinase mixture (4K mix) or 1:4-diluted MEE (1/4 MEE) for 2 h, and washed immunocomplexes were processed as described for (A). (D) IOE-Cdc25C was mock-treated or phosphorylated with p9-Cdk1 or MEE for 2 h, and washed immunocomplexes were processed as described for (A). (E) IOE-Cdc25C was mock-treated or phosphorylated with MEE in the presence or absence of the 4 specific kinase inhibitors (4KI) for 2 h, and washed immunocomplexes were processed as described for (A). (F) WT or T3V mutant form of IOE-Cdc25C was mock-treated or phosphorylated with MEE for 2 h, and washed immunocomplexes were processed as described for (A). (G) IOE-Cdc25C was mock-treated or phosphorylated with MEE for 1 h with or without subsequent dephosphorylation with λ phosphatase (λP) for 1 h. In conjunction, immunoprecipitated myc-Cdc25C 62DE was treated with MEE for 1 h. Washed immunocomplexes were processed as described for (A).

Article Snippet: The expression constructs for GST-tagged Cdc25C proteins were transformed into competent BL21 E. coli (Thermo Scientific, EC0114) by heat shock at 42°C.

Techniques: Activation Assay, Western Blot, Activity Assay, De-Phosphorylation Assay, Mutagenesis, Immunoprecipitation

Journal: iScience

Article Title: Revisiting phosphoregulation of Cdc25C during M-phase induction

doi: 10.1016/j.isci.2024.111603

Figure Lengend Snippet: Biphasic regulation of Cdc25C during M-phase induction (A) Xenopus oocytes were stimulated with progesterone for indicated hour, and oocyte extracts were both immunoblotted with indicated antibodies and assayed for phosphorylation of histone H1 or MBP by 32 P incorporation. (B) Xenopus oocytes were pretreated with U0126 for 1 h, and washed oocytes were processed as described for (A). (C) Xenopus oocytes ectopically expressing HA-Cdc25Cˆ were matured with progesterone, unstimulated or injected with a sub-threshold level of mRNA for myc-CA-MEK1. Oocyte extracts and proteins immunoprecipitated with anti-HA antibodies were immunoblotted with indicated antibodies. (D) Xenopus oocytes ectopically expressing myc-Cdc25Cˆ were stimulated with progesterone for indicated hour in the continued presence of U0126 or its inactive analog U0124. Oocyte extracts and proteins immunoprecipitated with anti-myc antibodies were immunoblotted with indicated antibodies. (E) Xenopus oocytes were injected with a threshold level of mRNA for Cdk1-AF and cultured for indicated hour in the continued presence of 50 μM U0126. Untreated and progesterone-matured oocytes were used as controls. Oocyte extracts were immunoblotted with indicated antibodies and assayed for phosphorylation of histone H1 by 32 P incorporation. (F) Interphase-arrested Xenopus egg extract (IE) was incubated with a threshold level of GST-ΔCycB for indicated min, and samples were immunoblotted with indicated antibodies and assayed for phosphorylation of histone H1 by 32 P incorporation. In each subfigure, the ± symbol indicates a slight gel mobility shift of endogenous or ectopically expressed Cdc25C prior to M-phase onset, whereas the ‡ symbol indicates the M-phase supershift of endogenous or ectopically expressed Cdc25C at M-phase onset.

Article Snippet: The expression constructs for GST-tagged Cdc25C proteins were transformed into competent BL21 E. coli (Thermo Scientific, EC0114) by heat shock at 42°C.

Techniques: Expressing, Injection, Immunoprecipitation, Cell Culture, Incubation, Mobility Shift

Journal: iScience

Article Title: Revisiting phosphoregulation of Cdc25C during M-phase induction

doi: 10.1016/j.isci.2024.111603

Figure Lengend Snippet:

Article Snippet: The expression constructs for GST-tagged Cdc25C proteins were transformed into competent BL21 E. coli (Thermo Scientific, EC0114) by heat shock at 42°C.

Techniques: Western Blot, Immunoprecipitation, Sequencing, Modification, Mass Spectrometry, Recombinant, Protease Inhibitor, Mutagenesis, Purification, Expressing

Journal: PLoS ONE

Article Title: Platelet Activating Factor Blocks Interkinetic Nuclear Migration in Retinal Progenitors through an Arrest of the Cell Cycle at the S/G2 Transition

doi: 10.1371/journal.pone.0016058

Figure Lengend Snippet: A, B: Analysis of cyclin B1 content in BrdU-labeled cells. A: Cytofluorograms gated on BrdU-labeled cells dissociated from either control or PAF-treated retinal explants shows displacement of PAF-treated cells towards lower levels of cyclin B1; BKG = controls without primary cyclin B1 antibody; B: Mean and S.E.M. of the median cyclin B1 fluorescent intensity in BrdU-labeled cells, averaged among 4 independent experiments. Data were normalized to the respective controls in each experiment; * = p<0.01. C: Treatment of retinal explants with 0.3 nM PAF for 3 h induces an increase in the activity of Chk1, as shown by increased phosphorylation of a target cdc25C peptide (n = 2 with identical results) D: SB218078, a Chk1 inhibitor, blocks the effect of PAF (Data are means ± S.E.M., from n = 5 independent experiments in duplicate); ** = p<0.001.

Article Snippet: GST-Cdc25C 200–256 fusion protein was affinity-purified using a glutathione sepharose 4B matrix (GS4B – Amersham Pharmacia) following a batch method described by the manufacturers.

Techniques: Labeling, Activity Assay