Journal: The Journal of Cell Biology

Article Title: Regulation of the Cell Cycle by Focal Adhesion Kinase

doi:

Figure Lengend Snippet: Regulation of the expression levels of components of G1/S cyclin-Cdk complexes by exogenous FAK and mutants. ( A ) α-p21, α-cyclin D1, α-Cdk2, α-cyclin E, α-cyclin A, or α-p27 Western blot of whole cell lysates prepared from cells expressing the indicated proteins under uninduced (lanes U ) and induced (lanes I ) conditions. ( B ) Serum-starved cells expressing ΔC14 were incubated in media containing 10% CS in the presence ( U ) or absence ( I ) of tetracycline. At the indicated times, lysates were prepared and analyzed by Western blot with α-p21, α-cyclin D1, α-Cdk2, α-cyclin E, or α-cyclin A.

Article Snippet: The following antibodies were purchased as indicated: α-BrdU mouse mAb from Sigma (St. Louis, MO); α-phosphotyrosine mouse mAb PY20 from Transduction Laboratories (Lexington, KY); rabbit α-Src, α-Fyn, α-Erk, α-cyclin A, α-cyclin E, α-Cdk2, α-His (His-Probe), and mouse mAb α-Shc from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); rabbit α-Shc from UBI (Lake Placid, NY); and rabbit α-phospho-MAPK from New England Biolabs, Inc. (Beverly, MA).

Techniques: Expressing, Western Blot, Incubation

Journal:

Article Title: Spy1 Interacts with p27 Kip1 to Allow G 1 /S Progression

doi: 10.1091/mbc.E02-12-0820

Figure Lengend Snippet: Human p27 interacts with human Spy1 in vitro. (A) In vitro–translated p27 (TNT-p27) was incubated for 2 h with either in vitro–translated pCS3-MT empty vector (TNT-mock) or in vitro–translated myc-tagged Spy1 (TNT-Spy1). The top panel demonstrates the amount of input TNT-Spy1 protein (lane 2), the middle panel demonstrates the input TNT-p27 protein (lanes 1 and 2). Both samples were immunoprecipitated with α-myc sera, and proteins were separated by 10% SDS-PAGE and immunoblotted with p27 sera (bottom panel). (B) In vitro–translated pCS3-MT (TNT-mock), p27 (TNT-p27), myc-Spy1 (TNT-Spy1), or CDK2 (TNT-CDK2) were separated by 10% SDS-PAGE and immunoblotted with α-CDK2 sera as a control. (C) Purified GST-p27 or GST-control protein conjugated to GST beads were incubated in binding buffer with either in vitro–translated (TNT) mock or Spy1 protein (top panel), both of which were previously immunodepleted for any CDK2 protein as an additional control. A GST pull-down assay was performed. Proteins were separated by 12.5% SDS-PAGE and immunoblotted with α-myc sera to detect immunoprecipitation of GST proteins with myc-tagged proteins (bottom panel). α-GST immunoblot is presented to demonstrate the GST proteins present in the GST pull-down experiment. Different exposures of the same immunoblot are presented in the middle panel because of the high amount of protein in the GST-control lanes.

Article Snippet: Lysates (1 mg protein) were precleared with 40 μl Protein A-Sepharose beads and incubated with 2 μg of α-flag (Sigma), α-myc (9E10; Santa Cruz Biotechnology, Santa Cruz, CA), α-p27 (Zymed, South San Francisco, CA, or Santa Cruz) or α-CDK2 (Santa Cruz) overnight at 4°C.

Techniques: In Vitro, Incubation, Plasmid Preparation, Immunoprecipitation, SDS Page, Purification, Binding Assay, Pull Down Assay, Western Blot

Journal:

Article Title: Spy1 Interacts with p27 Kip1 to Allow G 1 /S Progression

doi: 10.1091/mbc.E02-12-0820

Figure Lengend Snippet: p27 interacts with Spy1 and CDK2. 293T cells were transfected with the indicated constructs. The lysates were immunoprecipitated with α-p27 or α–CDK2 sera and immunoblotted for Spy1, CDK2, and p27. Lane 8 demonstrates that flag-Spy1 (top panel), endogenous CDK2 (middle panel), and p27 (bottom panel) can coimmunoprecipitate. The top panel of lane 11 shows that Spy1 interacts with CDK2 and that this interaction is increased when p27 is overexpressed as demonstrated in lane 12.

Article Snippet: Lysates (1 mg protein) were precleared with 40 μl Protein A-Sepharose beads and incubated with 2 μg of α-flag (Sigma), α-myc (9E10; Santa Cruz Biotechnology, Santa Cruz, CA), α-p27 (Zymed, South San Francisco, CA, or Santa Cruz) or α-CDK2 (Santa Cruz) overnight at 4°C.

Techniques: Transfection, Construct, Immunoprecipitation

Journal:

Article Title: Spy1 Interacts with p27 Kip1 to Allow G 1 /S Progression

doi: 10.1091/mbc.E02-12-0820

Figure Lengend Snippet: Spy1 enhances histone H1 kinase activity in the presence of p27. (A) 293T cells were transfected with the indicated constructs. The lysates were immunoprecipitated with α-CDK2 sera and subjected to an in vitro kinase assay. Lane 2 shows that p27-transfected cells have decreased kinase activity, but Spy1-transfected cells exhibit kinase activity higher than mock-transfected cells (lane 3). When both Spy1 and p27 are cotransfected, CDK2 histone H1 kinase activity is increased as shown in lane 4 compared with p27 alone. (B) The lysates were analyzed by 10% SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with flag-Spy1, CDK2, and p27 antisera to demonstrate equivalent protein expression.

Article Snippet: Lysates (1 mg protein) were precleared with 40 μl Protein A-Sepharose beads and incubated with 2 μg of α-flag (Sigma), α-myc (9E10; Santa Cruz Biotechnology, Santa Cruz, CA), α-p27 (Zymed, South San Francisco, CA, or Santa Cruz) or α-CDK2 (Santa Cruz) overnight at 4°C.

Techniques: Activity Assay, Transfection, Construct, Immunoprecipitation, In Vitro, Kinase Assay, SDS Page, Expressing

Journal:

Article Title: Spy1 Interacts with p27 Kip1 to Allow G 1 /S Progression

doi: 10.1091/mbc.E02-12-0820

Figure Lengend Snippet: Spy1-enhanced proliferation is dependent on endogenous p27. (A) MEF–/– cells were transfected with the indicated constructs, medium was changed 24 h posttransfection, and cells were counted via trypan blue exclusion 48 h posttransfection. The graphed data indicate that exogenous p27 slows proliferation in the MEF–/– cells and that Spy1 can overcome this inhibition of proliferation. Spy1 does not enhance proliferation over mock control in cells lacking endogenous p27. The data are one representative experiment of four. Error bars indicate the SEM of three separate transfections. (B) Lysates were analyzed by 10% SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with α-myc-Spy1, and α-p27 sera to demonstrate protein expression. (C) Endogenous Spy1 was immunoprecipitated from either NIH3T3 cells null for p27 (3T3–/–) or NIH3T3 wt (3T3wt). Both lysate and immunoprecipitated samples were separated by 10% SDS-PAGE and immunoblotted with α-CDK2 sera (top panel) or α-p27 sera (bottom panel). (D) Endogenous CDK2 was immunoprecipitated from both 3T3wt and 3T3–/– cell types. Lysate samples and the immunoprecipitated samples were analyzed by 10% SDS-PAGE, and immunoblotting was carried out with α-Spy1 sera.

Article Snippet: Lysates (1 mg protein) were precleared with 40 μl Protein A-Sepharose beads and incubated with 2 μg of α-flag (Sigma), α-myc (9E10; Santa Cruz Biotechnology, Santa Cruz, CA), α-p27 (Zymed, South San Francisco, CA, or Santa Cruz) or α-CDK2 (Santa Cruz) overnight at 4°C.

Techniques: Transfection, Construct, Inhibition, SDS Page, Expressing, Immunoprecipitation, Western Blot

Journal:

Article Title: Zbtb4 represses transcription of P21CIP1 and controls the cellular response to p53 activation

doi: 10.1038/emboj.2008.85

Figure Lengend Snippet: Depletion of Zbtb4 causes resistance to vincristine treatment through upregulation of P21CIP1. (A) Vincristine induces activation of p53. The panels show immunoblots of SH-EP cells infected with either a control vector (left) or a vector expressing a dominant-negative allele of p53 (p53DD; right) probed with the indicated antibodies. (B) Transient depletion of Zbtb4 upregulates P21CIP1 mRNA levels. The panels document the RQ–PCR analysis of relative mRNA expression level of ZBTB4, P21CIP1, 14-3-3σ, BTG2, GADD45, PUMA, BAX and NOXA in SH-EP and SH-SY5Y neuroblastoma cells transfected with either control or siRNA targeting ZBTB4. Cells were harvested 48 h after transfection and expression levels were determined by RQ–PCR. (C) Stable depletion of Zbtb4 upregulates p21Cip1 protein. The panels show immunoblots of p21Cip1 and Cdk2 protein levels in indicated SH-EP cell clones stably infected with either control vectors or vectors expressing shZBTB4. (D) Ectopic expression of Zbtb4 represses P21CIP1. The panels document the RQ–PCR analysis of the relative mRNA expression level of ZBTB4, P21CIP1, PUMA and NOXA in SH-EP cells stably infected with HA-tagged ZBTB4 vector or a control vector. (E) P21CIP1 is required for protection of SH-EP cells from vincristine-induced apoptosis by depletion of Zbtb4. The indicated SH-EP cell clones stably expressing either shZBTB4 or control shRNA were transiently transfected with vectors expressing shP21CIP1 or a scrambled shRNA. At 48 h after transfection, cells were plated in triplicate for vincristine treatment. Cells were treated with either vincristine or DMSO for 3 days; subsequently, the relative cell number was determined. Error bars represent standard error of the mean.

Article Snippet: Antibodies The following antibodies were used: α-HA (HA.11; Covance), α-p21Cip1 (N-20), α-p53 (DO-1), α-Cdk2 (M2), α-mSin3b (H4) (all Santa Cruz Biotechnology), α-β-tubulin (MAB3408; Chemicon), α-Miz1 (10E2, Herold et al , 2002 ; H190, Santa Cruz Biotechnology) and α-Zbtb4 ( Filion et al , 2006 ).

Techniques: Activation Assay, Western Blot, Infection, Control, Plasmid Preparation, Expressing, Dominant Negative Mutation, Transfection, Clone Assay, Stable Transfection, shRNA

Journal: International Journal of Molecular Sciences

Article Title: HMGA1 Regulates the Expression of Replication-Dependent Histone Genes and Cell-Cycle in Breast Cancer Cells

doi: 10.3390/ijms24010594

Figure Lengend Snippet: HMGA1 regulates cyclin E2 and Cdk2 expressions in MDA-MB-231 cells. Western blot analyses assess cyclinE2 and Cdk2 protein expression in MDA–MB–231 cells silenced for HMGA1 (siA1_3) or treated with control siRNA (siCTRL). Representative WB analyses, on the left, are shown together with red ponceau stained membrane to verify total protein normalization. The histogram graph on the right refers to densitometric analysis of western blot (siCTRL and siA1_3). Bars indicate the mean. Standard deviations are shown ( n = 3). Statistical significance was assessed with Student’s t -test (*: p ≤ 0.05; ***: p ≤ 0.001).

Article Snippet: Western blot analyses were carried out under standard procedures using nitrocellulose membranes (GE HealthCare, Chicago, IL, USA) and the following primary antibodies: α-HMGA1 [ ], α-GFP (GTX113617, Genetex, Irvine, CA, USA), α-SLBP (ab181972, Abcam, Cambridge, UK), α-NPAT (LS-C289483, LSBio, Seattle, WA, USA), CyclinE2 (ab226388, Abcam, Cambridge, UK), and α-Cdk2 (ab235941, Abcam, Cambridge, UK).

Techniques: Western Blot, Expressing, Control, Staining, Membrane

Journal: bioRxiv

Article Title: Phosphoregulation of RAD51AP1 function in homology-directed repair

doi: 10.1101/2025.09.10.675389

Figure Lengend Snippet: A , Schematic of the RAD51AP1 protein (isoform 2) with predicted CDK1/2 sites (GPS 6.0). The F3 fragment (residues 188-335) and the bi-partite DNA binding domain (grey) are indicated. Predicted CDK1/2 sites in F3 (S224, S277 and S310) were changed to alanine. B , Results from kinase reaction of purified His 6 -F3-FLAG and mutants (S224/310A and S224/277/310A) with CDK2/Cyclin E2 and ɣ- 32 P-ATP. Upper: Phosphorimage; lower: Western blot with ⍺-FLAG antibody. C , Results from kinase reaction of purified His 6 -F3-FLAG and mutants (S224/310A and S224/277/310A) with CDK1/Cyclin B1 and ɣ- 32 P-ATP. Upper: Phosphorimage; lower: Western blot with ⍺-FLAG antibody. D , Results from kinase reaction of stably expressed (in HeLa RAD51AP1 KO cells) and precipitated FLAG-RAD51AP1 and the S2A (S277/282A) mutant with CDK2/Cyclin E2 and ɣ- 32 P-ATP. Upper: Phosphorimage; lower: Western blot with ⍺-RAD51AP1 antibody. The product of the kinase reaction with purified (from E. coli ) His6-RAD51AP1-FLAG protein is shown for comparison purposes (lane 1). E , Results from kinase reaction of transiently expressed (in HeLa RAD51AP1 KO cells) and precipitated FLAG-F3 and mutants (S224/310A and S224/277/310A) with CDK2/Cyclin E2 and ɣ- 32 P-ATP. Upper: Phosphorimage; middle and lower: Western blot with ⍺-FLAG antibody. The product of the kinase reaction with purified (from E. coli ) His 6 -F3-FLAG is shown for comparison purposes (lane 1). Pink boxes: to highlight phosphorylated F3. F , Results from kinase reaction of transiently expressed (in HeLa RAD51AP1 KO cells) and precipitated FLAG-F3 with CDK2/Cyclin E2 and ɣ- 32 P-ATP without and with the addition of 1 µM Roscovitine (+ R; lanes 6 and 7, respectively). The product of the kinase reaction with purified (from E. coli ) His 6 -F3-FLAG is shown for comparison purposes (lane 1). G , CDK2 and Cyclin A are present in ⍺-FLAG precipitates of FLAG-RAD51AP1 (WT) stably expressed in HeLa RAD51AP1 KO cells, synchronized by a double thymidine block (DTB) and released into early S phase (lane 3). CDK2 and Cyclin A are not present FLAG-RAD51AP1 (WT) precipitates from cells arrested in late G2/M phase by treatment with nocodazole (NCO; lane 4). Note: In G2/M phase cells (lane 4), a partial mobility shift is observed for RAD51AP1 that – based on our preliminary results – is likely to be unrelated to phosphorylation (data not shown). H , CDK1 is not present in FLAG-RAD51AP1 (WT) precipitates of cells synchronized in early S (lane 3) or late G2/M phase (lane 4). I , CDK2 and Cyclin A are present in precipitates of transiently expressed FLAG-F3 and mutants in RAD51AP1 KO cells synchronized by a DTB and released into early S phase (lanes 2-4).

Article Snippet: The primary antibodies that were used are: α-RAD51AP1 (( ); 11255-1-AP; ProteinTech; 1:10,000); α-RAD51 (Ab-1; EMD Millipore; 1:3,000); α-PARP1 (ab6079; Abcam; 1:1,000); α-MSH2 (ab52266; Abcam; 1:10,000); α-Tubulin (DM1A; Santa Cruz Biotechnology; 1:1,000); α-FLAG (F3165; Sigma; 1:1,000); α-MBP (PAI-989; Thermo Fisher Scientific; 1:5,000); α-RAD54L (F-11; sc-374598; Santa Cruz Biotechnology; 1:500); α-NUCKS1 (( ); 1:10,000); α-GST (ab19256; Abcam; 1:10,000); α-UAF1 (ab122473; Abcam; 1:100); α-CDK1 (10762-1-AP; ProteinTech; 1:2,000); α-CDK2 (10122-1-AP; ProteinTech; 1:20,000); α-Cyclin A (H-432; Santa Cruz Biotechnology; 1:1,000).

Techniques: Binding Assay, Purification, Western Blot, Stable Transfection, Mutagenesis, Comparison, Blocking Assay, Mobility Shift, Phospho-proteomics

Journal: bioRxiv

Article Title: Phosphoregulation of RAD51AP1 function in homology-directed repair

doi: 10.1101/2025.09.10.675389

Figure Lengend Snippet: Model to illustrate how RAD51AP1-S277/S282 phosphorylation may lead to RAD51 activation in synapsis and D-loop formation. A , S277/S282 phosphorylation may occur prior to RAD51AP1’s association with the presynaptic filament. Alternatively, S277/S282 phosphorylation may occur when RAD51AP1 is bound to the presynaptic filament. Phosphorylation may allow RAD51AP1 to engage transiently and hierarchically in synapsis and strand exchange. It is unclear how RAD51AP1 leaves the D-loop. RAD51AP1 is dephosphorylated prior to its renewed engagement in HDR and when CDK2 is active. The kinase that targets S282 and the phosphatases involved remain to be identified. B , The RAD51AP1-S277/282A mutant displays enhanced proficiency in DNA binding, synapsis and D-loop formation, but is defective in promoting the continuous reaction due to its lack in flexibility and compromised dynamics. An HDR defective phenotype is observed. Created with BioRender.com.

Article Snippet: The primary antibodies that were used are: α-RAD51AP1 (( ); 11255-1-AP; ProteinTech; 1:10,000); α-RAD51 (Ab-1; EMD Millipore; 1:3,000); α-PARP1 (ab6079; Abcam; 1:1,000); α-MSH2 (ab52266; Abcam; 1:10,000); α-Tubulin (DM1A; Santa Cruz Biotechnology; 1:1,000); α-FLAG (F3165; Sigma; 1:1,000); α-MBP (PAI-989; Thermo Fisher Scientific; 1:5,000); α-RAD54L (F-11; sc-374598; Santa Cruz Biotechnology; 1:500); α-NUCKS1 (( ); 1:10,000); α-GST (ab19256; Abcam; 1:10,000); α-UAF1 (ab122473; Abcam; 1:100); α-CDK1 (10762-1-AP; ProteinTech; 1:2,000); α-CDK2 (10122-1-AP; ProteinTech; 1:20,000); α-Cyclin A (H-432; Santa Cruz Biotechnology; 1:1,000).

Techniques: Phospho-proteomics, Activation Assay, Mutagenesis, Binding Assay