α cdk1 cdk2 an21 2  (Santa Cruz Biotechnology)

Journal: The Journal of Biological Chemistry

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

doi: 10.1016/j.jbc.2026.111149

Figure Lengend Snippet: S277 is a CDK2 target in cells . A , schematic of the RAD51AP1 protein (isoform 2) with predicted CDK1/2 sites (GPS 6.0). The F3 fragment (residues 188–335) and the bipartite DNA binding domain ( gray ) 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 FLAG-RAD51AP1-S2A (S277/282A) mutant with CDK2/Cyclin E2 or CDK1/Cyclin B1 and ɣ- 32 P-ATP. Upper : Phosphorimage; middle : 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 (rRAD51AP1; 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 (rF3; 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 (rF3; 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 unrelated to S277/S282 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; 1:3000); α-PARP1 (ab6079; Abcam; 1:1000); α-MSH2 (ab52266; Abcam; 1:10,000); α-Tubulin (DM1A; Santa Cruz Biotechnology; 1:1000); α-FLAG (F3165; Sigma; 1:1000); α-MBP (PAI-989; Thermo Fisher Scientific; 1:5000); α-RAD54 L (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; 1:2000); α-CDK2 (10122-1-AP; ProteinTech; 1:20,000); α-Cyclin A (H-432; Santa Cruz Biotechnology; 1:1000).

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

Journal: The Journal of Biological Chemistry

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

doi: 10.1016/j.jbc.2026.111149

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; 1:3000); α-PARP1 (ab6079; Abcam; 1:1000); α-MSH2 (ab52266; Abcam; 1:10,000); α-Tubulin (DM1A; Santa Cruz Biotechnology; 1:1000); α-FLAG (F3165; Sigma; 1:1000); α-MBP (PAI-989; Thermo Fisher Scientific; 1:5000); α-RAD54 L (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; 1:2000); α-CDK2 (10122-1-AP; ProteinTech; 1:20,000); α-Cyclin A (H-432; Santa Cruz Biotechnology; 1:1000).

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

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