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Adipogen treatment with nocodazole
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 <t>nocodazole</t> (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).
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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).

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: For treatment with Nocodazole (NCO; AdipoGen Life Sciences), cells were released into regular growth medium for 4 h after the DTB before NCO (80 ng/ml) was added, and cells were incubated for another 14 h before cell harvest and lysis.

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