Human/Mouse/Rat Phospho-CDC2/CDK1 (Y15) Antibody

Detection of Mouse and Human Phospho-CDC2 (Y15) by Western Blot. Western blot shows lysates of L-929 mouse fibroblast cell line and Jurkat human acute T cell leukemia cell line untreated (-) or treated (+) with 0.2 µg/mL nocodazole or 12 µM aphidicolin or 1 mM hydroxurea for 18 hours. PVDF membrane was probed with 0.2 µg/mL of Rabbit Anti-Human/Mouse/Rat Phospho-CDC2 (Y15) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF888), followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). A specific band was detected for Phospho-CDC2 (Y15) at approximately 34 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Human CDC2/CDK1 by Western Blot Cooperative induction of DNA damage in vivo by WEE1 and CHK1 inhibitors. LoVo xenograft tumor-bearing mice were treated with 60 mpk MK-1775 BID for 2 days, 60 mpk MK-8776 BID for 2 days, or the combination of MK-1775 and MK-8776 each at 60 mpk BID for 2 days. Tumors were collected at 2, 24, and 48 hours following the final dose. A, LoVo tumor lysates were analyzed by Western blot for pCHK1S345. B, Tumor sections were fixed and analyzed by immunohistochemistry (IHC). Representative images for gamma H2AX at 2 hours and 48 hours post final dose are shown. C, Quantitative analysis of IHC for both phospho-CHK1S345 and gamma H2AX (n=3); one-way ANOVA analyses *P<0.05. **P<0.01, ***P<0.001. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/23148684), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human CDC2/CDK1 by Western Blot DNA damage response incurred by MK-1775 and MK-8776 is dependent on CDK activity.A, Resistant (H460) or sensitive (LoVo) cells were treated with concentrations of MK-1775 and MK-8776 described for Figure 4, or 1 uM nocodazole for control. After 24 hours, cells were harvested and lysates analyzed by Western blot for caspase-dependent cleaved PARP (PARP*). B, A2058, HT-29, and LoVo cells were treated for 30 minutes with either DMSO or the indicated concentration of CDK inhibitor (SCH-727965). Following this pretreatment, further DMSO or concentrations of MK-1775 and MK-8776 used in Figures 3 and 4 (125 nM MK-1775 plus 150 nM MK-8776 in A2058; 125 nM MK-1775 plus 300 nM MK-8776 in HT-29, and 40 nM MK-1775 plus 75 nM MK-8776 in LoVo) were added to the cells for an additional 2 hours before cells were harvested and lysates analyzed by Western blot for phosphorylated CHK1S345, indicative of activated DNA damage response. C, LoVo cells were treated for 2 hours with 75 nM MK-1775 alone or in combination with 150 nM MK-8776, as indicated. Cells were harvested and lysates analyzed by Western blot for the proteins and phosphoproteins indicated. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/23148684), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human CDC2/CDK1 by Western Blot DNA damage response incurred by MK-1775 and MK-8776 is dependent on CDK activity.A, Resistant (H460) or sensitive (LoVo) cells were treated with concentrations of MK-1775 and MK-8776 described for Figure  4, or 1 uM nocodazole for control. After 24 hours, cells were harvested and lysates analyzed by Western blot for caspase-dependent cleaved PARP (PARP*). B, A2058, HT-29, and LoVo cells were treated for 30 minutes with either DMSO or the indicated concentration of CDK inhibitor (SCH-727965). Following this pretreatment, further DMSO or concentrations of MK-1775 and MK-8776 used in Figures  3 and 4 (125 nM MK-1775 plus 150 nM MK-8776 in A2058; 125 nM MK-1775 plus 300 nM MK-8776 in HT-29, and 40 nM MK-1775 plus 75 nM MK-8776 in LoVo) were added to the cells for an additional 2 hours before cells were harvested and lysates analyzed by Western blot for phosphorylated CHK1S345, indicative of activated DNA damage response. C, LoVo cells were treated for 2 hours with 75 nM MK-1775 alone or in combination with 150 nM MK-8776, as indicated. Cells were harvested and lysates analyzed by Western blot for the proteins and phosphoproteins indicated. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/23148684), licensed under a CC-BY license. Not internally tested by R&D Systems.