Human/Mouse/Rat Phospho-Chk1 (S317) Antibody

Detection of Human Phospho-Chk1 (S317) by Western Blot. Western blot shows lysates of HeLa human cervical epithelial carcinoma cell line untreated (-) or exposed (+) to 50 J/m²UV-C for the indicated time. PVDF membrane was probed with 1 µg/mL Rabbit Anti-Human/Mouse/Rat Phospho-Chk1 (S317) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2054) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). A specific band for Phospho-Chk1 (S317) was detected at approximately 56 kDa (as indicated). The phospho-specificity of this antibody was supported by decreased labeling following treatment with 600 U lambda-phosphatase (lambda-PPase) for 1 hour. This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Human Phospho-Chk1 (S317) by Simple Western^TM. Simple Western lane view shows lysates of HeLa human cervical epithelial carcinoma cell line untreated (-) or treated (+) with 50 J/m²ultraviolet light (UV) for 2 hours, loaded at 0.2 mg/mL. A specific band was detected for Phospho-Chk1 (S317) at approximately 60 kDa (as indicated) using 10 µg/mL of Rabbit Anti-Human/Mouse/Rat Phospho-Chk1 (S317) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2054). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Detection of Phospho-Chk1 (S317) by Western Blot NF-kappa B reiterating miR146a function in the FA pathway and replication fork restart(A) Two days after transfection of cells with p65 cDNA and anti-miR146a in combination as indicated, cells were treated with 5 mM HU for 5 h. The levels of FANCM and p63 proteins, and FANCD2 ubiquitination were measured by western blotting. (B) After undergoing the same treatment as (A) HeLa cells were analyzed for FANCD2 foci formation. Results are shown as the mean ± SD (n = 3); **P < 0.01. (C) Under the same conditions described in (A) cell lysates were analyzed by western blotting with antibodies against pCHK1-S317, CHK1, pRPA-S4/8, RPA, and GFP. Right, quantitation of pCHK1-S317 and pRPA-S4/8 expression. Results are shown as the mean ± SD (n = 3). (D) GES-1 cells transfected with p65 construct were applied to DNA fiber analysis. Resulting images of DNA fibers of p65-overexpressing cells compared with control cells (top). CldU tract length distribution was determined (bottom). (E) HeLa or GES-1 cells with the same treatment as A were subjected to gamma -H2AX detection. Results are shown as the mean ± SD (n = 3); **P < 0.01. (F) HeLa and GES-1 cells were transfected with p65 construct and Anti-miR146a in combinations as indicated and were treated with different doses of HU for 5 hr. Cell survival thereafter was measured using clonogenic survival assay. Results are shown as the mean ± SD (n = 3); **P < 0.01. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/27351285), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Phospho-Chk1 (S317) by Western Blot Effect of miR146a on FANCD2 monoubiquitination and the FA pathway(A and B) HeLa cells were transfected with miR146a in the absence or presence of anti-miR146a (A) or miR146a-insensitive FANCM cDNA (B). After a 48 h transfection, the cells were treated with 5 mM HU for 5 h. The protein levels of FANCM and FANCD2 were measured by western blotting. Monoubiquitinated FANCD2 is indicated as the upper band of doublet protein bands corresponding to FANCD2. (C and D) After HeLa cells underwent the same treatment as (A and B) cells were analyzed for FANCD2 foci formation. DAPI was used for nuclear staining. Results are shown as the mean ± SD (n = 3); **P < 0.01. (E and F) Indicated cells were treated with 5 mM HU for 5 hr. Cell lysates were analyzed by Western blotting with antibodies against pCHK1-S317, CHK1, pRPA-S4/8, and RPA. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/27351285), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Phospho-Chk1 (S317) by Western Blot Depletion of HERC2 inhibit ATR-mediated phosphorylation of RPA2 induced by low-level replication stress. (a–d) HeLa-shHERC2 cells were induced or not with Dox, treated or not with the indicated genotoxic agents, and subjected to immunoblotting with the indicated antibodies. (e–h) HeLa-shHERC2 cells were transfected with siRNA specific to ATR (e,f) or RFWD3 (g,h) induced or not with Dox, treated or not with indicated concentration of HU (e,g) or APH (f,h), and subjected to immunoblotting with the indicated antibodies. The asterisks indicates non-specific bands. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/31582797), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Phospho-Chk1 (S317) by Western Blot Effect of miR146a on FANCD2 monoubiquitination and the FA pathway(A and B) HeLa cells were transfected with miR146a in the absence or presence of anti-miR146a (A) or miR146a-insensitive FANCM cDNA (B). After a 48 h transfection, the cells were treated with 5 mM HU for 5 h. The protein levels of FANCM and FANCD2 were measured by western blotting. Monoubiquitinated FANCD2 is indicated as the upper band of doublet protein bands corresponding to FANCD2. (C and D) After HeLa cells underwent the same treatment as (A and B) cells were analyzed for FANCD2 foci formation. DAPI was used for nuclear staining. Results are shown as the mean ± SD (n = 3); **P < 0.01. (E and F) Indicated cells were treated with 5 mM HU for 5 hr. Cell lysates were analyzed by Western blotting with antibodies against pCHK1-S317, CHK1, pRPA-S4/8, and RPA. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/27351285), licensed under a CC-BY license. Not internally tested by R&D Systems.