Journal: eLife

Article Title: PP2A/B55α substrate recruitment as defined by the retinoblastoma-related protein p107

doi: 10.7554/eLife.63181

Figure Lengend Snippet:

Article Snippet: Antibody , Phospho-CDK Substrate Motif(rabbit monoclonal) , CST , 9477S , WB(1:1000).

Techniques: Produced, Agarose Gel Electrophoresis, Recombinant, Sequencing, Variant Assay, Purification, Transfection, Construct, Stable Transfection, Phosphatase Assay, Plasmid Preparation, Binding Assay, Generated, Mutagenesis, Software

Journal: Aging Cell

Article Title: CDK2 phosphorylation of Werner protein (WRN) contributes to WRN’s DNA double‐strand break repair pathway choice

doi: 10.1111/acel.13484

Figure Lengend Snippet: CDK2‐CycE phosphorylates WRN. (a) FLAG/His‐tagged WRN protein was purified from baculovirus‐infected insect cells. The purified WRN protein was in vitro phosphorylated with the indicated CDK‐Cyc combinations and probed by Western blot for the antigens shown. Phosphorylated serine/threonine blot intensities were quantified by normalizing with WRN and basal phosphorylation without CDKs. Asterisks indicated directly above the columns represent p‐value calculations compared to controls (basal phosphorylation without CDKs). (b) To remove the basal phosphorylation seen in A, purified WRN proteins were treated with λPPase, then immunoprecipitated with FLAG beads and subjected to in vitro phosphorylation with CDK2‐CycA or CDK2‐Cyc2 combinations, followed by Western blot analysis. Phosphorylated serine blot intensity was quantified by normalizing with WRN. Error bars represent standard deviation. p ‐value, *, <0.05; **, <0.01; ***, <0.001

Article Snippet: Proteins were resolved in 4 to 15% Mini‐PROTEAN TGX gels (BioRad, Hercules, CA, USA), electroblotted to nitrocellulose membrane, and visualized using antibodies against anti‐WRN (in house), anti‐phospho‐CDK substrate (Cell Signaling, 9477), anti‐CDK1 (Abcam, ab133327), anti‐CDK2 (Abcam, ab32147), anti‐CycA2 (Abcam, ab38), anti‐CycE (Abcam, ab133266), anti‐DNA‐PKcs (BD, 610804), anti‐KU70 (ThermoFisher, PA5‐27538), and anti‐RPA (Calbiochem, NA18).

Techniques: Purification, Infection, In Vitro, Western Blot, Immunoprecipitation, Standard Deviation

Journal: Frontiers in Oncology

Article Title: Ceramide Regulates Anti-Tumor Mechanisms of Erianin in Androgen-Sensitive and Castration-Resistant Prostate Cancers

doi: 10.3389/fonc.2021.738078

Figure Lengend Snippet: Erianin induces cell cycle arrest at the M phase in PC3 cells. (A, B) PC3 cells were treated with erianin at indicated concentrations for 24 h. (A) Cell cycle phase was determined by flow cytometry with propidium iodide (PI) staining. G 0 /G 1 , S and G 2 /M phase populations were estimated using Flowjo software and are indicated in purple, yellow and green, respectively. (B) Percentages of cells at each phase was quantified by Flowjo software. Data are expressed as mean ± SD. n=5. * p < 0.05; *** p < 0.001, versus untreated control. (C) PC3 cells were treated with erianin at 50 nM for indicated times. Cyclin b1 and phosphorylated CDK1 substrates were examined by Western blotting. (D, E) Cells were treated with S-trityl-L-cysteine (STLC) at 10 µM for 16 h to synchronize cells at the prometaphase. Then STLC was withdrawn and cell cycle progression to metaphase, telophase and interphase were monitored in the presence ot absence of erianin at 100 nM for 1 h. Confocal images were captured. Bar = 10 µm (D) . Percentages of cells at each cell cycle phase were quantified (E) .

Article Snippet: Immunoblot analyses were conducted according to standard protocol with the following antisera: caspase-3 (#9662), PARP (#9542), β-actin (#3770), IRE1α (#3294), p-eIF2α (#3597), t-eIF2α (#2103), p-P38 MAPK (#4511), t-P38 MAPK (#8690), p-JNK1/2 (#4668), t-JNK1/2 (#9252), p-ERK1/2 (#9101), t-ERK1/2 (#9102), CHOP (#5554), Bim (#2933), Bcl-2 (#4223), Bax (#5023), p-Akt (#4060), t-Akt (#9272), GAPDH (#5174), cyclin b1 (#12231), p-CDK1 substrates (#9477) and FLAG (#14793) from Cell Signaling Technology; p62 (#ab56416) and Mcl-1 (#ab31948) antibodies were purchased from Abcam, while LC3 antibody (#NB100-2220) was obtained from Novus Biologicals.

Techniques: Flow Cytometry, Staining, Software, Western Blot