Journal: PLoS ONE

Article Title: c-Rel Deficiency Increases Caspase-4 Expression and Leads to ER Stress and Necrosis in EBV-Transformed Cells

doi: 10.1371/journal.pone.0025467

Figure Lengend Snippet: ( A ) Pt1- (dashed line) and control D11-LCL tet cells (solid line) grown in the absence of Tc for 5 days were plated at 1×10 6 cells/mL in Tc media and cultured in parallel for 6 days. Cells were withdrawn at days 1, 3 and 6 cells and counted using Trypan-Blue exclusion to determine the total number of viable cells. ( B ) 1×10 6 Pt1- and control D11- and C2-LCL tet cells were stained with CFSE and cultured in Tc media (pink line), absence of Tc (for 5 days prior to CFSE staining) (grey peak) or absence of Tc plus sCD40L (500 ng/mL) (green line) for 6 days. At day 4 (upper panel) and day 6 (lower panel) cells were analyzed for proliferation by flow cytometry. ( C ) Pt1- and control D11-LCL tet cells (2×10 5 ) grown in Tc media were analyzed for the expression of phosphorylated-CDC2 by intra-cellular staining. Numbers shown are the mean fluorescence intensity (MFI) of cells expressing high levels of phosphorylated CDC2. Graph is representative of three independent experiments.

Article Snippet: Anti-S6 mAb, rabbit polyclonal anti-caspase 4 and rabbit polyclonal anti-phospho-CDC2 were purchased from Cell Signaling.

Techniques: Cell Culture, Staining, Flow Cytometry, Expressing, Fluorescence

Journal: PLoS ONE

Article Title: Kaposi Sarcoma Herpes Virus Latency Associated Nuclear Antigen Protein Release the G2/M Cell Cycle Blocks by Modulating ATM/ATR Mediated Checkpoint Pathway

doi: 10.1371/journal.pone.0100228

Figure Lengend Snippet: (A) BJAB cells transfected with the pA3M vector or pA3M-LANA were treated with nocodazole (200 ng/mL) for 24 hours. The cells were then harvested, total cell lysate were prepared and western blot was performed using antibodies against cyclin B1, Cdc2, Tyr15-phosphorylated Cdc2 [Cdc2 (Tyr15)], and Histone H3 as a protein loading control. (B) BJAB cells transfected with the pA3M vector or pA3M-LANA were grown in 6 well culture plates and treated with nocodazole and observed for colocolaization in a confocal microscope. The result shown is the representative of 3 independent experiments. +, present; −, absent.

Article Snippet: Polyclonal rabbit anti-phospho-Tyr15 Cdc2, mouse monoclonal Cdc2, mouse monoclonal cyclin B1, anti-HA rabbit polyclonal antibody, anti-Myc mouse monoclonal antibody, rabbit polyclonal anti-β–actin, HRP conjugated mouse and rabbit polyclonal antibodies were all purchased from Santa Cruz Biotechnology (USA), IMGENEX Corporation (USA) or Cell Signaling Technology, Inc. (USA).

Techniques: Transfection, Plasmid Preparation, Western Blot, Microscopy

Journal: PLoS ONE

Article Title: Kaposi Sarcoma Herpes Virus Latency Associated Nuclear Antigen Protein Release the G2/M Cell Cycle Blocks by Modulating ATM/ATR Mediated Checkpoint Pathway

doi: 10.1371/journal.pone.0100228

Figure Lengend Snippet: Nocodazole treatment reduces the level of phosphorylated Cdc2. The viral nuclear antigen LANA binds directly to Chk2, which may result in the phosphorylation of Cdc25c and sequester it in the cytoplasm. Thus, it may be unable to regulate the phosphorylation of nuclear Cdc2 resulting the activation of cyclin B-Cdc2 and progression through the G2/M phase, releasing the nocodozole induced block.

Article Snippet: Polyclonal rabbit anti-phospho-Tyr15 Cdc2, mouse monoclonal Cdc2, mouse monoclonal cyclin B1, anti-HA rabbit polyclonal antibody, anti-Myc mouse monoclonal antibody, rabbit polyclonal anti-β–actin, HRP conjugated mouse and rabbit polyclonal antibodies were all purchased from Santa Cruz Biotechnology (USA), IMGENEX Corporation (USA) or Cell Signaling Technology, Inc. (USA).

Techniques: Activation Assay, Blocking Assay

Journal: iScience

Article Title: Blockage of lamin-A/C loss diminishes the pro-inflammatory macrophage response

doi: 10.1016/j.isci.2022.105528

Figure Lengend Snippet: Pro-inflammatory macrophage activation induces Lamin-A/C phosphorylation and degradation (A) Immunoblot shows total levels of Lamin-A and Lamin-C in Untreated (UT), and LPS-treated BMDMs. α-tubulin served as a loading control. (B) Time course quantifications of Lamin-A and Lamin-C normalized with α-tubulin over three independent immunoblotting experiments. Calculated values were further normalized to the UT BMDM condition to find the fold change. Data are presented as Mean ± S.E. (C) Longer exposure of immunoblots against Lamin-A/C in UT, and LPS-treated BMDMs to detect lower fragmented Lamin-A/C bands (50kDa and 40kDa). α-tubulin served as a loading control. (D) Box plots show degraded Lamin-A/C fragments (50kDa and 40kDa) normalized to α-tubulin between UT and LPS-treated BMDMs. Calculated values were further normalized to the UT BMDM condition to find the fold change. (E) Immunoblots against Lamin-A/C in UT, LPS-treated, LPS Wash-out BMDMs to detect fragmented Lamin-A/C bands (∼50kDa and ∼40kDa). α-tubulin served as a loading control. (F) Immunoblot shows total levels of p-(Ser22)-Lamin-A and p-(Ser22)-Lamin-C along with a phosphorylated fragmented band of Lamin-A/C (∼28kDa) in UT, and LPS-treated BMDMs. α-tubulin served as a loading control. (G) Time course quantification of total p-(Ser22)-Lamin-A+C normalized to the total Lamin-A/C over three independent immunoblotting experiments. Calculated values were further normalized to the UT BMDM condition to find the fold change. (H) (Top) Schematic shows the direct or indirect CDK1-mediated Lamin-A/C phosphorylation, which is known from other cells to be blocked by PP2A during the cell cycle. Also shown are the drugs used in this study to selectively inhibit the activity of the respective enzymes. (Bottom) Schematic shows Caspase-6 mediated Lamin-A/C degradation as inhibited by Z-VEID-FMK. (I) Box plots show the lowering of pro-inflammatory gene expression (qPCR) (Left) and secreted cytokine levels (ELISA) (Right) in LPS-treated BMDMs, also treated with RO-3306 or Z-VEID-FMK to inhibit Lamin-A/C phosphorylation and degradation, respectively. Levels were normalized to the 6 h LPS-treated BMDM condition to find the fold change. (J) Box plots show an increase in the pro-inflammatory gene expression (qPCR) (Left) and secreted cytokine levels (ELISA) (Right) in LPS-treated BMDMs, also treated with LB-100, a PP2A inhibitor, as compared to BMDMs treated with only LPS for 24 h. Levels were normalized to the 24 h LPS-treated BMDM condition to find the fold change. For all the plots p-values were obtained with the two-sided Student’s t -test. In all the box plots, the boxes show 25th and 75th percentiles, the middle horizontal lines show the median, small open squares show the mean, and whiskers indicate S.D. All the experiments were independently repeated three or more times.

Article Snippet: Rabbit polyclonal anti-phospho-CDK1 (Thr161) antibody , Abcam , Cat#ab194874.

Techniques: Activation Assay, Western Blot, Activity Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Blockage of lamin-A/C loss diminishes the pro-inflammatory macrophage response

doi: 10.1016/j.isci.2022.105528

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-phospho-CDK1 (Thr161) antibody , Abcam , Cat#ab194874.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, In Vitro, Derivative Assay, Software

Journal: bioRxiv

Article Title: A truncated form of the p27 CDK inhibitor translated from pre-mRNA causes cell cycle arrest at G2 phase

doi: 10.1101/2022.01.12.476115

Figure Lengend Snippet: (A-D) Two hours after release from a double thymidine block (DTB), synchronized HeLa S3 cells were treated with MeOH or the indicated concentrations of Pla-B. Black triangles indicate the time points of cell harvest and sample preparation (A). Cell cycle was analyzed at the indicated time points by cytometry (B). Morphology of the cells was observed under a microscope and round cells were counted at the indicated time points (C). Protein samples were prepared at the indicated time points. The protein levels of indicated proteins and phosphorylation status of Cdk1 were analysed by immunoblotting. Protein levels of α-tubulin were analysed as an internal control (D). Error bars indicate s.d. (n = 3).

Article Snippet: Mouse monoclonal anti-Cyclin A2 (#4656), rabbit polyclonal anti-Cyclin B1 (#4138), mouse monoclonal anti-cdc2 (#9116), rabbit polyclonal anti-phospho-cdc2 (Tyr15) (#9111), mouse monoclonal anti-Cyclin E1 (#4129), rabbit monoclonal anti-Plk2 (#14812), rabbit monoclonal anti-Wee1 (#13084) and rabbit monoclonal anti-p27 (#3686) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Blocking Assay, Sample Prep, Cytometry, Microscopy, Western Blot

Journal: bioRxiv

Article Title: A truncated form of the p27 CDK inhibitor translated from pre-mRNA causes cell cycle arrest at G2 phase

doi: 10.1101/2022.01.12.476115

Figure Lengend Snippet: (A) Cells expressing Flag-p27* under the control of tetracycline were synchronized by a double thymidine block. The cells were treated with 1 μg/ml DOX at the same time as release from the double thymidine block. The cells were harvested at 8 h after release from the double thymidine block (G2/M phase) and then immunoprecipitation was performed using anti-DDDDK (Flag) antibodies. Flag-tagged and coimmunoprecipitated proteins were analysed by immunoblotting. (B, C) Purified Flag-tagged proteins were applied to an in vitro kinase assay reaction and kinase activities of Cyclin A2/Cdk1 (B) and Cyclin B1/Cdk1 (C) were measured. Statistical significance was assessed by the one-way ANOVA and Dunnett’s test (*: P < 0.05; **: P < 0.01; ***: P < 0.01). Error bars indicate s.d. (n = 3).

Article Snippet: Mouse monoclonal anti-Cyclin A2 (#4656), rabbit polyclonal anti-Cyclin B1 (#4138), mouse monoclonal anti-cdc2 (#9116), rabbit polyclonal anti-phospho-cdc2 (Tyr15) (#9111), mouse monoclonal anti-Cyclin E1 (#4129), rabbit monoclonal anti-Plk2 (#14812), rabbit monoclonal anti-Wee1 (#13084) and rabbit monoclonal anti-p27 (#3686) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Blocking Assay, Immunoprecipitation, Western Blot, Purification, In Vitro, Kinase Assay

Journal: Cell Reports

Article Title: Crosstalk between Drp1 phosphorylation sites during mitochondrial remodeling and their impact on metabolic adaptation

doi: 10.1016/j.celrep.2021.109565

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-phospho-Cdk1 (Tyr15) (1:500) , Millipore , Cat# 219440; RRID: AB_564425.

Techniques: Recombinant, Plasmid Preparation, Knock-In, Clone Assay, Software