Journal: Journal of Advanced Research

Article Title: Multi-omics approach reveals TGF-β signaling-driven senescence in periodontium stem cells

doi: 10.1016/j.jare.2024.12.037

Figure Lengend Snippet: TGF-β1-induced ROS accumulation leads to G2 arrest through ATM signaling. (A). RT-qPCR analysis of ATM signaling in TGF-β1 and/or its inhibitor-treated PDLSCs; (B). Western blot analysis of ATM signaling in TGF-β1 and/or its inhibitor-treated PDLSCs; (C). Western blot analysis of ATM signaling phosphorylated activation in the condition of ATM or CHK2 knockdown; (D). Western blot analysis of ATM signaling phosphorylated activation under N-acetyl-l-cysteine (NAC) treatment; (E). Western blot analysis of ATM signaling phosphorylated activation under decitabine treatment. Reagent doses and duration: TGF-β1 (10 ng/ml, 48 h), NAC (4 mM, 48 h), decitabine (10uM, 48 h). Predicted molecular weight: ATM 350 kDa, pATM 370 kDa, CDC25C&pCDC25C 60 kDa, CHK1&pCHK1 54 kDa, CHK2&pCHK2 62 kDa, β-Tubulin 55 kDa; GAPDH 37 kDa. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, ns denotes not significant by one-way analysis of variance followed by the Tukey’s test. Error bars represent means ± SEM of at least three independent experiments.

Article Snippet: Antibodies used in this study were listed as follows: antibodies from Abmart: anti-β-Tubulin ( M20005 ); antibodies from Huabio: anti-GAPDH recombinant rabbit monoclonal antibody (ET1601-4), anti-DNMT1 recombinant rabbit monoclonal antibody (ET1702-77), anti-DNMT3A recombinant rabbit monoclonal antibody (ET1609-31), anti-DNMT3B recombinant rabbit monoclonal antibody (ET1605-9), anti-p16 INK4A recombinant rabbit monoclonal antibody (ET1608-62), anti-AMPKγ1 recombinant mouse monoclonal antibody (EM2001-06), anti-AMPK alpha 1 recombinant rabbit monoclonal antibody (ET1608-40), anti-phospho-AMPK alpha 1 (S496) recombinant rabbit monoclonal antibody (ET1612-72), HRP-conjugated goat anti-rabbit IgG goat polyclonal antibody (HA1001), HRP-conjugated goat anti-mouse IgG polyclonal antibody (HA1006); antibodies from Abcam: anti-ATM antibody (ab32420), anti-phospho-ATM (S1981) antibody (ab81292), anti-CHK1 antibody (ab40866), anti-phospho-CHK1 (S296) antibody (ab79758), anti-CDC25C antibody (ab32444), anti-p21 antibody (ab109199), anti-CHK2 antibody (ab109413), anti-histone H3 (tri-methyl K9) (ab176916), anti-phospho-γ-H2AX (S139) antibody (ab81299), anti-HMGB1 antibody (ab79823); antibodies from CST: anti-Ki67 (D3B5) rabbit mAb (#9129), anti-phospho-CHK2 (Thr68) rabbit mAb (#2197), anti-phospho-CDC25C (Ser216) rabbit mAb (#4901); antibodies from Bioss: goat anti-rabbit IgG antibody (H + L), FITC-conjugated (bs-0295G-FITC); goat anti-rat IgG antibody (H + L), cyanine 3-conjugated (bs-0293G-Cy3); antibodies from Biolegend: APC anti-human CD34 antibody (#343509), FITC anti-human CD146 antibody (#361011).

Techniques: Quantitative RT-PCR, Western Blot, Activation Assay, Knockdown, Molecular Weight

Journal: Leukemia & lymphoma

Article Title: Synergistic cytotoxicity of busulfan, melphalan, gemcitabine, panobinostat, and bortezomib in lymphoma cells

doi: 10.3109/10428194.2016.1157871

Figure Lengend Snippet: List of primary antibodies, their sources, dilutions and molecular weights.

Article Snippet: The primary antibodies, their sources, dilutions, and antigen molecular weights are listed in (Supplementary Materials). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibodies Source (Catalogue #) Clone type Dilutions Molecular weight (kDa) AcH3K9 Active Motif/39137 pAb 3500 17 AKT Cell Signaling/4691 pAb 2000 60 ATM Santa Cruz/23921 mAb 800 250 BCL-2 DAKO/124 mAb 1500 26 c-MYC Cell Signaling/9402 pAb 2500 57–70 Cleaved Caspase 3 Cell Signaling/9661 pAb 2000 17,19 Caspase 8 Cell Signaling/9746 mAb 2000 43 Caspase 9 Cell Signaling/9502 pAb 2000 35–47 CHK2 Cell Signaling/2662 pAb 1000 62 HDAC4 Cell Signaling/5392 mAb 3000 140 KAP1 Bethyl Lab A300-275A pAb 2500 100–117 MCL-1 Santa Cruz/819 pAb 700 40 mTOR Cell Signaling/2983 pAb 2500 289 NOXA Calbiochem/0180 mAb 1500 15 p-ATM (Ser1981) Rockland/200–301-400 mAb 2000 250 p-AKT (Ser 473) Cell Signaling/4060 pAb 2500 60 p-CHK2 (Thr68) Cell Signaling/2661 pAb 2000 62 p-KAP1 (Ser824) Cell Signaling/4127 pAb 2500 100 p-mTOR (Ser2448) Cell Signaling/5536 pAb 1500 289 p-P53 (Ser 15) Cell Signaling/9284 pAb 2000 53 p-PI3K (Tyr199/Tyr458) Cell Signaling /4228 pAb 2500 60, 85 p-SMC1 (Ser957) Novus Biologicals/100–205 pAb 1500 140 P53 Santa Cruz/126 pAb 2000 53 PARP1 Santa Cruz /8007 mAb 1000 116 PI3K Santa Cruz/423 pAb 500 85 SMC1 Cell Signaling/4802 pAb 2500 140 XIAP Cell Signaling/2045 pAb 2000 53 α-Tubulin Cell Signaling/2144 pAb 1000 57 β-ACTIN Sigma/A5316 mAb 10000 42 γ-H2AX Millipore/2554898 mAb 3000 15 3MeH3K27 Active Motif/39155 pAb 3500 17 Open in a separate window List of primary antibodies, their sources, dilutions and molecular weights.

Techniques: Molecular Weight

Journal: Cancer Research

Article Title: γ-Irradiation–Induced DNA Damage Checkpoint Activation Involves Feedback Regulation between Extracellular Signal-Regulated Kinase 1/2 and BRCA1

doi: 10.1158/0008-5472.can-07-5818

Figure Lengend Snippet: Figure 5. The effect of BRCA1 expression on IR-induced ATM and ATR signalings. A, top, MCF-7 cells expressing Luc-shRNA (Control) or BRCA1-shRNA (B2-6) were treated with 10-Gy IR, incubated for 15 min, and assessed for ATM-Ser1981 phosphorylation and total ATM by immunoblotting. Bottom, cells were treated as described above, incubated for the indicated times, and then analyzed for phosphorylation of Chk2-Thr68 and p53-Ser20 by immunoblotting. ATM, Chk2, p53, and actin levels in the lysate samples were determined by immunoblotting. B, top, Cells were exposed to 10-Gy IR and incubated for the indicated times. ATR kinase was immunoprecipitated from 500-Ag cell lysate and analyzed for ATR activity using recombinant p53 protein as substrate (ATR activity, lanes 1–8), as described in Materials and Methods. As a negative control, ATR activity in irradiated cells was assayed using GST recombinant protein as substrate (ATR activity, lane 9). ATR levels in all immunoprecipitates were assessed by immunoblotting using anti-ATR antibody N-19 (ATR IP-WB). Bottom, control (Control) and BRCA1-shRNA–expressing cells (B2-6 and B2-4) were exposed to 10-Gy IR, incubated for the indicated times, and analyzed for ATR protein levels by immunoblotting (ATR). C, cells treated with or without 10-Gy IR were incubated for 1 h and then analyzed for levels of Chk1-Ser317 phosphorylation and total Chk1.

Article Snippet: These include mouse IgG for phosphorylated ATM (Ser1981; Rockland Immunochemical for Research), BRCA1 (D-9), BRCA1 (Ab-1; EMD Biosciences), phosphorylated ERK1/2 (pERK1/2; E-4), Chk1 (G-4), Chk2 (B-4), and p53 (DO-1); rabbit IgG for ATM (Ab-3; EMD Biosciences), BRCA1 (I-20), phosphorylated Chk1 (Ser317; Cell Signaling Technology), phosphorylated Chk2 (Thr68; Cell Signaling Technology), ERK1/2 (C-14-R), pERK1/2 (20G11; Cell Signaling Technology), phosphorylated p53 (Ser20; Cell Signaling Technology), and ubiquitin (FL-76); and goat IgG for ERK1/2 (C-14-G), ATR (N-19), and actin (I-19).

Techniques: Expressing, shRNA, Control, Incubation, Phospho-proteomics, Western Blot, Immunoprecipitation, Activity Assay, Recombinant, Negative Control, Irradiation

Journal: Cancer Research

Article Title: γ-Irradiation–Induced DNA Damage Checkpoint Activation Involves Feedback Regulation between Extracellular Signal-Regulated Kinase 1/2 and BRCA1

doi: 10.1158/0008-5472.can-07-5818

Figure Lengend Snippet: Figure 6. A model of DNA damage checkpoint that involves both BRCA1 and ERK1/2. The current understanding of protein signaling pathways involved in DNA damage induced G2-M checkpoint control are indicated by solid lines. Dashed lines indicate a feedback regulatory loop between ERK1/2 and BRCA1 in the cellular response to IR exposure, as identified by the studies presented in this report. Results in this report also suggest that both BRCA1 and ERK1/2 play important roles in the activation of ATR after IR exposure. In addition, studies in this report indicate an essential role for BRCA1 in IR-induced Chk2 activation.

Article Snippet: These include mouse IgG for phosphorylated ATM (Ser1981; Rockland Immunochemical for Research), BRCA1 (D-9), BRCA1 (Ab-1; EMD Biosciences), phosphorylated ERK1/2 (pERK1/2; E-4), Chk1 (G-4), Chk2 (B-4), and p53 (DO-1); rabbit IgG for ATM (Ab-3; EMD Biosciences), BRCA1 (I-20), phosphorylated Chk1 (Ser317; Cell Signaling Technology), phosphorylated Chk2 (Thr68; Cell Signaling Technology), ERK1/2 (C-14-R), pERK1/2 (20G11; Cell Signaling Technology), phosphorylated p53 (Ser20; Cell Signaling Technology), and ubiquitin (FL-76); and goat IgG for ERK1/2 (C-14-G), ATR (N-19), and actin (I-19).

Techniques: Protein-Protein interactions, Control, Activation Assay