α total chk2  (Cell Signaling Technology Inc)

Journal: iScience

Article Title: Lipopolysaccharide induces placental mitochondrial dysfunction in murine and human systems by reducing MNRR1 levels via a TLR4-independent pathway

doi: 10.1016/j.isci.2022.105342

Figure Lengend Snippet: ROS generated by NOX2 activates ATM kinase in LPS-treated placental cells (A–C) Left , Equal amounts of HTR cells treated with control (water) or LPS were separated on an SDS-PAGE gel and probed for NOX2. Tubulin was probed as a loading control. Right , NOX2 levels relative to tubulin are shown (n = 4 biological replicates). (B) Left , Equal amounts of HTR cells were treated for 24 h with control (water) or LPS and, for second blot, 25 μM NOX2 inhibitor (using DMSO in control); lysates were separated on an SDS-PAGE gel and probed for MNRR1. Actin was probed as a loading control. Right , Relative MNRR1 levels are shown for each lane (n = 4 biological replicates). (C) HTR cells were treated with control (water) or LPS for the times shown, and ROS levels were measured as in Figure 1 C. Total ROS, black; mitochondrial ROS, red; total ROS with ATM inhibitor, gray (n = 2 biological replicates). (D) Equal amounts of HTR cells were treated with control (water) or H 2 O 2 for 16 h and lysates separated on an SDS-PAGE gel and probed for phospho-CHK2, total CHK2, and ATM kinase. Actin was probed as a loading control. (E) Left, Equal amounts of HTR cells treated with control (water) or LPS with either Vehicle (DMSO) or 100 μM N-acetyl cysteine for 24 h were separated on an SDS-PAGE gel and probed for YME1L1. Actin was probed as a loading control. Right , Relative YME1L1 levels are shown for each condition (n = 4 biological replicates). (F) TNFα and PTGS2 transcript levels relative to Actin were measured in HTR cells treated with Control (water), LPS, or LPS +20 μM MitoTempo (n = 4 biological replicates). (G) TNFα and PTGS2 relative transcript levels in HTR cells treated with Control (DMSO), LPS (LPS + DMSO) or LPS +25 μM NOX2 inhibitor (n = 4 biological replicates). (H) Equal amounts of YME1L1 −/− cells overexpressing WT or various mutants of YME1L1 were treated as control (water) or with LPS (1 μg/mL). Lysates were separated on an SDS-PAGE gel and probed for MNRR1, STARD7, and YME1L1. Actin was probed as a loading control.

Article Snippet: α-total CHK2 , Cell Signaling Technology , Cat # 6334; RRID: AB_11178526.

Techniques: Generated, Control, SDS Page

Journal: iScience

Article Title: Lipopolysaccharide induces placental mitochondrial dysfunction in murine and human systems by reducing MNRR1 levels via a TLR4-independent pathway

doi: 10.1016/j.isci.2022.105342

Figure Lengend Snippet:

Article Snippet: α-total CHK2 , Cell Signaling Technology , Cat # 6334; RRID: AB_11178526.

Techniques: Immunostaining, Recombinant, Luciferase, Reporter Assay, Plasmid Preparation, Purification, Fractionation, cDNA Synthesis, Mutagenesis, Isolation, Transfection, Generated, Software

Journal: Molecular cancer

Article Title: MutS homologue hMSH4: interaction with eIF3f and a role in NHEJ-mediated DSB repair.

doi: 10.1186/1476-4598-12-51

Figure Lengend Snippet: Figure 7 Immunoblotting analysis of IR-induced AKT (Ser473) activation. (A) The levels of AKT activation were measured by AKT Ser473 phosphorylation in 293T, 293T/eIF3f and 293T/eIF3f-hMSH4 cells treated with 10 Gy IR in comparison to untreated controls. Levels of p53 Ser15 phosphorylation and the total protein levels of AKT, hMRE11 and p53 were also analyzed. α-tubulin was used as a loading control. (B) The levels of Chk2 activation (Chk2 Thr68 phosphorylation) in 293T, 293T/eIF3f and 293T/eIF3f-hMSH4 cells in response to 10 Gy IR. Cell lysates were prepared at 1 hr post IR treatment. Untreated cells were analyzed as controls, while α-tubulin was used as a loading control.

Article Snippet: Antibodies used in this study include: α-Flag M2 (Sigma), α-α-tubulin (Sigma), α-GST (GE Healthcare Life Sciences, Piscataway, NJ), α-eIF3f (Rockland Immunochemical Inc.), α-Myc (Clontech), and α-hMSH4 [20], α-p-AKT (Ser473) (Cell Signaling Technology, Danvers, MA), α-pS15-p53 (Cell Signaling Technology), α-total p53 (Cell Signaling Technology), α-phospho-Chk2 Thr68 (Cell Signaling Technology). α-hMRE11 (Novus Biologicals, Littleton, CO), α-HDAC3 (Abcam), α-prefolding 3 (VBP1) (K-13, Santa Cruz), α-Rad51 (ab-1) (Novus).

Techniques: Western Blot, Activation Assay, Phospho-proteomics, Comparison, Control