rabbit monoclonal anti histone h2ax total  (Cell Signaling Technology Inc)

Journal: Biology Open

Article Title: PIWIL2 downregulation in colon cancer promotes transposon activity and pro-tumorigenic phenotypes

doi: 10.1242/bio.061942

Figure Lengend Snippet: PIWIL2 depletion promotes DNA damage via LINE-1 transposon activation. (A) γH2AX increases in PIWIL2 KO cells compared to Caco2 WT cells by immunofluorescence. Representative confocal images shown. (B) Quantification of γH2AX foci per cell [ n =nine fields (three biological replicates, three fields per replicate), mean±s.e., t -test * P =0.0151]. (C) Representative western blot of total H2AX, phosphorylated γH2AX, and β-actin (actin) loading control in Caco2 and PIWIL2 KO cells. (D) LINE-1 GFP reporter assay staining for eGFP and γH2AX co-localization as indicated by white arrows. (E) Quantification of the average % positive GFP and γH2AX expressing cells normalized to transfection efficiency for each cell line. ( n =3 biological replicates with three fields taken per condition and averaged, mean±s.e., two-way ANOVA with Bonferroni correction for multiple comparisons, Caco2 WT LINE-1 inactive (-) versus PIWIL2 KO LINE-1 GFP, * P =0.0351.

Article Snippet: H2AX total , Santa Cruz Biotechnology , sc517336 , Ms , 1 to 1000 , -.

Techniques: Activation Assay, Immunofluorescence, Western Blot, Control, Reporter Assay, Staining, Expressing, Transfection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Fructose-2,6-bisphosphate restores DNA repair activity of PNKP and ameliorates neurodegenerative symptoms in Huntington's disease.

doi: 10.1073/pnas.2406308121

Figure Lengend Snippet: Fig. 4. F2,6BP mediated in cell rescue of the PNKP activity and TC-NHEJ repair in HD mice striatum-derived neuronal cells (Q-111). (A) Upper panel: Representative gel image of 3′-phosphatase activity of PNKP in the nuclear extract of Q-7 (lanes 2 and 3) and Q-111 (lanes 4 to 9) mice striatum-derived neuronal cells with mock treatment (Mock 1; lanes 2 and 4), treatment with K16ApoE carrier peptide alone (25 µM; Mock 2; lanes 3 and 5) or supplemented with F2,6BP (200 µM; 48 and 72 h) (lanes 6 and 7), or F1,6BP (lanes 8 and 9, 200 µM) in presence of carrier peptide. Lane 1: substrate only. Lane 10: purified PNKP (2 ng). Lower panel: Quantitation of the % released phosphate in the indicated lanes. Error bars show ±SD of the mean; n = 3, **P < 0.01; ***P < 0.005 between groups as indicated or compared to lanes 2 and 3. (B) Upper panel: The western blot shows the levels of PFKFB3 in the cytosolic (CE) and nuclear extracts (NE) of Q-7 and Q-111 cells. GAPDH: cytosolic loading control; HDAC2: used as nuclear loading control. Lower panel: Quantitation of the relative PFKFB3 levels after normalization with respective loading controls; n = 3, ***P < 0.005. (C) Upper panel: Amplification of each long amplicon (6 to 8 kb) and a small amplicon (~200 to 300 bp) of the transcribed (Tubb, Enolase, Neurod) genes to assess DNA strand break accumulation. Lower panel: The bar diagram represents the normalized (with short amplicon) relative band intensity with the mock-treated Q-7 sample arbitrarily set as 100. (D) Upper panel: Amplification of each long amplicon (6 to 8 kb) and a small amplicon (~200 to 300 bp) of the nontranscribed (Myh4, Myh6, and Myod) genes to assess DNA strand break accumulation. Lower panel: The bar diagram represents the normalized (with short amplicon) relative band intensity with the mock-treated Q-7 sample arbitrarily set as 100. For both C and D, Error bars show ±SD of the mean; n = 3, ***P < 0.005; ns P > 0.05 compared to lanes 1 and 2. (E) Crystal violet (CV)-stained Q-7 (Upper panel) and Q-111 (Lower panel) cells following mock treatment with carrier peptide (Left panels) and supplemented with F2,6BP (200 µM) in the presence of carrier peptide (Right panels) at 48 h post F2,6BP delivery. (F) Similar Crystal violet staining of Q-7 and Q-111 cells at 72 h post F2,6BP delivery. (G) The bar diagram shows the quantification of cell viability represented as CV-stained cell number/sq. mm area selected from five representative microscopic images of different fields captured independently. Error bars show ±SD of the mean; n = 3, ***P < 0.005 between indicated groups. (H) Left panel: The western blots show the levels of various proteins (indicated on the Right) in the nuclear extracts of Q-7 and Q-111 cells under indicated treatment conditions. HDAC2: used as nuclear loading control. Right panel: Quantitation of the relative p53BP1 and γH2AX levels after normalization with nuclear loading control HDAC2; n = 3, ***P < 0.005 between indicated groups.

Article Snippet: The membranes were blocked with 5% w/v skimmed milk in TBST buffer (1X TrisBuffered Saline, 0.1% Tween 20), then immunoblotted with appropriate antibodies [PNKP, PFKFB3, γ- H2AX (S139 residue; #9718S, Cell Signaling Technology), p53BP1 (S1778, #2675S Cell Signaling Technology), 53BP1, HDAC2, H2AX (total) (#2595S; Cell Signaling technology)].

Techniques: Activity Assay, Derivative Assay, Purification, Quantitation Assay, Western Blot, Control, Amplification, Staining

Journal: bioRxiv

Article Title: The Hippo kinases control inflammatory Hippo signaling and restrict bacterial infection in eukaryotic phagocytes

doi: 10.1101/2023.12.08.570858

Figure Lengend Snippet: A, B and E . RNA and protein expression in WT and Mst1/2 −/− iBMDMs. Top: Average RNA transcript reads for Ctsk, Bcl2 and Maf from the RNAseq data of Mst1/2 −/− N5 clone and WT iBMDMs. RNA average reads from five independent repeats. Student’s t-test, two tailed, unpaired, **: p < 0.01; ***: p <0.001. Bottom: Protein levels of CTSK, BCL2 and MAF in cell lysate of WT iBMDMs and two Mst1/2 −/− iBMDM clones were detected by immunoblotting. C and F . Levels of the indicated proteins in WT, Mst1/2 −/− , MST1-expressing ( Mst1+ ), and MST2-expressing ( Mst2+ ) iBMDMs were determined by immunoblots. D and G : WT or Mst1/2 −/− iBMDMs were treated with or without 10 µM Truli for 24 hours. Cells were then lysed, and protein samples were collected and analyzed by immunoblots. Vinculin, GAPDH, and Total histone H2AX were used as internal loading controls in immunoblots. All blots are representative of three independent repeats. H . Schematic model of the regulatory activities of MST1 and MST2 through inflammatory Hippo signaling to control expression of Ctsk , Bcl2 and Maf in macrophages.

Article Snippet: MST1 (14946S), cleaved caspase-3 (9661S), PARP1 (9542S), phospho-S139 H2AX (2577S), total H2AX (2595S), MOB1 (13730S), phospho-T35 MOB1 (8699S), and LATS1 (3477S) from Cell Signaling.

Techniques: Expressing, Two Tailed Test, Clone Assay, Western Blot