Journal: Cell Death and Differentiation

Article Title: O-GlcNAcylation of UGDH regulates its activity and remodels the extracellular matrix to facilitate tumor growth

doi: 10.1038/s41418-025-01591-8

Figure Lengend Snippet: A Immunoblotting analysis of endogenous OGT depletion using shRNAs in A549 and NCI-H1299 cells. B ELISA analysis of HA concentrations in genetically engineered cells. C Immunofluorescence staining of HA accumulation from A549 cells upon UGDH depletion. HABP (HA stain in green) and DAPI (nuclear stain in blue). Scale bar, 10 μm. D Competitive ELISA analysis of the absolute concentration of HA in A549 cells with or without UGDH depletion. E Pathological staining and quantitative statistical analysis of the ECM contents. Masson’s trichrome staining for collagen or IHC staining for HA and α-SMA. The statistical data are shown as mean ± SEM ( n = 12–16). Statistical analyses were performed by an unpaired two-tailed Student’s t -test. F IHC analysis was performed with anti-UGDH, anti-HA, anti-collagen, anti-α-SMA, and anti-CD8 antibodies on specimens from patients with lung cancer. Representative images of IHC staining graphs from two patients are shown. Scale bar, 200 μm. G A higher level of UGDH O-GlcNAcylation, α-SMA, collagen, HA, and a lesser infiltration of CD8 + T cells in tumor tissues were observed compared to adjacent peritumoral tissues in a cohort of 18 patient samples. H The deposition of HA, α-SMA, and collagen increased with the level of UGDH O-GlcNAcylation, but not for CD8 + T cells. I The correlation between CD8 + T cell infiltration and the expression of HA. J The effect of CD8α mAb treatment on tumor growth. K Representative IHC images and quantification of CD8 + T cells in the tumors after CD8α mAb treatment. Scale bar, 100 μm. L 4-MU treatment diminished HA accumulation and abolished the effect of UGDH O-GlcNAcylation on tumor development.

Article Snippet: The mouse monoclonal antibody against Flag (catalog number F1804, 1:5,000 dilution for immunoblotting), alpha-smooth muscle actin (catalog number A5228, 1:200 dilution for IHC), and biotinylated hyaluronic acid-binding protein (catalog number 385911, 1:1000 dilution for IHC or ELISA) were all acquired from Merck.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Competitive ELISA, Concentration Assay, Immunohistochemistry, Two Tailed Test, Expressing

Journal: Oncology Letters

Article Title: Downregulation of FHL1 protein in glioma inhibits tumor growth through PI3K/AKT signaling

doi: 10.3892/ol.2020.11476

Figure Lengend Snippet: FHL1 affects glioma cell growth through PI3K/Akt signaling. (A) Western blot analysis for the phosphorylation levels of Akt and total AKT expression in the two types of stable cell lines and corresponding control cell lines, with β-actin as an internal control. (B) FHL1 interacts with AKT in U87 cells. U87 cells were transfected with 0.5 µg FLAG-FHL1 expression vector or 0.5 µg FLAG empty vector. The IP analysis was performed with anti-FLAG antibody, and the WB was performed with anti-AKT antibody and anti-p110α antibody. FHL1, four-and-a-half LIM domains 1; p, phosphorylated; siRNA, small interfering RNA; IP, immunoprecipitation; WB, western blot analysis.

Article Snippet: Co-IP was performed as previously described , with an anti-FLAG monoclonal antibody (Merck KGaA; cat. no. F1804; 1:1,000.).

Techniques: Western Blot, Phospho-proteomics, Expressing, Stable Transfection, Control, Transfection, Plasmid Preparation, Small Interfering RNA, Immunoprecipitation

Journal: EMBO Reports

Article Title: CDK9‐mediated phosphorylation controls the interaction of TIP60 with the transcriptional machinery

doi: 10.15252/embr.201744311

Figure Lengend Snippet: Antibodies

Article Snippet: Supplier FLAG M2 1:1,000 F1804 Sigma‐Aldrich P‐ERK 1:2,000 #9101 Cell Signaling Technology P‐AKT 1:1,000 #4058 Cell Signaling Technology PARP 1:1,000 #9542 Cell Signaling Technology TIP60 C7 1:1,000 sc‐166323 Santa Cruz Biotechnology TIP60 1:2,000 ab151432 Abcam gH2AX 1:100,000 ab81299 Abcam CDK9 (C12F7) 1:1,000 #2316 Cell Signaling Technology GSK3a/b (1H8) 1:1,000 sc‐56913 Santa Cruz Biotechnology Ku80 1:1,000 #2753 Cell Signaling Technology mouse p21 (M‐19) 1:200 sc‐471 Santa Cruz Biotechnology Human p21 1:1,000 556430 BD Pharmingen BRD4 (E2A7X) 1:1,000 #13440 Cell Signaling Technology RNA Pol II (F‐12) 1:200 sc‐55492 Santa Cruz Biotechnology RNA Pol II (N‐20) 1:500 sc‐899 Santa Cruz Biotechnology pS2 RNA Pol II (3E10) 1:2,000 # 04‐1571‐l Upstate/Millipore pS2/S5 RNA Pol II 1:1,000 #4735 Cell Signaling Technology EPC1 1:1,000 ab5514 Abcam H3 (1B1B2) 1:1,000 #14269 Cell Signaling Technology H4 (L64C1) 1:1,000 #2935 Cell Signaling Technology Acetyl‐H4 Lsy16 (E2B8W) 1:1,000 #13534 Cell Signaling Technology Acetylated lysine (Ac‐K‐103) 1:1,000 #9681 Cell Signaling Technology RIPK1 1:1,000 D94C12, #3493 Cell Signaling Technology Lamin B1 (H‐90) 1:500 sc‐20682 Santa Cruz Biotechnology MYC (9E10) 1:500 sc‐40 Santa Cruz Biotechnology Open in a separate window Antibodies Antibody Dilution Epitope Supplier pS90 (‐12) 1:1,000 RPG(pS)PERE ProSci pS90 (‐72) 1:1,000 GSRPG(pS)PEREV ProSci pS86 (‐69) 1:1,000 G(pS)RPGSPEREV ProSci Open in a separate window Custom‐made antibodies In vitro kinase assay FLAG‐TIP60 WT , FLAG‐TIP60 S90A or empty pcDNA3.1 were transiently expressed in HEK293T cells.

Techniques: