Journal: Indian Journal of Pharmaceutical Sciences

Article Title: Protective Effects of Ginsenoside Rb1 in Rats with Diabetic Cardiomyopathy

doi: 10.36468/pharmaceutical-sciences.967

Figure Lengend Snippet: Fig. 4: Ginsenoside Rb1 attenuated inflammation and suppressed the HB-EGF pathway in DCM rats, (A): RNA expressions of IL6 as determined by quantitative real-time RT-PCR; (B): Protein expressions of HB-EGF in serum as determined by ELISA and (C): Protein expressions of HB-EGF in heart tissue as determined by Western blot Note: Compared to NC, ***p<0.001 and compared to DCM, ###p<0.001

Article Snippet: Human Heparin-Binging Epidermal Growth Factor (HB-EGF) Enzyme-Linked Immunosorbent Assay (ELISA): A Human HB-EGF ELISA kit (Elabscience, China) was used to test for the activity of HB-EGF in plasma, according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: International Journal of General Medicine

Article Title: Characterization of Gastric Mucinous Carcinoma: Clinicopathological Insights and Vascular Features via MSCT

doi: 10.2147/IJGM.S567353

Figure Lengend Snippet: Immunohistochemistry (IHC) detection of vascular endothelial growth factor A (VEGF-A), hypoxia-inducible factor-1α (HIF-1α), and epidermal growth factor (EGF) in carcinomas and corresponding paracancerous normal tissues of MGC, LEMPC, and NMGC. ( A – C ) Representative immunohistochemical staining of VEGF-A ( A ), HIF-1α ( B ), EGF( C ) in carcinomas and normal tissues (×400, bar = 50μm). ( D – F ) IHC score differences (cancer tissue score - normal tissue score) for VEGF-A ( D ) HIF-1α ( E ) EGF ( F ) among the three groups. Statistical significance is indicated as follows: P < 0.05; P < 0.001.

Article Snippet: Sections were stained for CD34 (1:2500, Abcam, ab81289), VEGF-A (1:800, Proteintech, 66828-1-IG), HIF-1α (1:200, Proteintech, 20960-1-AP), and EGF (1:200, Proteintech, 27141-1-AP).

Techniques: Immunohistochemistry, Immunohistochemical staining, Staining

Journal: Journal of Biological Chemistry

Article Title: Suppressors of T-cell Receptor Signaling Sts-1 and Sts-2 Bind to Cbl and Inhibit Endocytosis of Receptor Tyrosine Kinases

doi: 10.1074/jbc.m403759200

Figure Lengend Snippet: FIG. 4. Sts-2 stabilizes activated EGF receptors in a UBA- and SH3-domain dependent manner. A, CHO cells were transfected with EGFR and Cbl together with Sts-2, Sts-2 mutants, Sts-1, or GFP as a control as indicated. Receptor internalization was induced with 50 ng/ml EGF for the indicated times. Thereafter, cell-surface remaining EGFR was determined by incubation with 125I-EGF as described under “Experimental Procedures.” Results were plotted as percentage of cell surface receptor in non-stimulated cells. The levels of different proteins were monitored in TCLs using anti-FLAG antibodies (right). B, COS-1 cells overexpressing indicated FLAG-tagged Sts-2 constructs were stimulated with rhodamine- labeled EGF and subjected to immunofluorescence analysis as described under “Experimental Procedures.” C, HEK293T cells were transfected with EGFR, FLAG-Ub, and, where indicated, with Sts-1 or Sts-2 (right). Cells were stimulated with EGF for indicated periods, and the levels of different proteins were monitored in TCLs using indicated antibodies. D, the experimental procedure was the same as in C, except that either the wild-type or mutant Sts-2 constructs were used for receptor degradation assays. The graph shows quantification of receptor degradation from three independent experiments and the expression levels of the different forms of Sts-2 are displayed in the blot at bottom. E, left, HEK293T cells were transfected with EGFR, FLAG-Ub, Cbl, and, where indicated, Sts-2. Cells were stimulated with EGF for the indicated periods and the levels of different proteins were monitored in TCLs using indicated antibodies. Right, HEK293T cells were transfected with EGFR, ubiquitin, Cbl, and either the control vector or indicated Sts-2 constructs. Cells were left unstimulated or stimulated with EGF for 30 min, and the effects of Sts-2 constructs on MAP kinase pathway activation were monitored with the use of anti-pERK antibodies. The graph shows quantification of densitometric analysis of the immunoblots from three independent experiments. IP, immunoprecipitation; WB, Western blotting.

Article Snippet: Surface-bound 125I-EGF was determined by lysis of cells in 1 M NaOH and analysis in a -counter (1470 Wizard; PerkinElmer).

Techniques: Transfection, Control, Incubation, Cell Surface Receptor Assay, Construct, Labeling, Immunofluorescence, Mutagenesis, Expressing, Ubiquitin Proteomics, Plasmid Preparation, Activation Assay, Western Blot, Immunoprecipitation

Journal: Drug delivery

Article Title: In vivo antitumor activity of PHT-427 inhibitor-loaded polymeric nanoparticles in head and neck squamous cell carcinoma.

doi: 10.1080/10717544.2024.2449376

Figure Lengend Snippet: Figure 3. NP-427 downregulates EGFR expression in the HNSCC model. (A) EGFR protein expression was determined by ELISA. (B) Fluorescence micro graphs of representative immunostaining of EGFR-Alexa Fluor546 conjugate in tumor tissue (red) and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (blue) (x40), Scale bar 100 µm. (C) EGFR immunofluorescence intensity was ana lyzed by ImageJ software. Experimental groups: control group (CTR) treated with 0.9% saline solution; unloaded nanoparticles group (NP-Ø) with a concen tration of 1 mg/mL; PHT-427 inhibitor-free at concentrations of 0.5 and 1 mg/ mL group (PHT-0.5 and PHT-1, respectively) and loaded nanoparticles with the PHT-427 inhibitor at the concentrations of 0.5 and 1 mg/mL groups (NP-0.5 and NP-1, respectively) for 21 days. The diagrams for EGFR represent the mean ± SD of ng/mL for a and arbitrary units (AU) for B. p < .05 versus CTR and NP-Ø groups. (n = 32).

Article Snippet: Protein extracts were assayed by a different human enzyme-linked immunosorbent assay (elisa) kits by the following proteins: eGFR was quantified using an elisa kit (catalog number: csB-e12124h; cusabio, houston, Usa); akt(ps473) and total akt levels were measured using abcam elisa kit (catalog number: ab126433; abcam, cambridge, UK); Phospho-PDK1 (s241) and total PDK1 by the elisa Kit (catalog number: ab279889) of abcam (cambridge, UK), and human Pi3K by the elisa Kit (catalog number: csB-e08417h, cusabio, houston, Usa) according to the protocol’s instructions. the plates were assessed on the FlUOstar Omega (BMG labtech, Ortenberg, Germany) to 450 nm. these experiments were performed in triplicate for each samples (N = 31) (ctR group (n = 4); NP-Ø group (n = 5); Pht-0.5 group (n = 5); Pht-1 (n = 5); NP-0.5 (n = 6); and NP-1 group (n = 6)).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Fluorescence, Immunostaining, Immunofluorescence, Software, Control, Saline

Journal: Journal of Biological Chemistry

Article Title: The Caenorhabditis elegansmRNA 5′-Capping Enzyme

doi: 10.1074/jbc.m212101200

Figure Lengend Snippet: FIG. 3. The CEL-1 GTase domain can replace Ceg1 in S. cerevi- siae. A, complementation of a double deletion mutant (ceg1cet1) lacking both the GTase and RTPase. The CEG1/CET1 shuffling strain YSB719 (11) was transformed with the following LEU2/2-m plasmids: vector (pAD5); MCE (pGL-MCE, expressing mouse capping enzyme MCE1 from the GPD promoter); CEL-1 (pAD5-CEL1, expressing CEL-1 from the ADH1 promoter); CEL-1-(1–585) (pAD5-CEL-1-(1–585), ex- pressing residues 1–585 of CEL-1 from the ADH1 promoter). Leu

Article Snippet: When overexpressed in ceg1 cet1 strain in combination with MCE-(211–597), cells containing Cet1 formed colonies after 1 day, whereas cells containing either CEL-1-(13–221) or MCE-(1–210) formed colonies after 3 days (Fig. 4A).

Techniques: Mutagenesis, Transformation Assay, Plasmid Preparation, Expressing

Journal: Journal of Biological Chemistry

Article Title: The Caenorhabditis elegansmRNA 5′-Capping Enzyme

doi: 10.1074/jbc.m212101200

Figure Lengend Snippet: FIG. 4. The isolated metazoan RTPase domain can replace Cet1 in S. cerevisiae. A, complementation by plasmid shuffling. YSB719 was transformed with pDB20H-MCE-(211–597). A His isolate was subsequently transformed with the following LEU2/2-m plasmids: vector (pRS425 (81)); CET1 (pAD5-CET1); CEL-1 (13–221) (pAD5-CEL-1-(13–221), expressing CEL-1-(13–221) tagged with an HA epitope from the ADH1 promoter); MCE (1–210) (pAD5-MCE-(1–210), expressing MCE-(1–210) tagged with the HA epitope from the ADH1 promoter). Leu His

Article Snippet: When overexpressed in ceg1 cet1 strain in combination with MCE-(211–597), cells containing Cet1 formed colonies after 1 day, whereas cells containing either CEL-1-(13–221) or MCE-(1–210) formed colonies after 3 days (Fig. 4A).

Techniques: Isolation, Plasmid Preparation, Transformation Assay, Expressing

Journal: Journal of Biological Chemistry

Article Title: The Caenorhabditis elegansmRNA 5′-Capping Enzyme

doi: 10.1074/jbc.m212101200

Figure Lengend Snippet: FIG. 5. The addition of the Ceg1-interacting region from Cet1 onto CEL-1 RTPase domain allows it to function in the presence of Ceg1. YSB719 was transformed with (left) pRSH-CEG1 or (right) pDB20H-MCE-(211–597). His isolates were subsequently transformed with the following LEU2/2 m plasmids: vector (pAD5); CET1 (pAD5- CET1); CET1-(1–225)-CEL-1-(13–221) (pAD5-CET1 (1–225)-CEL-1-(13– 221), expressing a fusion protein containing residues 1–225 of Cet1 and CEL-1-(13–221) tagged with the HA epitope); CET1-(1–265)-CEL-1-(13– 221) (pAD5-CET1 (1–265)-CEL-1-(13–221)); cet1–446-(1–265)-CEL-1-(13– 221) (pAD5-cet1–446-(1–265)-CEL-1-(13–221), expressing a fusion pro- tein consisting of residues 1–265 from the cet1–446 mutant (11) and CEL-1-(13–221) tagged with the HA epitope); and CEL-1-(13–221) (pAD5- CEL-1-(13–221)). Leu His transformants were grown in the presence of 5-FOA, and the plates were incubated for 3 days at 30 °C.

Article Snippet: When overexpressed in ceg1 cet1 strain in combination with MCE-(211–597), cells containing Cet1 formed colonies after 1 day, whereas cells containing either CEL-1-(13–221) or MCE-(1–210) formed colonies after 3 days (Fig. 4A).

Techniques: Transformation Assay, Plasmid Preparation, Expressing, Mutagenesis, Incubation

Journal: Journal of Biological Chemistry

Article Title: The Caenorhabditis elegansmRNA 5′-Capping Enzyme

doi: 10.1074/jbc.m212101200

Figure Lengend Snippet: FIG. 6. RTPase activities of CEL-1-(13–221) mutants. A, purification of recombinant polyhistidine-tagged CEL-1-(13–221) protein. The peak fractions of the heparin-Sepharose CL-6B column were analyzed by SDS-PAGE and visualized by Coomassie Brilliant Blue staining (top panel) or immunoblotting with monoclonal anti-polyhistidine antibody (bottom panel). 400 and 50 ng of protein were loaded for the top and bottom panels, respectively. Lane 1, wild-type CEL-1-(13–221); lane 2, C136S; lane 3, R142K; lane 4, D76N; lane 5, E111Q; lane 6, D112N. For reasons that are not clear, the D76N mutant shows slightly altered mobility. B, RTPase assay. The wild-type and mutated CEL-1-(13–221) proteins were incubated at 30 °C for 10 min with 1 M termini of a [-32P]GTP-labeled 65 nucleotide RNA. Reaction mixtures were analyzed by thin layer chromatography (TLC) on polyethyleneimine-cellulose plates. Released phosphate was detected by autoradiography, and radioactive spots were cut out and quantitated by liquid scintillation counting. Relative amounts of released phosphate were plotted versus protein amount. C, in vivo analysis of CEL-1-(13–221) mutants by plasmid shuffling. YSB719 carrying pDB20H-MCE-(211–597) was transformed with the vector pAD5 or derivatives expressing the indicated CEL-1 alleles. Leu His transformants were tested for growth in the presence of 5-FOA. Plates were incubated for 4 days at 30 °C. D, immunoblot analysis of S. cerevisiae whole-cell extracts. YSB719 cells carrying pDB20H-MCE-(211–597) and CEL-1-(13–221) derivatives were grown in selective media but without shuffling out the CEG1/CET1 plasmid. Extracts were prepared, and 10-g protein was analyzed by SDS-PAGE and immunoblotting with the anti-HA antibody12CA5. Lane 1, pAD5; lane 2, pAD5-CEL-1-(13–221); lane 3, pAD5-CEL- 1-(13–221)C136S; lane 4, pAD5-CEL-1-(13–221)C136A; lane 5, pAD5-CEL-1-(13–221)R142K; lane 6, pAD5-CEL-1-(13–221)R142A; lane 7, pAD5- CEL-1-(13–221)D76N; lane 8, pAD5-CEL-1-(13–221)E111Q; lane 9, pAD5-CEL-1-(13–221)D112N.

Article Snippet: When overexpressed in ceg1 cet1 strain in combination with MCE-(211–597), cells containing Cet1 formed colonies after 1 day, whereas cells containing either CEL-1-(13–221) or MCE-(1–210) formed colonies after 3 days (Fig. 4A).

Techniques: Purification, Recombinant, SDS Page, Staining, Western Blot, Mutagenesis, Incubation, Labeling, Thin Layer Chromatography, Autoradiography, In Vivo, Plasmid Preparation, Transformation Assay, Expressing