Journal: European Journal of Medicinal Chemistry Reports

Article Title: Synthesis of new para-aminobenzoic acid derivatives, in vitro biological evaluation and preclinical validation of DAB-2-28 as a therapeutic option for the treatment of bladder cancer

doi: 10.1016/j.ejmcr.2022.100069

Figure Lengend Snippet: Fig. 3. Representative images (a) and graphical analysis (b) showing the immunodetection of phos- phorylated STAT3 (p-STAT3) in MB49-I cells pre- treated for 30 min with vehicle (DMSO) or DAB-1, DAB-2-28, DAB-2-31A and DAB-2-31B molecules at 15 and 30 μM, and then washed and recovered after 15 min of activation with 100 ng/mL IL6. The ratio of phosphorylated/unphosphorylated proteins was calculated from densitometric analysis of each sample to evaluate the relative activation of p-STAT3. *p < 0.05 and **p < 0.01 denote significant differences compared with vehicle control group.

Article Snippet: The antibodies against pSTAT3 (pY705; #9145), STAT3 (#4904), p-IκB (# 9246), iNOS (# 2977), and COX-2 (#12282) were purchased from Cell Signaling Technology (Danvers, MA) and β-actin (# A3854) was from Sigma Chemical Company (Oakville, Canada).

Techniques: Immunodetection, Activation Assay, Control

Journal: European Journal of Medicinal Chemistry Reports

Article Title: Synthesis of new para-aminobenzoic acid derivatives, in vitro biological evaluation and preclinical validation of DAB-2-28 as a therapeutic option for the treatment of bladder cancer

doi: 10.1016/j.ejmcr.2022.100069

Figure Lengend Snippet: Fig. 5. Representative images (a) and graphical analysis (b) showing the immunodetection of phos- phorylated STAT3 (p-STAT3) in MB49-I cells pre- treated for 30 min with vehicle (DMSO) or DAB-1, DAB-3-27, and DAB-3-33 molecules at 10, 20 and 30 μM, and then washed and recovered after 15 min of activation with 100 ng/mL IL6. The ratio of p-STAT3/ STAT3 proteins was calculated from densitometric analysis of each sample to evaluate the relative acti- vation of p-STAT3. *p < 0.05 and **p < 0.01 denote significant differences compared with vehicle control group.

Article Snippet: The antibodies against pSTAT3 (pY705; #9145), STAT3 (#4904), p-IκB (# 9246), iNOS (# 2977), and COX-2 (#12282) were purchased from Cell Signaling Technology (Danvers, MA) and β-actin (# A3854) was from Sigma Chemical Company (Oakville, Canada).

Techniques: Immunodetection, Activation Assay, Control

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Expression of Signal Transducer and Activator of Transcription 3 and Suppressor of Cytokine Signaling 3 in Urothelial Carcinoma

doi: 10.1016/s1607-551x(09)70569-8

Figure Lengend Snippet: Figure 1. Immunohistochemical staining of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) Tyr705 and cytokine signaling 3 (SOCS3) in urothelial carcinomas. Nuclear staining of p-STAT3 (Tyr705): (A) low intensity grade (insert in A is normal urothelium); (B) high intensity grade. Cytoplasmic staining of SOCS3: (C) low intensity grade; (D) high intensity grade. Original magnification, 400×.

Article Snippet: The membranes were blocked with 5% non-fat dried milk in Tris-buffered Saline (pH 7.4) with Tween-20, and then incubated with either anti-phospho-STAT3 (Tyr705) (1:1,000 dilution; Cell Signaling Technology) or SOCS3 rabbit polyclonal primary antibody (Santa Cruz Biotechnology Inc.).

Techniques: Immunohistochemical staining, Staining

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Expression of Signal Transducer and Activator of Transcription 3 and Suppressor of Cytokine Signaling 3 in Urothelial Carcinoma

doi: 10.1016/s1607-551x(09)70569-8

Figure Lengend Snippet: Figure 2. Western blotting of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) Tyr705 and cytokine signaling 3 (SOCS3) protein in low-grade and high-grade UCs. p-STAT3 (Tyr705) expression was low and high in low- and high-grade UCs, respectively. Lane 1 = low-grade UC; lane 2=high-grade UC. However, the expression of SOCS3 was simi- lar in low- and high-grade UCs. GADPH protein expression was used as an internal control.

Article Snippet: The membranes were blocked with 5% non-fat dried milk in Tris-buffered Saline (pH 7.4) with Tween-20, and then incubated with either anti-phospho-STAT3 (Tyr705) (1:1,000 dilution; Cell Signaling Technology) or SOCS3 rabbit polyclonal primary antibody (Santa Cruz Biotechnology Inc.).

Techniques: Western Blot, Expressing, Control

Journal:

Article Title: Interleukin-6-Specific Activation of the C/EBP? Gene in Hepatocytes Is Mediated by Stat3 and Sp1

doi:

Figure Lengend Snippet: Binding of nuclear proteins to the Sp1(−117) site. (A) Supershift analysis using an antibody (Ab) against Sp1. Nuclear extracts were prepared from control or IL-6-treated HepG2 cells by a method that maximizes the extraction of Sp1 (see Materials and Methods). The extracts were incubated with either NRS or an Sp1-specific antibody and the δAPRE/Sp1 or δAPRE probe. The supershift generated by the Sp1 antibody in lanes 2 and 4 is indicated. (B and C) Binding of recombinant Sp1 to the δAPRE/Sp1 (B) and δAPRE (C) probes. Recombinant human Sp1 (50 ng) was used alone (lanes 5 and 10) or mixed with 8 μg of HepG2 nuclear extract (optimized for Stat protein extraction) from control or IL-6-treated cells. Stat3 antibody was added to the indicated reactions. The lower panel is a longer exposure of the top portion of the gel to emphasize the Stat3 antibody supershift complex.

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology, Inc.: Stat1 p84/p91 (E-23), Stat3 (C-20), Sp1 (PEP2), and normal rabbit immunoglobulin G (normal rabbit serum [NRS]).

Techniques: Binding Assay, Incubation, Generated, Recombinant, Protein Extraction

Journal:

Article Title: Interleukin-6-Specific Activation of the C/EBP? Gene in Hepatocytes Is Mediated by Stat3 and Sp1

doi:

Figure Lengend Snippet: Replacement of the C/EBPδ APRE with SBEs from ICAM-1 or C/EBPβ renders the promoter responsive to IFN-γ. (A) The C/EBPβ promoter contains an SBE that binds Stat1 and Stat3. A probe containing the putative SBE from the C/EBPβ promoter and Stat1- or α-Stat3-specific antibody (Ab) (lanes 3 and 4, respectively) were added to nuclear extracts from control (lane 1) or IL-6 treated (lanes 2 to 4) HepG2 cells and the reactions analyzed by EMSA. Antibody supershift species are indicated. (B) Comparison of SBE sequences from the C/EBPδ, C/EBPβ, and ICAM-1 promoters. Bases that differ from the C/EBPδ APRE sequence are underlined. (C) IL-6 and IFN-γ responsiveness of SBE swap mutants. Constructs in which the C/EBPδ APRE was exchanged with SBEs from the C/EBPβ or ICAM-1 genes were generated. These constructs and (−127)-Luc were cotransfected with pRSV β-gal into Hep3B cells and assayed for basal expression and IL-6 or IFN-γ inducibility. The values represent the average of three independent experiments. Relative basal expression was normalized to the (−127)-Luc level.

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology, Inc.: Stat1 p84/p91 (E-23), Stat3 (C-20), Sp1 (PEP2), and normal rabbit immunoglobulin G (normal rabbit serum [NRS]).

Techniques: Sequencing, Construct, Generated, Expressing

Journal:

Article Title: Interleukin-6-Specific Activation of the C/EBP? Gene in Hepatocytes Is Mediated by Stat3 and Sp1

doi:

Figure Lengend Snippet: The C/EBPδ APRE competes for binding of Stat3 to the α2-m APRE. (A) EMSA using the rat α2-m APRE, nuclear extracts (6.5 μg) from HepG2 cells, and NRS or Stat3-specific antibody (Ab) as indicated. The HepG2 cells were treated with IL-6 for 15 min. An upper complex (u) and a lower complex (l) appear in the IL-6-treated extracts (lane 3). The Stat3 antibody supershift complex (lane 4) is indicated. (B) Competition for Stat3 binding by the C/EBPδ APRE. Nuclear extracts (10 μg) from IL-6 treated HepG2 cells were incubated with Stat3-specific antibody and 10× (lanes 3 and 6), 30× (lanes 4 and 7), or 100× (lanes 5 and 8) molar excess of unlabeled wild-type (δAPRE) or mutant (δAPREm) binding site, as indicated. The rat α2-m APRE was used as a probe. The film was overexposed to emphasize the supershifted complex.

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology, Inc.: Stat1 p84/p91 (E-23), Stat3 (C-20), Sp1 (PEP2), and normal rabbit immunoglobulin G (normal rabbit serum [NRS]).

Techniques: Binding Assay, Incubation, Mutagenesis

Journal:

Article Title: Interleukin-6-Specific Activation of the C/EBP? Gene in Hepatocytes Is Mediated by Stat3 and Sp1

doi:

Figure Lengend Snippet: Selective binding of Stat3 to the C/EBPδ APRE. Nuclear extracts from control or IL-6-treated HepG2 cells were analyzed by EMSA using the wild-type or mutant C/EBPδ APRE probes and control antiserum or Stat1- or Stat3-specific antibody (Ab), as indicated. The film was overexposed to emphasize the supershift signal.

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology, Inc.: Stat1 p84/p91 (E-23), Stat3 (C-20), Sp1 (PEP2), and normal rabbit immunoglobulin G (normal rabbit serum [NRS]).

Techniques: Binding Assay, Mutagenesis

Journal:

Article Title: Interleukin-6-Specific Activation of the C/EBP? Gene in Hepatocytes Is Mediated by Stat3 and Sp1

doi:

Figure Lengend Snippet: Stat3 mediates IL-6-induced expression from the C/EBPδ promoter. (A) Stat3 but not Stat1 transactivates the C/EBPδ promoter. The (−127)-Luc construct was cotransfected into Hep3B cells with expression vectors for Stat1 or Stat3 or the parental pCDNA1 vector, together with pRSV β-gal as an internal standard, and tested for basal and IL-6-induced luciferase expression. (B) Stat3 transactivation of C/EBPδ promoter mutants. The indicated deletion and point mutants (Fig. ​(Fig.33 and ​and4)4) were cotransfected with the Stat3 expression vector into Hep3B cells and tested for basal and IL-6-inducible luciferase expression. (C) Stat3 transactivates a heterologous promoter containing the C/EBPδ APRE. The indicated TK promoter-luciferase reporter constructs (Fig. ​(Fig.8)8) were cotransfected with the Stat3 expression plasmid into Hep3B cells and tested for basal and IL-6-inducible expression. The cell extracts were assayed for luciferase and β-galactosidase activities as described in Fig. ​Fig.3.3. The values represent the averages of three to six independent experiments.

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology, Inc.: Stat1 p84/p91 (E-23), Stat3 (C-20), Sp1 (PEP2), and normal rabbit immunoglobulin G (normal rabbit serum [NRS]).

Techniques: Expressing, Construct, Plasmid Preparation, Luciferase

Journal:

Article Title: Interleukin-6-Specific Activation of the C/EBP? Gene in Hepatocytes Is Mediated by Stat3 and Sp1

doi:

Figure Lengend Snippet: Sequences and Stat binding properties of the C/EBPδ APRE and several known SBEs

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology, Inc.: Stat1 p84/p91 (E-23), Stat3 (C-20), Sp1 (PEP2), and normal rabbit immunoglobulin G (normal rabbit serum [NRS]).

Techniques: Binding Assay