Journal: iScience

Article Title: DNA methylation of the IL1R2 gene is associated with porcine placental development and birth weight

doi: 10.1016/j.isci.2026.115055

Figure Lengend Snippet: RT-qPCR analysis of DNMTs and IL1R2 expression, along with IHC staining (A) RT-qPCR was used to quantitatively analyze the expression profiles of DNMTs and IL1R2 in HBW and LBW placentas ( DNMT3A : p = 0.0264; DNMT3L : p = 0.0016; DNMT1 : p = 0.0069; IL1R2 : p = 0.0156). (B and D) IHC was performed to analyze the protein expression intensity of DNMT3A in HBW and LBW placentas (DNMT3A: p = 0.0059). Scale bars, 50 μm. (C and E) IHC was performed to analyze the protein expression intensity of IL1R2 in HBW and LBW placentas (IL1R2: p = 0.0007). Scale bars, 50 μm. Data are represented as mean ± SEM. Statistical significance was assessed by paired Student’s t test, with n = 6 (3 samples in the LBW group and 3 samples in the HBW group).

Article Snippet: Rabbit anti-Homo sapiens (Human) IL1R2 Polyclonal antibody , Cusabio Biotech , Cat# CSB-PA011622ESR1HU; RRID: AB_3719906.

Techniques: Quantitative RT-PCR, Expressing, Immunohistochemistry

Journal: iScience

Article Title: DNA methylation of the IL1R2 gene is associated with porcine placental development and birth weight

doi: 10.1016/j.isci.2026.115055

Figure Lengend Snippet: MethPrimer-based prediction of CpG islands in the IL1R2 promoter region and subsequent analysis of the methylation status in placental tissues (A) Bioinformatics analysis predicted CpG islands in the promoter region of IL1R2 . (B) MS-PCR measured IL1R2 methylation levels in HBW and LBW placentas ( p = 0.0062). Data are represented as mean ± SEM. Statistical significance was assessed by unpaired Student’s t test, with n = 6 (3 samples in the LBW group and 3 samples in the HBW group).

Article Snippet: Rabbit anti-Homo sapiens (Human) IL1R2 Polyclonal antibody , Cusabio Biotech , Cat# CSB-PA011622ESR1HU; RRID: AB_3719906.

Techniques: Methylation

Journal: iScience

Article Title: DNA methylation of the IL1R2 gene is associated with porcine placental development and birth weight

doi: 10.1016/j.isci.2026.115055

Figure Lengend Snippet: Effects of 5-Aza treatment on IL1R2 gene expression and methylation levels in PTr2 cells (A) The mRNA expression of IL1R2 in 5-Aza-treated PTr2 cells was measured using RT-qPCR (20 μM: p = 0.0199). “ns” denotes non-significant results ( p > 0.05). (B) MS-PCR measured the methylation levels of the IL1R2 promoter in 5-Aza-treated PTr2 Cells ( p = 0.0005). (C) BS-PCR measured the methylation levels of the IL1R2 promoter in 5-Aza-treated PTr2 cells ( p = 0.0011). Data are represented as mean ± SEM. Statistical significance was assessed by unpaired Student’s t test, n = 3.

Article Snippet: Rabbit anti-Homo sapiens (Human) IL1R2 Polyclonal antibody , Cusabio Biotech , Cat# CSB-PA011622ESR1HU; RRID: AB_3719906.

Techniques: Gene Expression, Methylation, Expressing, Quantitative RT-PCR

Journal: iScience

Article Title: DNA methylation of the IL1R2 gene is associated with porcine placental development and birth weight

doi: 10.1016/j.isci.2026.115055

Figure Lengend Snippet: IL1R2 knockdown promotes PTr2 Cell proliferation and migration (A) RT-qPCR analysis of IL1R2 knockdown efficiency and its effect on proliferation and apoptosis markers in PTr2 cells ( IL1R2 : p = 0.0002; Ki67 : p = 0.0043; BAX : p = 0.0155; CASP3 : p = 0.0088; CASP9 : p = 0.0331). “ns” denotes non-significant results ( p > 0.05). (B) Cell proliferation assessed using CCK-8 assay after IL1R2 knockdown (48 h: p = 0.0224; 72 h: p = 0.0035). (C and D) Cell proliferation evaluated using EdU assay ( p = 0.0083). Scale bars, 100 μm. (E and F) Cell migration was analyzed by scratch assay ( p = 0.0462). Scale bars, 500 μm. Data are represented as mean ± SEM. Statistical significance was assessed by unpaired Student’s t test, n = 3.

Article Snippet: Rabbit anti-Homo sapiens (Human) IL1R2 Polyclonal antibody , Cusabio Biotech , Cat# CSB-PA011622ESR1HU; RRID: AB_3719906.

Techniques: Knockdown, Migration, Quantitative RT-PCR, CCK-8 Assay, EdU Assay, Wound Healing Assay

Journal: iScience

Article Title: DNA methylation of the IL1R2 gene is associated with porcine placental development and birth weight

doi: 10.1016/j.isci.2026.115055

Figure Lengend Snippet: IL1R2 overexpression inhibits PTr2 Cell proliferation and migration (A) RT-qPCR analysis of IL1R2 overexpression efficiency and its effect on proliferation and apoptosis markers in PTr2 cells ( IL1R2 : p = 0.0003; PCNA : p = 0.0256; BAX : p = 0.0497). “ns” denotes non-significant results ( p > 0.05). (B) Cell proliferation was assessed using CCK-8 assay following IL1R2 overexpression (48 h: p = 0.0027; 72 h: p = 0.0011). (C and D) Cell proliferation evaluated using EdU assay ( p = 0.0452). Scale bars, 100 μm. (E and F) Cell migration was analyzed by scratch assay ( p = 0.0078). Scale bars, 500 μm. Data are represented as mean ± SEM. Statistical significance was assessed by unpaired Student’s t test, n = 3.

Article Snippet: Rabbit anti-Homo sapiens (Human) IL1R2 Polyclonal antibody , Cusabio Biotech , Cat# CSB-PA011622ESR1HU; RRID: AB_3719906.

Techniques: Over Expression, Migration, Quantitative RT-PCR, CCK-8 Assay, EdU Assay, Wound Healing Assay

Journal: iScience

Article Title: DNA methylation of the IL1R2 gene is associated with porcine placental development and birth weight

doi: 10.1016/j.isci.2026.115055

Figure Lengend Snippet: IL1R2 positively regulates TNF- α expression in PTr2 cells (A) IL1R2 knockdown suppresses TNF-α mRNA levels ( p = 0.0442). (B) IL1R2 overexpression elevates TNF-α mRNA levels ( p = 0.0057). (C - E) IL1R2 knockdown reduces TNF- α protein abundance, with confirmation of knockdown efficiency (IL1R2: p = 0.0012; TNF-α: p = 0.0394). (F - H) IL1R2 overexpression increases TNF- α protein levels, with confirmation of knockdown efficiency (IL1R2: p = 0.0011; TNF-α: p = 0.0014). Data are represented as mean ± SEM. Statistical significance was assessed by unpaired Student’s t test, n = 3.

Article Snippet: Rabbit anti-Homo sapiens (Human) IL1R2 Polyclonal antibody , Cusabio Biotech , Cat# CSB-PA011622ESR1HU; RRID: AB_3719906.

Techniques: Expressing, Knockdown, Over Expression, Quantitative Proteomics

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 1 Effect of IL1B on mbIL1R, mbIL1R2 and IL1RA expression in KLE cells. Confluent KLE cell cultures were treated with 0.1 ng/ml IL1B for varying periods of time (0–24 h) (A) or with different concentrations of IL1B (0, 0.1, 1 and 10 ng/ml) for 12 h (B). Cells were recovered to evaluate IL1R1, IL1R2 and IL1RA protein expression in total cell protein extracts by western blot. a-Tubulin was also probed on the same membranes to ensure equal protein loading. The intensity of mbIL1Rs, IL1RA and corresponding a-tubulin bands was evaluated by densitometric analysis (C and D). Values were normalized to a-tubulin band intensity and expressed as % of control (ratio of normalized mbIL1R1, mbIL1R2 or IL1RA band intensity detected following treatment with IL1B to that detected following incubation with the control culture medium alone for an equivalent period of time). *P , 0.05 and **P , 0.01 as compared with control. Data are from three different experiments. MM, minimal medium; mb, membrane-bound; s, soluble.

Article Snippet: IL1R2 ELISA is based on the use of a mouse monoclonal anti-human IL1R2 antibody for capture and a goat polyclonal anti-human IL1R2 antibody for detection (R&D systems).

Techniques: Expressing, Western Blot, Control, Incubation, Membrane

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 3 Immunocytofluorescence of IL1R1, IL1R2 and IL1RA in KLE cells. Cells cultured in chamber slides were incubated for 24 h with the culture medium alone or containing 0.1 ng/ml IL1B. Detection of IL1R1 (A and C), IL1R2 (E and G) and IL1RA (I and K) was performed by immunocytochemical staining using specific monoclonal mouse antibodies. Note the increase in IL1R1 (C), IL1R2 (G) and IL1RA (K) immunofluorescent signal in KLE cells exposed to IL1B by comparison with non-stimulated cells (A, E and I, respectively). No immunofluorescence was observed in the absence of primary antibodies (B, F and J) or the presence of mouse IgGs (D, H and L) (controls). Data are representative of four different experiments.

Article Snippet: IL1R2 ELISA is based on the use of a mouse monoclonal anti-human IL1R2 antibody for capture and a goat polyclonal anti-human IL1R2 antibody for detection (R&D systems).

Techniques: Cell Culture, Incubation, Staining, Comparison

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 4 Effect of IL1B on IL1R1, IL1R2 and IL1RA mRNA expression in KLE cells. Confluent KLE cell cultures were treated with 0.1 ng/ml IL1B for varying periods of time (0–24 h) (A, B and C, respectively) or with different con- centrations of IL1B (0, 0.1, 1 and 10 ng/ml) for 12 h (D, E and F, respectively). Total RNA was extracted and reverse transcribed, IL1R1, IL1R2, IL1RA and GAPDH cDNAs were amplified by Real-Time PCR as described in Materials and Methods and IL1R1, IL1R2 and IL1RA mRNA levels were normalized to GAPDH mRNA levels. Data were expressed as % of control (ratio of IL1R1, IL1R2 or IL1RA mRNA levels found in cells incubated with IL1 to those found in cells incubated with the control basal culture medium for an equivalent period of time). *P , 0.05, **P , 0.01 and ***P , 0.001 as compared with control. Data are from three different experiments.

Article Snippet: IL1R2 ELISA is based on the use of a mouse monoclonal anti-human IL1R2 antibody for capture and a goat polyclonal anti-human IL1R2 antibody for detection (R&D systems).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Incubation

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 5 Effect of IL1B on IL1R1, IL1R2 and IL1RA mRNA stability and gene expression in KLE cells. Confluent KLE cell cultures were stimulated with IL1B (1 ng/mll) for 12 h. Actinomycin D (10 mg/ml) was added to stop the de novo RNA synthesis, and cells were harvested after 0, 2, 4 and 8 h of incubation with actinomycin D. Total RNA was extracted and reverse transcribed. cDNA was analyzed by real-time PCR with specific primers for IL1R1, IL1R2, IL1RA and GAPDH. Levels of IL1R1, IL1R2 and IL1RA mRNA were normalized to those of GAPDH to assess the kinetics of IL1R1 (A), IL1R2 (B) and IL1RA (C) mRNA degradation. Data were expressed as % of control (ratio of IL1R1, IL1R2 or IL1RA mRNA levels found at different periods of time following the arrest of de novo mRNA transcription to that found at the time of arrest); data are from three different experiments. To evaluate IL1R1, IL1R2 and IL1RA transcriptional activation (D), confluent KLE cell cultures were stimulated with IL1B (1 ng/ml) for 6 h, cell nuclei were isolated and nuclear mRNA transcription was analyzed by nuclear run-on as described in Materials and Methods. DNA samples immobilized onto nylon membranes were as follows: lane 1, IL1R1 cDNA; lane 2, IL1R2 cDNA; lane 3, IL1RA; lane 4, 28S cDNA; and lane 5, pBluescript plasmid DNA. Radioactive transcripts were from KLE cells stimulated in MM or with 1 ng/ml IL1B. Data are repre- sentative of four different experiments.

Article Snippet: IL1R2 ELISA is based on the use of a mouse monoclonal anti-human IL1R2 antibody for capture and a goat polyclonal anti-human IL1R2 antibody for detection (R&D systems).

Techniques: Gene Expression, Incubation, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Activation Assay, Isolation, Plasmid Preparation

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 6 Effect of IL1B on mbIL1R1, mbIL1R2 and IL1RA expression in endometrial epithelial cells. Confluent endometrial epithelial cell cultures were treated with different concentrations of IL1B (0, 0.1, 1 and 10 ng/ml) for 12 h. Cells were recov- ered to evaluate IL1R1, IL1R2 and IL1RA protein expression in total cell protein extracts by Western blot (A). a-Tubulin was probed on the same membranes to ensure equal protein loading. Culture supernatants were recovered to evaluate sIL1R2 release (C) and IL1RA secretion (D) by ELISA; data were from four different endometrial samples and expressed as % of control (ratio of sIL1R2 or IL1RA concentrations detected in the presence of IL1 to those detected in the control basal culture medium for an equivalent period of 12 h). *P , 0.05, as compared with control. The intensity of mbIL1Rs, IL1RA and corresponding a-tubulin bands was evaluated by densitometric analysis (B). Values were normalized to a-tubulin band intensity and expressed as % of control (ratio of normalized mbIL1R1, mbIL1R2 or IL1RA band intensity detected following treatment with IL1B to that detected following incubation with the control culture medium alone for an equivalent period of time). *P , 0.05 and **P , 0.01 as compared with control. Data were from three different endometrial samples. sIL1R1 and sIL1R2 release and IL1RA secretion from endometrial epithelial cell cultures was also analyzed by metabolic labeling with 35S-cysteine and immunoprecipitation using a goat anti-IL1R1, goat anti-IL1R2 or rabbit anti- IL1RA antibody (E). Immunoprecipitated proteins were then analyzed by SDS-PAGE as described in Materials and Methods (representative data from three different endometrial samples); mb, membrane-bound; s, soluble.

Article Snippet: IL1R2 ELISA is based on the use of a mouse monoclonal anti-human IL1R2 antibody for capture and a goat polyclonal anti-human IL1R2 antibody for detection (R&D systems).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Incubation, Labeling, Immunoprecipitation, SDS Page, Membrane

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 7 Effect of IL1B on IL1R1, IL1R2 and IL1RA mRNA expression in endometrial epithelial cells. Confluent cultures were treated with 0.1 ng/ml IL1B with different concentrations of IL1B (0, 0.1, 1 and 10 ng/ml) for 12 h. Total RNA was extracted and reverse transcribed, IL1R1, IL1R2, IL1RA and GAPDH cDNAs were amplified by Real-Time PCR as described in Materials and Methods and IL1R1 (A), IL1R2 (B) and IL1RA (C) mRNA levels were normalized to GAPDH mRNA levels. Data were from three different endometrial samples and expressed as % of control (ratio of IL1R1, IL1R2 or IL1RA mRNA levels found in cells incubated with IL1 to those found in cells incubated with the control basal culture medium for an equivalent period of 12 h). *P , 0.05 and **P , 0.01 as compared with control.

Article Snippet: IL1R2 ELISA is based on the use of a mouse monoclonal anti-human IL1R2 antibody for capture and a goat polyclonal anti-human IL1R2 antibody for detection (R&D systems).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Incubation

Journal: Reproduction

Article Title: Stable inhibition of interleukin 1 receptor type II in Ishikawa cells augments secretion of matrix metalloproteinases: possible role in endometriosis pathophysiology

doi: 10.1530/rep-06-0377

Figure Lengend Snippet: Figure 1 IL1R2 concentration as measured in cell lysates (A) and cell- free culture supernatants (B) by ELISA. Data from three independent experiments are shown as meansGS.E.M. and presented as percentage of control (IL1R2 concentration in the culture supernatant of non-transfected (NT) cells). **Significant diminution of IL1R2 expression in A08 and A17 when compared with NT cells in cell lysates (P!0.01). *Significant diminution of IL1R2 release in A17 when compared with NT cells in cell-free culture supernatants (P!0.05).

Article Snippet: Membranes were incubated for 3 h at room temperature with a polyclonal goat anti-human IL1R1 (R&D Systems; www.reproduction-online.org 2 mg/ml in blocking solution), a polyclonal goat anti-human IL1R2 (R&D Systems; 2 mg/ml in blocking solution), or a polyclonal goat anti-human IL1RAcP (R&D Systems; 2 mg/ml in blocking solution).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Control, Transfection, Expressing

Journal: Reproduction

Article Title: Stable inhibition of interleukin 1 receptor type II in Ishikawa cells augments secretion of matrix metalloproteinases: possible role in endometriosis pathophysiology

doi: 10.1530/rep-06-0377

Figure Lengend Snippet: Figure 2 Immunofluorescence analysis of IL1R1, IL1R2, and IL1RAcP expression in non-transfected Ishikawa cells (NT) and E03, E11, A08, and A17 clones. Note the com- parable intensity of IL1R1 and IL1RAcP immunofluorescent signals in NT, E03, and E11 clones, and the marked decline in IL1R2 immunofluorescent signal in A08 and A17 clones. No immunofluorescence was observedinnegativecontrols(c)in theabsence of primary antibodies (objective !40).

Article Snippet: Membranes were incubated for 3 h at room temperature with a polyclonal goat anti-human IL1R1 (R&D Systems; www.reproduction-online.org 2 mg/ml in blocking solution), a polyclonal goat anti-human IL1R2 (R&D Systems; 2 mg/ml in blocking solution), or a polyclonal goat anti-human IL1RAcP (R&D Systems; 2 mg/ml in blocking solution).

Techniques: Expressing, Transfection, Clone Assay

Journal: Reproduction

Article Title: Stable inhibition of interleukin 1 receptor type II in Ishikawa cells augments secretion of matrix metalloproteinases: possible role in endometriosis pathophysiology

doi: 10.1530/rep-06-0377

Figure Lengend Snippet: Figure 3 Representative Western blot analysis of IL1R1 (A), IL1R2 (B), and IL1RAcP (C) expression in non-transfected Ishikawa cells (NT) and E03, E11, A08, and A17 clones. Equal amounts of proteins were subjected to SDS-PAGE analysis and Western blotting. Recombinant IL1R1, IL1R2, and IL1RAcP proteins were taken as controls (rR). Blotting with anti-tubulin antibody demonstrates equal protein loading. Note the reduced intensity of IL1R2 bands corresponding to either the membrane-bound or the soluble forms of the receptor, when compared with NT cells, and the absence of noticeable change in IL1R1 and IL1RAcP expression; mb, membrane-bound; s, soluble.

Article Snippet: Membranes were incubated for 3 h at room temperature with a polyclonal goat anti-human IL1R1 (R&D Systems; www.reproduction-online.org 2 mg/ml in blocking solution), a polyclonal goat anti-human IL1R2 (R&D Systems; 2 mg/ml in blocking solution), or a polyclonal goat anti-human IL1RAcP (R&D Systems; 2 mg/ml in blocking solution).

Techniques: Western Blot, Expressing, Transfection, Clone Assay, SDS Page, Recombinant, Membrane

Journal: Molecular and Cellular Biology

Article Title: Selective Modulation of Hedgehog/GLI Target Gene Expression by Epidermal Growth Factor Signaling in Human Keratinocytes

doi: 10.1128/mcb.02317-05

Figure Lengend Snippet: FIG. 2. Direct activation of the IL1R2 promoter by combinatorial GLI1/EGF signaling. Bs, binding site; mBs, mutated binding site; RLU, relative light units. (A) Illustration of the localization of three putative GLI binding sites in the 255-bp promoter (prom) fragment of the IL1R2 upstream regulatory region. The sequence of the three binding sites is shown in the table (right panel). For mutated binding sites used in EMSA (mBs1, mBs2, and mBs3), two essential C nucleotides (underlined) were changed to two G nucleotides. (B) EMSA analysis of the three GLI binding sites showing that all three sites are bound by recombinant DNA binding domain of GLI1. To demonstrate specific binding, competition experiments (comp) with increasing amounts of oligonucleotide corresponding either to wild-type or mutated binding site sequences were done. Furthermore, poly(dI-dC) poly(dI-dC) (dIdC) was added to the binding reactions to test for unspecific binding. (C) Luciferase reporter assays showing that both GLI1 (left) and active GLI2* (right) efficiently stimulate reporter activity from the 255-bp IL1R2 promoter fragment. Note that mutation of any of the three GLI binding sites is sufficient to abrogate reporter activation. (D) Activation of the IL1R2 promoter by GLI1 and active GLI2* is enhanced by simultaneous activation of EGF signaling. Mutation of Bs1 in the IL1R2 promoter (IL1R2-mBs1) inhibits the stimulatory effect of EGF signaling. cont, control. Promoter (prom) activation of the known direct GLI target PTCH is unaffected by EGF signaling. Reporter assays shown in panels C and D were done in HaCaT keratinocytes.

Article Snippet: Following preincubation for 10 min at room temperature with rabbit (Novacastra) or goat normal serum (DakoCytomation), proteins were detected with polyclonal goat anti-IL1R2 antibody (1:25; RnD Systems), monoclonal mouse anti-EGFR antibody (1:20; DakoCytomation), and monoclonal mouse anti-phospho-p44/42 MAPK (1:50) (Thr202/Tyr204 [E10]; Cell Signaling Technology) antibody.

Techniques: Activation Assay, Binding Assay, Sequencing, Recombinant, Luciferase, Activity Assay, Mutagenesis, Control

Journal: Molecular and Cellular Biology

Article Title: Selective Modulation of Hedgehog/GLI Target Gene Expression by Epidermal Growth Factor Signaling in Human Keratinocytes

doi: 10.1128/mcb.02317-05

Figure Lengend Snippet: FIG. 4. Inhibition of EGFR and MEK/ERK signaling abrogates synergistic activation of GLI/EGF target genes. (A through D) qRT-PCR analysis of GLI/EGF (IL1R2, S100A9, and ARC [and EGR3 in panel A]) and EGF-insensitive GLI target genes (PTCH and BCL2 genes) in HaCaT keratinocytes expressing a doxycycline (Dox)-regulated conditional allele of GLI1 (A, B, and D) or GLI2* (C). (A) Inhibition of EGFR signaling by gefitinib (Gef) (at 0.5 M and 1.0 M) abrogates synergistic activation of GLI1/EGF target genes (IL1R2 [left graph] and ARC, EGR3, and S100A9 [right graph] genes) but does not affect the induction of PTCH mRNA. (Right panel) Control Western blot showing efficient inhibition of EGF-induced ERK phosphorylation by gefitinib (0.5 M and 1.0 M). (B and C) Synergistic induction of GLI/EGF target gene expression by EGF is abolished by simultaneous treatment with UO126, a selective inhibitor of MEK1/2 function that prevents activation and phosphorylation of ERK1/2 (pERK), respectively. mRNA expression of BCL2 was unaffected by EGF and UO126 treatment in GLI1-expressing cells (B) yet was reduced by UO126 in GLI2-expressing cells (C). Control Western blot analysis of transgene and activated ERK1/2 (pERK) expression in GLI1 (B) and GLI2 (C) HaCaT cells treated with EGF and/or UO126, showing that UO126 treatment efficiently blocked phosphorylation of ERK1/2. Note that the GLI2 protein level is decreased by UO126 treatment. Total ERK protein was monitored as loading control. (D) Inhibition of PI3K/AKT signaling by LY294002 treatment reduces neither synergistic activation of GLI1/EGF target genes nor GLI1-mediated activation of PTCH mRNA expression. (Right panel) Control Western blot showing inhibition of AKT phosphorylation (pAKT) by the PI3K inhibitor LY294002.

Article Snippet: Following preincubation for 10 min at room temperature with rabbit (Novacastra) or goat normal serum (DakoCytomation), proteins were detected with polyclonal goat anti-IL1R2 antibody (1:25; RnD Systems), monoclonal mouse anti-EGFR antibody (1:20; DakoCytomation), and monoclonal mouse anti-phospho-p44/42 MAPK (1:50) (Thr202/Tyr204 [E10]; Cell Signaling Technology) antibody.

Techniques: Inhibition, Activation Assay, Quantitative RT-PCR, Expressing, Control, Western Blot, Phospho-proteomics, Targeted Gene Expression