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(A) Relative mRNA expression of IGFBP3 and IGFBP5 in control, PAE-L, and PAE-M newborn mammary tissues determined by quantitative RT-PCR. (B) Representative Western blots of selected insulin/IGF- and growth-factor-associated signaling proteins, including p-IRS1(T895), p-IRS1(S612), IRS1, EGFR, ErbB3, IGF1R, p-Akt1(S473), Akt1, p-ERK1/2, <t>ERK2,</t> ERα, Sox2, and IGFBP5. (C) Densitometric analysis of Western blot data. Phosphorylated proteins were normalized to their corresponding total proteins, and total protein levels were normalized to β-Actin, as appropriate.
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(A) Relative mRNA expression of IGFBP3 and IGFBP5 in control, PAE-L, and PAE-M newborn mammary tissues determined by quantitative RT-PCR. (B) Representative Western blots of selected insulin/IGF- and growth-factor-associated signaling proteins, including p-IRS1(T895), p-IRS1(S612), IRS1, EGFR, ErbB3, IGF1R, p-Akt1(S473), Akt1, p-ERK1/2, <t>ERK2,</t> ERα, Sox2, and IGFBP5. (C) Densitometric analysis of Western blot data. Phosphorylated proteins were normalized to their corresponding total proteins, and total protein levels were normalized to β-Actin, as appropriate.
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(A) Relative mRNA expression of IGFBP3 and IGFBP5 in control, PAE-L, and PAE-M newborn mammary tissues determined by quantitative RT-PCR. (B) Representative Western blots of selected insulin/IGF- and growth-factor-associated signaling proteins, including p-IRS1(T895), p-IRS1(S612), IRS1, EGFR, ErbB3, IGF1R, p-Akt1(S473), Akt1, p-ERK1/2, <t>ERK2,</t> ERα, Sox2, and IGFBP5. (C) Densitometric analysis of Western blot data. Phosphorylated proteins were normalized to their corresponding total proteins, and total protein levels were normalized to β-Actin, as appropriate.
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SPRED1 regulates NHEK cell proliferation via indirect interaction with ERK (A) Co-immunoprecipitation (CoIP) analysis of SPRED1-ERK interaction in NHEK cells transfected with pLVX-Flag-spred1. (B) qRT-PCR analysis of ERK mRNA expression in five experimental groups treated with ERK agonist Ro67-7476 or inhibitors <t>PD98059/SCH772984.</t> Statistical significance was assessed by one-way analysis of variance (ANOVA). (C and D) Western blot analysis of p -ERK protein expression. Statistical significance was assessed by one-way analysis of variance (ANOVA). (E and F) Immunofluorescence staining of p -ERK. Statistical significance was assessed by Brown-Forsythe corrected ANOVA. Scale bars, 20 μm. (G and H) CCK-8 assays for keratinocyte viability in five groups. Statistical significance was assessed by one-way ANOVA followed by Bonferroni correction. (I and J) EdU staining for keratinocyte proliferation. Statistical significance was assessed by one-way analysis of variance (ANOVA). Scale bars, 200 μm. (K) qRT-PCR analysis of PCNA mRNA expression. Statistical significance was assessed by one-way analysis of variance (ANOVA). (L and M) Western blot analysis of PCNA protein expression. Statistical significance was assessed by one-way analysis of variance (ANOVA). (N and O) Immunofluorescence staining of PCNA. Statistical significance was assessed by Brown-Forsythe corrected ANOVA. Scale bars, 40 μm. All experiments were performed in triplicate. N = 6 for all. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.
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(A) Relative mRNA expression of IGFBP3 and IGFBP5 in control, PAE-L, and PAE-M newborn mammary tissues determined by quantitative RT-PCR. (B) Representative Western blots of selected insulin/IGF- and growth-factor-associated signaling proteins, including p-IRS1(T895), p-IRS1(S612), IRS1, EGFR, ErbB3, IGF1R, p-Akt1(S473), Akt1, p-ERK1/2, ERK2, ERα, Sox2, and IGFBP5. (C) Densitometric analysis of Western blot data. Phosphorylated proteins were normalized to their corresponding total proteins, and total protein levels were normalized to β-Actin, as appropriate.

Journal: bioRxiv

Article Title: Transcriptomic Analysis of the Newborn Mammary Gland Identifies Prenatal Ethanol-Induced Remodeling of Insulin Signaling and Enhanced Proliferative Programming

doi: 10.64898/2026.09.22.753596

Figure Lengend Snippet: (A) Relative mRNA expression of IGFBP3 and IGFBP5 in control, PAE-L, and PAE-M newborn mammary tissues determined by quantitative RT-PCR. (B) Representative Western blots of selected insulin/IGF- and growth-factor-associated signaling proteins, including p-IRS1(T895), p-IRS1(S612), IRS1, EGFR, ErbB3, IGF1R, p-Akt1(S473), Akt1, p-ERK1/2, ERK2, ERα, Sox2, and IGFBP5. (C) Densitometric analysis of Western blot data. Phosphorylated proteins were normalized to their corresponding total proteins, and total protein levels were normalized to β-Actin, as appropriate.

Article Snippet: Proteins examined included IRS1(#3407), phospho-IRS1 (Ser612) (#3203), phospho-IRS1 (Tyr895) (#3070), phospho-Akt1 (Ser473) (#4060), ErbB3 (#12708), phospho-ERK1/2 (Thr202/Tyr204) (#9101) were purchased from Cell signaling Technology, EGFR (sc-03), IGF1R (sc-712), Akt1 (sc-5298), Erk2 (sc-1647), ERα (sc-8002), Sox2 (sc-398254), IGFBP5 (sc-515116) and β-Actin (sc-47778) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot, Analysis

SPRED1 regulates NHEK cell proliferation via indirect interaction with ERK (A) Co-immunoprecipitation (CoIP) analysis of SPRED1-ERK interaction in NHEK cells transfected with pLVX-Flag-spred1. (B) qRT-PCR analysis of ERK mRNA expression in five experimental groups treated with ERK agonist Ro67-7476 or inhibitors PD98059/SCH772984. Statistical significance was assessed by one-way analysis of variance (ANOVA). (C and D) Western blot analysis of p -ERK protein expression. Statistical significance was assessed by one-way analysis of variance (ANOVA). (E and F) Immunofluorescence staining of p -ERK. Statistical significance was assessed by Brown-Forsythe corrected ANOVA. Scale bars, 20 μm. (G and H) CCK-8 assays for keratinocyte viability in five groups. Statistical significance was assessed by one-way ANOVA followed by Bonferroni correction. (I and J) EdU staining for keratinocyte proliferation. Statistical significance was assessed by one-way analysis of variance (ANOVA). Scale bars, 200 μm. (K) qRT-PCR analysis of PCNA mRNA expression. Statistical significance was assessed by one-way analysis of variance (ANOVA). (L and M) Western blot analysis of PCNA protein expression. Statistical significance was assessed by one-way analysis of variance (ANOVA). (N and O) Immunofluorescence staining of PCNA. Statistical significance was assessed by Brown-Forsythe corrected ANOVA. Scale bars, 40 μm. All experiments were performed in triplicate. N = 6 for all. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Journal: iScience

Article Title: Downregulation of SPRED1 drives keratinocyte hyperproliferation in psoriasis via activation of the ERK-RSK1 signaling axis

doi: 10.1016/j.isci.2026.116812

Figure Lengend Snippet: SPRED1 regulates NHEK cell proliferation via indirect interaction with ERK (A) Co-immunoprecipitation (CoIP) analysis of SPRED1-ERK interaction in NHEK cells transfected with pLVX-Flag-spred1. (B) qRT-PCR analysis of ERK mRNA expression in five experimental groups treated with ERK agonist Ro67-7476 or inhibitors PD98059/SCH772984. Statistical significance was assessed by one-way analysis of variance (ANOVA). (C and D) Western blot analysis of p -ERK protein expression. Statistical significance was assessed by one-way analysis of variance (ANOVA). (E and F) Immunofluorescence staining of p -ERK. Statistical significance was assessed by Brown-Forsythe corrected ANOVA. Scale bars, 20 μm. (G and H) CCK-8 assays for keratinocyte viability in five groups. Statistical significance was assessed by one-way ANOVA followed by Bonferroni correction. (I and J) EdU staining for keratinocyte proliferation. Statistical significance was assessed by one-way analysis of variance (ANOVA). Scale bars, 200 μm. (K) qRT-PCR analysis of PCNA mRNA expression. Statistical significance was assessed by one-way analysis of variance (ANOVA). (L and M) Western blot analysis of PCNA protein expression. Statistical significance was assessed by one-way analysis of variance (ANOVA). (N and O) Immunofluorescence staining of PCNA. Statistical significance was assessed by Brown-Forsythe corrected ANOVA. Scale bars, 40 μm. All experiments were performed in triplicate. N = 6 for all. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: ERK inhibitor PD98059 , MCE , Cat No. HY-12028.

Techniques: Immunoprecipitation, Transfection, Quantitative RT-PCR, Expressing, Western Blot, Immunofluorescence, Staining, CCK-8 Assay