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(A) To identify downstream signalling pathways activated by HIP1 overexpression membrane A of the Human phospho-kinase array <t>(ARY003)</t> used to de-termine the relative levels of phosphorylated proteins of 46 specific kinases and substrates. Lysates were prepared from PNT1A-empty vector (EV) and PNT1A-HIP1 cell lines grown in full serum. The intensity of array spots in duplicate were analysed with ImageQUANT™ software. (B) Immunoblot of PNT1a-EV and PNT1a-HIP1 lysates for HIP1, p-STAT3(Y705) and actin. Relative levels of phosphorylation of STAT3 in PNT1a-HIP1 normalised to the control cell line after correction for equal protein loading control is displayed below blots. (C) Immunoblot of HIP1, p-STAT3(Y705) upon transient ectopic expression of GFP-HIP1 in the PNT1a-EV control for 48 hours. (D) Immunoblots of HIP1 and pSTAT3 in NIH3T3 cells transiently overexpressing HIP1. (E) Immunoblots of HIP1, p-STAT3(Y705), and p-FGFR following stable knockdown of HIP1 in PNT1a-HIP1 using shRNA compared to scrambled control. Fold changes indicated were normalised to actin. (F) WP1066 pre-treatment blocked STAT3 phosphorylation upon FGF2 stimulation in HIP1 overexpressing cells. PNT1A-HIP1 and PNT1A-EV were serum starved for 24 hours, pre-treated with DMSO or kinase inhibitors for one hour and stimulated with FGF2 (20ng/ml). Comparison of treatments with PI3K inhibitor (LY294002), Jak2 inhibitor (WP1066), MEK1/2 inhibitor (U0126), FGFR phosphorylation inhibitor (PD173074). (G) Drug treatment of PNT1A cells cultured in full serum conditions. PNT1A-EV and PNT1A-HIP1 cells cultured in full serum were treated with JAK2 kinase inhibitor (WP1066), MEK1/2 kinase inhibitor (U1026) and FGFR phosphorylation inhibitor (PD173074).
Ary003, supplied by R&D Systems, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/ary003/product/R&D Systems
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(A) To identify downstream signalling pathways activated by HIP1 overexpression membrane A of the Human phospho-kinase array <t>(ARY003)</t> used to de-termine the relative levels of phosphorylated proteins of 46 specific kinases and substrates. Lysates were prepared from PNT1A-empty vector (EV) and PNT1A-HIP1 cell lines grown in full serum. The intensity of array spots in duplicate were analysed with ImageQUANT™ software. (B) Immunoblot of PNT1a-EV and PNT1a-HIP1 lysates for HIP1, p-STAT3(Y705) and actin. Relative levels of phosphorylation of STAT3 in PNT1a-HIP1 normalised to the control cell line after correction for equal protein loading control is displayed below blots. (C) Immunoblot of HIP1, p-STAT3(Y705) upon transient ectopic expression of GFP-HIP1 in the PNT1a-EV control for 48 hours. (D) Immunoblots of HIP1 and pSTAT3 in NIH3T3 cells transiently overexpressing HIP1. (E) Immunoblots of HIP1, p-STAT3(Y705), and p-FGFR following stable knockdown of HIP1 in PNT1a-HIP1 using shRNA compared to scrambled control. Fold changes indicated were normalised to actin. (F) WP1066 pre-treatment blocked STAT3 phosphorylation upon FGF2 stimulation in HIP1 overexpressing cells. PNT1A-HIP1 and PNT1A-EV were serum starved for 24 hours, pre-treated with DMSO or kinase inhibitors for one hour and stimulated with FGF2 (20ng/ml). Comparison of treatments with PI3K inhibitor (LY294002), Jak2 inhibitor (WP1066), MEK1/2 inhibitor (U0126), FGFR phosphorylation inhibitor (PD173074). (G) Drug treatment of PNT1A cells cultured in full serum conditions. PNT1A-EV and PNT1A-HIP1 cells cultured in full serum were treated with JAK2 kinase inhibitor (WP1066), MEK1/2 kinase inhibitor (U1026) and FGFR phosphorylation inhibitor (PD173074).
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(A) To identify downstream signalling pathways activated by HIP1 overexpression membrane A of the Human phospho-kinase array <t>(ARY003)</t> used to de-termine the relative levels of phosphorylated proteins of 46 specific kinases and substrates. Lysates were prepared from PNT1A-empty vector (EV) and PNT1A-HIP1 cell lines grown in full serum. The intensity of array spots in duplicate were analysed with ImageQUANT™ software. (B) Immunoblot of PNT1a-EV and PNT1a-HIP1 lysates for HIP1, p-STAT3(Y705) and actin. Relative levels of phosphorylation of STAT3 in PNT1a-HIP1 normalised to the control cell line after correction for equal protein loading control is displayed below blots. (C) Immunoblot of HIP1, p-STAT3(Y705) upon transient ectopic expression of GFP-HIP1 in the PNT1a-EV control for 48 hours. (D) Immunoblots of HIP1 and pSTAT3 in NIH3T3 cells transiently overexpressing HIP1. (E) Immunoblots of HIP1, p-STAT3(Y705), and p-FGFR following stable knockdown of HIP1 in PNT1a-HIP1 using shRNA compared to scrambled control. Fold changes indicated were normalised to actin. (F) WP1066 pre-treatment blocked STAT3 phosphorylation upon FGF2 stimulation in HIP1 overexpressing cells. PNT1A-HIP1 and PNT1A-EV were serum starved for 24 hours, pre-treated with DMSO or kinase inhibitors for one hour and stimulated with FGF2 (20ng/ml). Comparison of treatments with PI3K inhibitor (LY294002), Jak2 inhibitor (WP1066), MEK1/2 inhibitor (U0126), FGFR phosphorylation inhibitor (PD173074). (G) Drug treatment of PNT1A cells cultured in full serum conditions. PNT1A-EV and PNT1A-HIP1 cells cultured in full serum were treated with JAK2 kinase inhibitor (WP1066), MEK1/2 kinase inhibitor (U1026) and FGFR phosphorylation inhibitor (PD173074).
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(A) To identify downstream signalling pathways activated by HIP1 overexpression membrane A of the Human phospho-kinase array <t>(ARY003)</t> used to de-termine the relative levels of phosphorylated proteins of 46 specific kinases and substrates. Lysates were prepared from PNT1A-empty vector (EV) and PNT1A-HIP1 cell lines grown in full serum. The intensity of array spots in duplicate were analysed with ImageQUANT™ software. (B) Immunoblot of PNT1a-EV and PNT1a-HIP1 lysates for HIP1, p-STAT3(Y705) and actin. Relative levels of phosphorylation of STAT3 in PNT1a-HIP1 normalised to the control cell line after correction for equal protein loading control is displayed below blots. (C) Immunoblot of HIP1, p-STAT3(Y705) upon transient ectopic expression of GFP-HIP1 in the PNT1a-EV control for 48 hours. (D) Immunoblots of HIP1 and pSTAT3 in NIH3T3 cells transiently overexpressing HIP1. (E) Immunoblots of HIP1, p-STAT3(Y705), and p-FGFR following stable knockdown of HIP1 in PNT1a-HIP1 using shRNA compared to scrambled control. Fold changes indicated were normalised to actin. (F) WP1066 pre-treatment blocked STAT3 phosphorylation upon FGF2 stimulation in HIP1 overexpressing cells. PNT1A-HIP1 and PNT1A-EV were serum starved for 24 hours, pre-treated with DMSO or kinase inhibitors for one hour and stimulated with FGF2 (20ng/ml). Comparison of treatments with PI3K inhibitor (LY294002), Jak2 inhibitor (WP1066), MEK1/2 inhibitor (U0126), FGFR phosphorylation inhibitor (PD173074). (G) Drug treatment of PNT1A cells cultured in full serum conditions. PNT1A-EV and PNT1A-HIP1 cells cultured in full serum were treated with JAK2 kinase inhibitor (WP1066), MEK1/2 kinase inhibitor (U1026) and FGFR phosphorylation inhibitor (PD173074).
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(A) To identify downstream signalling pathways activated by HIP1 overexpression membrane A of the Human phospho-kinase array (ARY003) used to de-termine the relative levels of phosphorylated proteins of 46 specific kinases and substrates. Lysates were prepared from PNT1A-empty vector (EV) and PNT1A-HIP1 cell lines grown in full serum. The intensity of array spots in duplicate were analysed with ImageQUANT™ software. (B) Immunoblot of PNT1a-EV and PNT1a-HIP1 lysates for HIP1, p-STAT3(Y705) and actin. Relative levels of phosphorylation of STAT3 in PNT1a-HIP1 normalised to the control cell line after correction for equal protein loading control is displayed below blots. (C) Immunoblot of HIP1, p-STAT3(Y705) upon transient ectopic expression of GFP-HIP1 in the PNT1a-EV control for 48 hours. (D) Immunoblots of HIP1 and pSTAT3 in NIH3T3 cells transiently overexpressing HIP1. (E) Immunoblots of HIP1, p-STAT3(Y705), and p-FGFR following stable knockdown of HIP1 in PNT1a-HIP1 using shRNA compared to scrambled control. Fold changes indicated were normalised to actin. (F) WP1066 pre-treatment blocked STAT3 phosphorylation upon FGF2 stimulation in HIP1 overexpressing cells. PNT1A-HIP1 and PNT1A-EV were serum starved for 24 hours, pre-treated with DMSO or kinase inhibitors for one hour and stimulated with FGF2 (20ng/ml). Comparison of treatments with PI3K inhibitor (LY294002), Jak2 inhibitor (WP1066), MEK1/2 inhibitor (U0126), FGFR phosphorylation inhibitor (PD173074). (G) Drug treatment of PNT1A cells cultured in full serum conditions. PNT1A-EV and PNT1A-HIP1 cells cultured in full serum were treated with JAK2 kinase inhibitor (WP1066), MEK1/2 kinase inhibitor (U1026) and FGFR phosphorylation inhibitor (PD173074).

Journal: bioRxiv

Article Title: HIP1 mediates oncogenic transformation and cancer progression through STAT3 signalling

doi: 10.1101/2020.07.09.191734

Figure Lengend Snippet: (A) To identify downstream signalling pathways activated by HIP1 overexpression membrane A of the Human phospho-kinase array (ARY003) used to de-termine the relative levels of phosphorylated proteins of 46 specific kinases and substrates. Lysates were prepared from PNT1A-empty vector (EV) and PNT1A-HIP1 cell lines grown in full serum. The intensity of array spots in duplicate were analysed with ImageQUANT™ software. (B) Immunoblot of PNT1a-EV and PNT1a-HIP1 lysates for HIP1, p-STAT3(Y705) and actin. Relative levels of phosphorylation of STAT3 in PNT1a-HIP1 normalised to the control cell line after correction for equal protein loading control is displayed below blots. (C) Immunoblot of HIP1, p-STAT3(Y705) upon transient ectopic expression of GFP-HIP1 in the PNT1a-EV control for 48 hours. (D) Immunoblots of HIP1 and pSTAT3 in NIH3T3 cells transiently overexpressing HIP1. (E) Immunoblots of HIP1, p-STAT3(Y705), and p-FGFR following stable knockdown of HIP1 in PNT1a-HIP1 using shRNA compared to scrambled control. Fold changes indicated were normalised to actin. (F) WP1066 pre-treatment blocked STAT3 phosphorylation upon FGF2 stimulation in HIP1 overexpressing cells. PNT1A-HIP1 and PNT1A-EV were serum starved for 24 hours, pre-treated with DMSO or kinase inhibitors for one hour and stimulated with FGF2 (20ng/ml). Comparison of treatments with PI3K inhibitor (LY294002), Jak2 inhibitor (WP1066), MEK1/2 inhibitor (U0126), FGFR phosphorylation inhibitor (PD173074). (G) Drug treatment of PNT1A cells cultured in full serum conditions. PNT1A-EV and PNT1A-HIP1 cells cultured in full serum were treated with JAK2 kinase inhibitor (WP1066), MEK1/2 kinase inhibitor (U1026) and FGFR phosphorylation inhibitor (PD173074).

Article Snippet: The Prosphoproteome array was used according to the manufacturer’s instructions (R&D systems, # ARY003) with 400μg of protein used per membrane.

Techniques: Over Expression, Membrane, Plasmid Preparation, Software, Western Blot, Expressing, shRNA, Comparison, Cell Culture