primary antibodies against ifitm1  (Cell Signaling Technology Inc)

Journal: Cancers

Article Title: Enhanced IFNα Signaling Promotes Ligand-Independent Activation of ERα to Promote Aromatase Inhibitor Resistance in Breast Cancer

doi: 10.3390/cancers13205130

Figure Lengend Snippet: Inhibition of ERα directly affects IFITM1, a downstream target of IFNα signaling. MCF-7 and MCF-7:5C cells were transiently transfected for 48 h with siRNA against ERα and ( A ) immunoblotted for the proteins indicated; ( B ) analyzed by RT-PCR. ( C ) MCF-7:5C cells were transiently transfected for the IFITM1 promoter construct and siRNA against ERα for 48 h. ( D ) MCF-7 and MCF-7:5C cells were transfected with the ISRE reporter construct and siRNA against ERα for 48 h. Luciferase activity was read. ** p < 0.05 *** p < 0.001.

Article Snippet: Following fixing and permeabilization, cells were stained using primary antibodies against anti-IFITM1 (Santa Cruz Biotechnology, Cat#SC-374026) and anti-ERα (Santa Cruz Biotechnology, Cat#SC-544) followed by secondary antibodies, and mounted as previously described [ ].

Techniques: Inhibition, Transfection, Reverse Transcription Polymerase Chain Reaction, Construct, Luciferase, Activity Assay

Journal: Cancers

Article Title: Enhanced IFNα Signaling Promotes Ligand-Independent Activation of ERα to Promote Aromatase Inhibitor Resistance in Breast Cancer

doi: 10.3390/cancers13205130

Figure Lengend Snippet: ERα and STAT1 regulate IFITM1 through binding to ERE and ISRE elements in the promoter. ( A ) ChIP data from Tam-resistant breast cancer cells from the UCSC Genome Browser were analyzed for potential ERα binding sites. ( B ) Chromatin immunoprecipitation (ChIP) with antibodies against ERα, STAT1 or species-specific IgG control was performed and analyzed by qPCR and DNA gels on the isolated DNA using primers designed to amplify the ERE and ISRE regulatory regions. Recruitment of the indicated proteins to the ERE and ISRE site was compared to input DNA and displayed as mean ± SD of technical triplicates in two independent experiments. * p < 0.5, ** p < 0.05 and *** p < 0.001.

Article Snippet: Following fixing and permeabilization, cells were stained using primary antibodies against anti-IFITM1 (Santa Cruz Biotechnology, Cat#SC-374026) and anti-ERα (Santa Cruz Biotechnology, Cat#SC-544) followed by secondary antibodies, and mounted as previously described [ ].

Techniques: Binding Assay, Chromatin Immunoprecipitation, Control, Isolation

Journal: Cancers

Article Title: Enhanced IFNα Signaling Promotes Ligand-Independent Activation of ERα to Promote Aromatase Inhibitor Resistance in Breast Cancer

doi: 10.3390/cancers13205130

Figure Lengend Snippet: E 2 treatment inhibits IFITM1 expression and blocks ERα and STAT1 recruitment to the IFITM1 promoter. MCF-7:5C cells were treated for 48 h with E 2 and ( A ) immunoblotted for ERα, p-STAT1, STAT1, and IFITM1 expression; ( B ) analyzed by RT-PCR. ( C ) Chromatin immunoprecipitation (ChIP) with antibodies against ERα, STAT1 or species-specific IgG control was performed and analyzed by qPCR and DNA gels on the isolated DNA using primers designed to amplify the ERE and ISRE regulatory regions. Recruitment of the indicated proteins to the ERE and ISRE site was compared to input DNA and displayed as mean ± SD of technical triplicates in two independent experiments. * p < 0.5, ** p < 0.05 and *** p < 0.001.

Article Snippet: Following fixing and permeabilization, cells were stained using primary antibodies against anti-IFITM1 (Santa Cruz Biotechnology, Cat#SC-374026) and anti-ERα (Santa Cruz Biotechnology, Cat#SC-544) followed by secondary antibodies, and mounted as previously described [ ].

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, Control, Isolation

Journal: Cancers

Article Title: Enhanced IFNα Signaling Promotes Ligand-Independent Activation of ERα to Promote Aromatase Inhibitor Resistance in Breast Cancer

doi: 10.3390/cancers13205130

Figure Lengend Snippet: Proposed mechanism of enhanced IFNα signaling on ligand-independent expression of ERα in driving AI resistance and IFITM1 expression. Enhanced IFNα signaling seen in the AI-resistant MCF-7:5C cells upregulates JAK/STAT signaling and expression of not only ISGs but also ERα. This enhanced signaling also promotes ligand-independent activation of ERα through phosphorylation of the S167 residue. STAT1 and ERα then function as co-activators of not only ER-regulated genes but also of IFITM1 by binding directly to its promoter which increases survival signaling in AI-resistant cells. (Figure created with Biorender.com.)

Article Snippet: Following fixing and permeabilization, cells were stained using primary antibodies against anti-IFITM1 (Santa Cruz Biotechnology, Cat#SC-374026) and anti-ERα (Santa Cruz Biotechnology, Cat#SC-544) followed by secondary antibodies, and mounted as previously described [ ].

Techniques: Expressing, Activation Assay, Phospho-proteomics, Residue, Binding Assay