- MA1-23242 - Invitrogen Antibodies ID3 antibody

Submitted references Angiopoietin-like 4 induces a β-catenin-mediated upregulation of ID3 in fibroblasts to reduce scar collagen expression.

Teo Z, Chan JSK, Chong HC, Sng MK, Choo CC, Phua GZM, Teo DJR, Zhu P, Choong C, Wong MTC, Tan NS
Scientific reports 2017 Jul 24;7(1):6303

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot analysis of ID3 was performed by separating 30 µg of non-transfected (–) and transfected (+) 293T whole cell extracts by 15% SDS-PAGE. Proteins were transferred to a membrane and probed with a ID3 Monoclonal Antibody (2B11) (Product # MA1-23242) at a dilution of 1:1000. The HRP-conjugated anti-mouse IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunocytochemistry-Immunofluorescence analysis of ID3 was performed in HeLa cells fixed in 4% paraformaldehyde at RT for 15 min. Green: ID3 Monoclonal Antibody (2B11) (Product # MA1-23242) diluted at 1:100. Blue: Hoechst 33342 staining. Scale bar = 10 µm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 cANGPTL4 regulates COL1A2 and COL3A1 expression in fibroblasts via a bHLH-dependent signaling mechanism. ( a ) Immunoblot analysis of indicated proteins from fibroblasts treated with cANGPTL4 (12 ug/mL) in the presence or absence of TGFbeta1 (10 ng/mL). beta-tubulin, as loading control, was from the same samples. ( b,d ) Chromatin immunoprecipitation (ChIP) of SBE sites in the human COL1A2 gene promoter region and E-box sites in the human COL1A2 and COL3A1 gene promoter regions using phospho-Smad3 antibodies and scleraxis (ScxA) antibodies respectively. The gene sequences spanning the SBE and E-box sites and a random control sequence were analyzed by PCR from immunoprecipitated chromatin from fibroblasts treated with TGFbeta1 in the presence or absence of cANGPTL4 (cANG). Pre-immune serum was used as a control. PCR was performed using chromatin before immunoprecipitation as input. M, 100 bp DNA ladder. ( c ) A schematic diagram showing the relative positions of Enhancer boxes (E-Boxes) and Smad binding elements (SBE) on the human COL1A2 and COL3A1 proximal promoters. Sequences of E-Boxes and SBE are shown. ChIP PCR primers designed specifically to bind scleraxis along this proximal promoter region are indicated. ( e ) Relative mRNA expression of ID3 in fibroblasts treated with 12 ug/mL of cANGPTL4 and lysed for mRNA at the indicated time points post-treatment. ( f ) Relative mRNA expression of ID3 in control (F ctrl ) and ID3-knockdown (F ID3 ) fibroblasts treated fo

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 cANGPTL4 upregulates ID3 via a beta-catenin-dependent pathway. ( a ) Immunoblot of full-length cadherin-11 (CDH11) and splice variant CDH11 (CDH11 var ) in human fibroblasts. Fibroblasts were transfected with empty vector (F ctrl ) or expression vector containing cDNA encoding CDH11 var (F CDH11var ). beta-tubulin, as loading control, was from the same samples. Relative mRNA expression of ID3 in fibroblasts subjected to the indicated treatments. ( b-e ) Immunodetection and quantification of ID3 or beta-catenin proteins in fibroblasts subjected to the indicated treatments. beta-tubulin (cytosol) and c-JUN (nuclear) from the same samples were used as loading controls. Immunoblot images are cropped; full-length immunoblots can be found in Supplementary Fig. S4h-n . ( f , g ) Relative mRNA and protein expression of ID3 in fibroblasts treated with ICG001 and XAV939, two inhibitors of beta-catenin downstream signaling. Ribosomal protein L27 was used as housekeeping control for qPCR. beta-tubulin from the same samples was used as loading control for immunoblots. Values represent mean +- SD, n = 5 independent experiments. n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001 (Mann-Whitney U-test). ( h ) ChIP of TBE on the human ID3 gene using anti-beta-catenin antibody followed by re-ChIP with anti-LEF antibody. Gene sequence spanning the TBE II/III and a random control sequence were analyzed by PCR in the immunoprecipitated chromatin of fibroblasts subjected to the ind

MA1-23242

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