Human HTRA1/PRSS11 Antibody

Detection of Human HTRA1/PRSS11 by Western Blot. Western blot shows lysates of human placenta tissue. PVDF membrane was probed with 1 µg/mL of Mouse Anti-Human HTRA1/PRSS11 Monoclonal Antibody (Catalog # MAB2916) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF007). A specific band was detected for HTRA1/PRSS11 at approximately 51 kDa (as indicated). This experiment was conducted under non-reducing conditions and using Immunoblot Buffer Group 1.

Detection of Human HTRA1/PRSS11 by Western Blot HtrA1 Protein Expression in hBC cell lines.Cells (as indicated) were extracted into nuclear (N) and cytoplasmic (C) fractions as described (Materials & Methods), and proteins were analyzed by Immunoblot analyses using the polyclonal antibody against human HtrA1. beta -actin was used as a loading control, and DEK was used to assess the nuclear/cytoplasmic fractionation (DEK is exclusively nuclear). The larger Mr bands seen in the MCF12A and MCF10A/Flp cell lines are consistent with dimers, trimers, etc., although this was not confirmed. MCF10A/Flp is the parental Flp-in cell line which was used to produce the various MCF10A/siRNA or Htra1 cell lines. Results are from a representative experiment. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/22761798), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human HTRA1/PRSS11 by Western Blot Characterization of MCF10A cell lines carrying HtrA1 siRNA and overexpression vectors.A random antisense oligonucleotide library was used to identify optimally accessible sites in HtrA1 mRNA. SiRNAs (short hairpin RNAs) were designed to target these sites, and stably transfected cell lines were developed from MCF10A cells (4 independent cell lines, designated MCF10A/siRNA1-4). A control cell line expressing an irrelevant siRNA (designated MCF10A/HPVsh) was also developed. In parallel, we also developed a cell line over-expressing HtrA1 (designated MCF10A/HtrA1). (A). Target sites empirically identified in HtrA1 mRNA. Identified domains within HtrA1 include: SS, signal sequence; IGFBP, IGF binding site; KI, Kazal Type I protease inhibitor domain; Trypsin-like protease domain; PDZ, PDZ binding domain. (B) Immunoblot analysis for HtrA1 protein. Cytoplasmic and nuclear protein fractions were prepared from the developed cell lines (as indicated), and were probed with a polyclonal antibody preparation directed against a region in the trypsin-like protease domain. As is evident, the reductions in HtrA1 protein levels were >90% compared with the various MCF10A and MCF10A/HPVsh cells. Right panel shows HtrA1 in concentrated culture medium. (C) Cells were plated and growth was measured over a 6 day period. Both of the MCF10A cell lines tested (MCF10A/siRNA1 and siRNA4) grew significantly faster than the control cells (p<0.01 at days 4 and 6). Over-expression of HtrA1 in the MCF10A/HtrA1 cells had no effect on cell growth rate. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/22761798), licensed under a CC-BY license. Not internally tested by R&D Systems.