Anti-SEL1L (internal region) polyclonal antibody

Hrd1 is the core structural component of a large endoplasmic reticulum membrane-embedded protein complex that coordinates the destruction of folding-defective proteins in the early secretory pathway. Defining the composition, dynamics, and ultimately, the structure of the Hrd1 complex is a crucial step in understanding the molecular basis of glycoprotein quality control but has been hampered by the lack of suitable techniques to interrogate this complex under native conditions. In this study we used genome editing to generate clonal HEK293 (Hrd1. KI) cells harboring a homozygous insertion of a small tandem affinity tag knocked into the endogenous Hrd1 locus. We found that steady-state levels of tagged Hrd1 in these cells are indistinguishable from those of Hrd1 in unmodified cells and that the tagged variant is functional in supporting the degradation of well characterized luminal and membrane substrates. Analysis of detergent-solubilized Hrd1. KI cells indicates that the composition and stoichiometry of Hrd1 complexes are strongly influenced by Hrd1 expression levels. Analysis of affinity-captured Hrd1 complexes from these cells by size-exclusion chromatography, immunodepletion, and absolute quantification mass spectrometry identified two major high-molecular-mass complexes with distinct sets of interacting proteins and variable stoichiometries, suggesting a hitherto unrecognized heterogeneity in the functional units of Hrd1-mediated protein degradation.

SEL1L, the human orthologue of the Caenorhabditis elegans sel-1 gene, is differentially expressed in breast primary tumors and in normal breast tissues. Analysis of a series of human primary breast carcinomas, using a monoclonal antibody raised against a SEL1L recombinant protein, revealed down-modulation or absence of SEL1L expression in about two-thirds of the tumors as compared with normal breast epithelial cells. Overall survival analysis of breast carcinoma patients indicated a statistically significant correlation between SEL1L down-modulation and poor prognosis. MCF-7, human breast carcinoma cells, were transfected with a construct containing the entire SEL1L cDNA driven by an inducible promoter and showed a dramatic reduction in anchorage-dependent growth and colony formation in soft agar. Growth of the transfected cells in Matrigel, an extracellular matrix rich with laminin, restored colony-formation ability. These results point to the role for SEL1L in breast tumor growth and aggressiveness, possibly involving cell-matrix interactions.