PIN/DLC8 Overexpression Lysate

HEK293T cells in 10-cm dishes were transiently transfected with a non-lipid polymer transfection reagent specially designed and manufactured for large volume DNA transfection. Transfected cells were cultured for 48hrs before collection. The cells were lysed in modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, 1xProteinase inhibitor cocktail mix, 1mM PMSF and 1mM Na3VO4, and then centrifuged to clarify the lysate. Protein concentration was measured by BCA protein assay kit.

DLC8 protein bound efficiently to gephyrin in in vitro binding assays and colocalized with gephyrin during coexpression in HEK293 cells. The binding site for DLC8 was mapped to a fragment of 63 amino acids within the central linker domain of gephyrin. In hippocampal neurons, endogenous DLC8 protein was enriched at synaptic sites identified by synaptophysin and gephyrin immunostaining. Because DLC8 has been described as stoichiometric components of cytoplasmic dynein and myosin-Va complexes, results suggest that motor proteins are involved in the subcellular localization of gephyrin. DLC8 was identified as a transport molecule in the cytoplasm. DLC8 co-localizes with TRPS1 in dot-like structures in the cell nucleus. In an electrophoretic mobility shift assay it was shown that the interaction of DLC8 and TRPS1 lowers the binding of TRPS1 to the GATA consensus sequence. In addition DLC8 is able to suppress the transcriptional repression activity of TRPS1.

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Lysates are guaranteed for 6 months from date of receipt.

Array NBP2-10621

• PIN/DLC8 Antibodies
• PIN/DLC8 Lysates
• PIN/DLC8 Proteins