| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Synthesis | The gene from rat CRIP1a was codon-optimized for Escherichia coli by GenScript and subcloned into the NdeI-BamHI restriction sites of pET15b. C41(DE3) E. coli cells containing the expression plasmid were grown at 37 °C in LB broth containing ampicillin to an OD600 of ~0.8 with a 10L fermentor.Peptides containing the last 9 and 12 amino acids of the CB1R were synthesized by GenScript to contain a fluorescein molecule attached to the N-terminus. For pull-down assays, peptides representing the last 16 residues (149-164) of the C-terminal of CRIP1a, the last 14 residues (460-473) of the C-terminal of CB1R, or the CB1R C-terminal 14mer peptide phosphorylated at threonine 468, were each synthesized with an N-terminal cysteine residue (purity ≥ 98%) (GenScript), and immobilized onto agarose beads through irreversible thioether bonds (Pierce SulfoLink Immobilization kit). For CB1R C-terminal peptide competition co-immunoprecipitation, peptides were synthesized (GenScript) with or without phosphorylation on the underlined amino acids: Central: TAQPLDNSMGDSDCLHKH; Distal: VTMSVSTDTSAEAL. N18TG2 whole cell detergent lysates (300 μg protein) were incubated with a final concentration of 3 μM peptide in IP buffer for 1.5 h at 4oC. | Get A Quote |
Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB receptor (CBR) distal C-terminal-associated protein that alters CBR interactions with G-proteins. We tested the hypothesis that CRIP1a is capable of also altering CBR interactions with β-arrestin proteins that interact with the CBR at the C-terminus. Coimmunoprecipitation studies indicated that CBR associates in complexes with either CRIP1a or β-arrestin， but CRIP1a and β-arrestin fail to coimmunoprecipitate with each other. This suggests a competition for CRIP1a and β-arrestin binding to the CBR， which we hypothesized could attenuate the action of β-arrestin to mediate CBR internalization. We determined that agonist-mediated reduction of the... More
