CL200: HeLa Parkin Lysate Control Panel to Study Mitophagy

Specifications

PINK1 (PTEN-induced putative kinase protein 1) is crucial in the cellular process of protecting cells from stress-induced mitochondrial dysfunction. Healthy mitochondria have a normal membrane potential that allows PINK1 to be recruited and subsequently cleaved on the mitochondrial membrane. However, damaged mitochondria present an abnormal mitochondria membrane potential, which allows PINK1 to accumulate on the outer membrane3. The stabilized PINK1 on the outer mitochondrial membrane recruits the Parkin E3 Ligase (PARK2), phosphorylating Parkin on Ser65 of its UBL domain, partially activating it.

PINK1 also phosphorylates numerous other proteins, including ubiquitin on Ser65. Binding of pSer65-Ub to phosphorylated Parkin fully activates Parkin5. This signaling cascade is crucial in the cellular process of clearing damaged mitochondria, known as the process of mitophagy. The lysates derived from a stable Hela cells line that over expresses the PARK2 gene are an ideal cellular model for studying phosphoubiquitination of target proteins and mitophagy related pathways. CCCP (carbonyl cyanide m-chlorophenyl hydrazone) disrupts mitochondrial membrane potential and leads its depolarization and dysfunction of the mitochondria. Cells treated with CCCP treated cells can be used in phosphoubiquitination studies because it induces mitophagy and the accumulation of phosphorylated mitochondrial proteins. The lysate is available both with and without treatment with proteasome inhibitor MG-132, the mitochondrial uncoupler CCCP, and the macrolide antibiotic bafilomycin. Cell pellets were lysed using RIPA complete with proteasome, phosphatase, and DUB inhibitors.

This kit consists of HeLa Parkin Lysate treated with:

• DMSO
• CCCP 10µm
• DMSO, MG132, Bafilomycin
• CCCP, MG132, Bafilomycin.

This product is designed for use in investigating and research of the Parkin and Pink1 pathway and/or drug discovery.