GRP78/HSPA5 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.

Within the ER, misfolded proteins are detected by Bip (GPR78). In the presence of misfolded proteins, GPR78 disscociates from PERK, allowing it to homodimerize and autophosphoyate. In its dimerized phosphorylated form, PERK phosphorylates eIF2. The phosphorylation of eIF2 blocks the majority of translation to prevent the continued accumulation of protein in the ER. GRP78 also binds to IRE1 and ATF6 under normal ER conditions, but dissociates upon accumulation of misfolded proteins. Unbound ATF6 is translocated to the gogli where it is converted into a cleaved, active form. The cleaved form of ATF6 is then able to up regulate transcription of UPR genes including XBP1. Activated IRE1 acts as an endoribonuclease to an intron from XBP1 transcripts. The XBP1 splice variant codes for an active transcription factor which activates transcription of P58IPK. P58IPK is a HSP40 protein which binds to and inhibits PERK. Thus, if the accumulated misfolded proteins have been removed, P58IPK acts to shut down the UPR by removing the block to translation.

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 NBP2-10471

• GRP78/HSPA5 Antibodies
• GRP78/HSPA5 ELISA Kits
• GRP78/HSPA5 Lysate
• GRP78/HSPA5 Proteins