Journal: Molecular Biology of the Cell
Article Title: p38 MAP kinase–dependent phosphorylation of the Gp78 E3 ubiquitin ligase controls ER–mitochondria association and mitochondria motility
Figure Lengend Snippet: 3F3A mAb, but not anti-Gp78C, selectively recognizes dephosphorylated Gp78. (A) Cos7 cells were untransfected (Cos7 NT) or transfected with empty vector (PC3 EV), FLAG-tagged wild type (WT), S538A, phosphomimetic S538D or S538E, D536A mutant, or amino acid 533–541 deletion mutant (Δ3F3A) of Gp78. Cell lysates were immunoprecipitated with FLAG beads and probed with 3F3A, Gp78C, or FLAG mAbs. (B) Bacteria transformed with GST-tagged empty vector (GST-EV) or Gp78 C-terminal (GST-Gp78C) wild type (WT), S538A, S538D, S538E mutants, or amino acid 533–541 deletion mutant (Δ3F3A) were uninduced or induced by IPTG. Total bacterial lysates were dot blotted onto membranes and probed with 3F3A, Gp78C, or GST mAbs. (C) FLAG-tagged wild type of Gp78 (FLAG-Gp78-WT) or phosphorylation dominant-negative mutant (FLAG-Gp78 S538A) from Cos7 cells were immunoprecipitated and treated or not with CIAP and probed with 3F3A mAb, Gp78C, or FLAG mAb (±SEM; n = 3).
Article Snippet: GST-Gp78C protein expression was induced by 1 mM isopropyl-β-d -thiogalactoside (IPTG) at 37°C for 4 h. pE-SUMO His-Gp78 was purchased from LifeSensors and the His-hGp78C induced with 0.4 mM IPTG at 16°C overnight and purified with His60 Ni Superflow Resin (Clontech). pcDNA3-Flag dominant-negative MKK6(K82A) and constitutively active MKK6 (Glu) were gifts from Roger Davis (University of Massachusetts, Worcester, MA; Addgene plasmids 13519 and 13518; ).
Techniques: Transfection, Plasmid Preparation, Mutagenesis, Immunoprecipitation, Transformation Assay, Dominant Negative Mutation