Journal: The Journal of Biological Chemistry

Article Title: Myristoylated methionine sulfoxide reductase A is a late endosomal protein

doi: 10.1074/jbc.RA117.000473

Figure Lengend Snippet: Myristoylated MSRA is anchored to the membrane of late endosomes. A and C, COS7 cells were transiently transfected with three constructs, Stard3-FLAG, mRFP-Rab7, a late endosomal protein, and either (A) myristoylated MsrA-gfp or (C) nonmyristoylated MsrA-gfp. After 8 h, cells were treated with 20 mm NH4Cl for 16 h to alkalinize and enlarge late endosomes and lysosomes. The images within the white box of the upper panels are magnified in the lower panels. Scale bar, 10 μm. B and D, the merged images were scanned along the white arrow for relative fluorescence intensity with ZEN 2 software. The black arrows indicate the direction of scanning.

Article Snippet: The Met-307 and M307R/N311D mutants of the START domain were generated with the QuikChange kit. pDsRed2-Mito was purchased from Clontech (number 6975-1). mRFP-RAB7 was a gift from Ari Helenius (Addgene plasmid number 14436).

Techniques: Transfection, Construct, Fluorescence, Software

Journal: The Journal of Biological Chemistry

Article Title: Myristoylated methionine sulfoxide reductase A is a late endosomal protein

doi: 10.1074/jbc.RA117.000473

Figure Lengend Snippet: Subcellular fractionation demonstrates that myristoylated MSRA is enriched in the late endosome fraction. A, myristoylated MSRA is most prominent in fraction 1 from wildtype (WT) mouse liver and is not present in fractions prepared from the MSRA knockout liver (KO). Fraction 1 also contains most of the intact late endosomes. Each fraction was evaluated by immunoblot with these marker proteins: RAB7 (late endosome), mtHSP70 (mitochondria), myristoylated MSRA, total MSRA, and STARD3. The asterisk (*) indicates a nonspecific band. The molecular size markers of protein are indicated in kDa. The specificity of the mAb for the myristoylated form of MSRA is shown in panel B. 100 ng of purified, recombinant human myristoylated or nonmyristoylated MSRA were subjected to SDS-PAGE followed by immunoblotting by either the anti-myristoylated MSRA antibody or the general anti-MSRA antibody. As indicated under “Experimental procedures,” the concentration of protein was determined spectrophotometrically. However, to assure that equal amounts of the two proteins were loaded for immunoblotting, we also subjected 1 μg of each protein SDS-PAGE followed by staining with Coomassie Brilliant Blue. C, the anti-myristoylated MSRA and general anti-MSRA antibodies are specific for MSRA. This was demonstrated by immunoblotting homogenates of liver and kidney from WT and MSRA knockout mice. The asterisk (*) marks nonspecific bands detected by the general anti-MSRA antibody. Tubulin served as a loading control.

Article Snippet: The Met-307 and M307R/N311D mutants of the START domain were generated with the QuikChange kit. pDsRed2-Mito was purchased from Clontech (number 6975-1). mRFP-RAB7 was a gift from Ari Helenius (Addgene plasmid number 14436).

Techniques: Fractionation, Knock-Out, Western Blot, Marker, Purification, Recombinant, SDS Page, Concentration Assay, Staining