pcmv6 gfp msra fl  (OriGene)

Journal: Drug metabolism and disposition: the biological fate of chemicals

Article Title: Methionine Sulfoxide Reductase A in Human and Mouse Tissues is Responsible for Sulindac Activation, Making a Larger Contribution than the Gut Microbiota.

doi: 10.1124/dmd.122.000828

Figure Lengend Snippet: Fig. 4. Contribution of MSRA to sulindac reductase activity in human tissue cytosol. (A) Relative MSRA protein abundance in HIC, HLC, HRC, and purified recom- binant human MSRA. HIC, HLC, and HRC (20 lg) and purified recombinant human MSRA (0.035 lg) was separated by electrophoresis using a 15% SDS-polyacryl- amide gel. (B) The amount of sulindac sulfide produced by purified recombinant human MSRA in the absence and presence of tissue cytosols. Human tissue cytosols (0.25 mg/ml) and purified recombinant human MSRA (0.75 mg/ml) were incubated with 20 mM of sulindac for 30 minutes in the presence of 10 mM of DTT. Each column represents the mean ± SD of three experiments. (C) Effects of TXN on the sulindac reductase activity of purified recombinant human MSRA. Purified recom- binant human MSRA (0.75 mg/ml) was incubated with 20 mM sulindac for 30 minutes in the presence of 10 mM of DTT and recombinant TXN. D, E Relative TXN protein abundance (D) and activity (E) in HIC, HLC, HRC, and recombinant TXN. HIC, HLC, HRC (50 mg/ml), or recombinant human TXN (4 mg/ml) were incu- bated with 300 mM of insulin for 30 minutes in the presence of an NADPH-generating system and recombinant human TXN reductase. HIC, HLC, and HRC (30 lg) and recombinant TXN (0.4 lg) was separated by electrophoresis using a 15% SDS-polyacrylamide gel. Each column represents the mean ± SD of three experiments.

Article Snippet: Polyclonal rabbit anti-human MSRA (14547- 1-AP), TXN (sc-20146), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, NB100-56875) antibodies were purchased from Proteintech (Chicago, IL), Santa Cruz Biotechnology (Santa Cruz, CA), and IMGENEX (San Diego, CA), respectively.

Techniques: Activity Assay, Quantitative Proteomics, Purification, Recombinant, Electrophoresis, Produced, Incubation

Journal: Drug metabolism and disposition: the biological fate of chemicals

Article Title: Methionine Sulfoxide Reductase A in Human and Mouse Tissues is Responsible for Sulindac Activation, Making a Larger Contribution than the Gut Microbiota.

doi: 10.1124/dmd.122.000828

Figure Lengend Snippet: Fig. 5. Effects of MSRA knockdown on sulindac reductase activity in HepG2 cells. Relative MSRA mRNA (A) and protein (B) levels and sulindac reductase activity (C) were measured 48 hours after the transfection of 10 nM of siRNA targeting MSRA (siMSRA) or control (siControl). Cell homogenates (MSRA: 40 mg, GAPDH: 3 mg) were separated by electrophoresis using a 15% SDS-polyacrylamide gel. Each column represents the mean ± SD (n 5 4). ***P < 0.001.

Article Snippet: Polyclonal rabbit anti-human MSRA (14547- 1-AP), TXN (sc-20146), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, NB100-56875) antibodies were purchased from Proteintech (Chicago, IL), Santa Cruz Biotechnology (Santa Cruz, CA), and IMGENEX (San Diego, CA), respectively.

Techniques: Knockdown, Activity Assay, Transfection, Control, Electrophoresis

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: Neither myristoylated MSRA nor nonmyristoylated MSRA is localized to mitochondria or the endoplasmic reticulum. A and B, COS7 cells were cotransfected with either myristoylated MsrA-gfp (A) or nonmyristoylated MsrA (B) and DsRed-Mito, a mitochondrial marker protein. C and D, HeLa cells expressing either myristoylated MSRA-GFP (C) or nonmyristoylated MSRA (D) were stained with anti-KDEL, an endoplasmic reticulum marker protein. Scale bars, 20 μm.

Article Snippet: Human MSRA with a C-terminal GFP tag, pCMV6-AC-GFP-MSRA was purchased from Origene (accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_012331","term_id":"1519312561"}} NM_012331 , catalogue number RG 208916) and amplified by PCR to generate constructs for myristoylated MSRA and nonmyristoylated MSRA (G24A).

Techniques: Marker, Expressing, Staining

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: Human MSRA with a C-terminal GFP tag, pCMV6-AC-GFP-MSRA was purchased from Origene (accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_012331","term_id":"1519312561"}} NM_012331 , catalogue number RG 208916) and amplified by PCR to generate constructs for myristoylated MSRA and nonmyristoylated MSRA (G24A).

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: Knocking down STARD3 decreases MSRA on late endosomes. A, HeLa cells were transfected for 48 h with nontargeting siRNA (siCont) or 2 different Stard3 targeting oligomers (siSTARD3 #1 and siSTARD3 #2). STARD3 was quantitated by immunoblotting with anti-STARD3, with tubulin serving as a loading control. The asterisk (*) marks a nonspecific band. B, quantitation of 3 replicates of the experiment shown in panel A, run on separate days. The fluorescence intensity of the STARD3 with control siRNA was set to 1.0. The mean ± S.D. are shown as lines. Compared with the control using the paired two-tailed t test, *, p < 0.0001 and **, p < 0.0003. C, 30 h after transfection with siRNA, HeLa cells were transfected with myristoylated MsrA-gfp. 18 h later they were stained with anti-STARD3 antibody. Scale bar, 20 μm. The dashed lines outline STARD3-depleted cells. 40 such depleted cells were measured for each siRNA and the results shown in panel D. D, relative fluorescence intensity of myristoylated MSRA and STARD3 was determined using ZEN 2 software (Carl Zeiss) from control and STARD3-depleted cells. 40 cells were analyzed for each sample in 3 independent experiments performed on separate days. The mean ± S.D. are shown as lines. Compared with the control using the paired two-tailed t test; *, p < 0.0001 and **, p < 0.0002. E, colocalization of myristoylated MSRA and STARD3. 40 cells were analyzed for each sample in 3 independent experiments performed on separate days, and the Pearson's correlation coefficient determined with ImageJ software. The mean ± S.D. are shown as lines. Compared with the control siRNA using the paired two-tailed t test, *, p < 0.0001.

Article Snippet: Human MSRA with a C-terminal GFP tag, pCMV6-AC-GFP-MSRA was purchased from Origene (accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_012331","term_id":"1519312561"}} NM_012331 , catalogue number RG 208916) and amplified by PCR to generate constructs for myristoylated MSRA and nonmyristoylated MSRA (G24A).

Techniques: Transfection, Western Blot, Quantitation Assay, Fluorescence, Two Tailed Test, Staining, 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: Transfection with higher amounts of DNA causes cytosolic localization of myristoylated MSRA. A–D, COS7 cells were transfected with the indicated amount of myristoylated MsrA-gfp. After 24 h, cells were stained for the late endosomal marker, LAMP1. Scale bars, 10 μm.

Article Snippet: Human MSRA with a C-terminal GFP tag, pCMV6-AC-GFP-MSRA was purchased from Origene (accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_012331","term_id":"1519312561"}} NM_012331 , catalogue number RG 208916) and amplified by PCR to generate constructs for myristoylated MSRA and nonmyristoylated MSRA (G24A).

Techniques: Transfection, Staining, Marker