lightcycler probe design2 software Search Results


cdna  (Addgene inc)
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Addgene inc cdna
<t>(A)</t> <t>HEK-293T</t> cells were transfected with pcDNA3 empty vector or plasmids coding for S-OGA or L-OGA. S-OGA and L-OGA in total cell lysates were detected as bands of apparent molecular weight of 95 kDa and 130 kDa by western-blotting using anti-OGA Novus antibody. (B) HEK-293T cells were transfected with plasmids coding for GFP-tagged S-OGA or L-OGA. GFP-S-OGA and GFP-L-OGA (left panel) were detected as bands of apparent molecular weights of 130 kDa and 160 kDa by western-blotting with an anti-GFP antibody. In mitochondria-enriched fractions from these cells, recovery of transfected GFP-S-OGA was higher than GFP-L-OGA, whereas GFP-L-OGA recovery was higher in the cytosolic enriched fraction. Mitochondrial and cytosol fractions were controlled using anti-ATP5A and anti-GAPDH antibodies. (C) Densitometric analysis of the 130 kDa and 160 kDa GFP-OGA bands in HEK-293T cells. The results are the mean ± SEM of the ratio of the 130 kDa to 160 kDa GFP-OGA bands detected in cytosol- and mitochondria-enriched fractions (n=3; *: p<0.05). (D) MEF were transfected with <t>cDNA</t> coding for either GFP alone, GFP-S-OGA (130 kDa) or GFP-L-OGA (160 kDa). (A) Total cell lysate (TCL), mitochondria (Mito) and cytosolic (Cyto) enriched fractions from these cells were submitted to western-blotting using an anti-GFP antibody. Mitochondrial and cytosol enrichment was controlled using anti-ATP5A and anti-α-tubulin antibodies. In mitochondria-enriched fractions, recovery of transfected GFP-S-OGA was higher than GFP-L-OGA, whereas GFP-L-OGA recovery was higher in the cytosolic enriched fraction. (E) Densitometric analysis of the 130 kDa and 160 kDa GFP-OGA bands in HEK-293T cells. The results are the mean ± SEM of the ratio of the 130 kDa to 160 kDa GFP-OGA signals detected in TCL, cytosol- and mitochondria-enriched fractions (n=3; *: p<0.05).
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(A) HEK-293T cells were transfected with pcDNA3 empty vector or plasmids coding for S-OGA or L-OGA. S-OGA and L-OGA in total cell lysates were detected as bands of apparent molecular weight of 95 kDa and 130 kDa by western-blotting using anti-OGA Novus antibody. (B) HEK-293T cells were transfected with plasmids coding for GFP-tagged S-OGA or L-OGA. GFP-S-OGA and GFP-L-OGA (left panel) were detected as bands of apparent molecular weights of 130 kDa and 160 kDa by western-blotting with an anti-GFP antibody. In mitochondria-enriched fractions from these cells, recovery of transfected GFP-S-OGA was higher than GFP-L-OGA, whereas GFP-L-OGA recovery was higher in the cytosolic enriched fraction. Mitochondrial and cytosol fractions were controlled using anti-ATP5A and anti-GAPDH antibodies. (C) Densitometric analysis of the 130 kDa and 160 kDa GFP-OGA bands in HEK-293T cells. The results are the mean ± SEM of the ratio of the 130 kDa to 160 kDa GFP-OGA bands detected in cytosol- and mitochondria-enriched fractions (n=3; *: p<0.05). (D) MEF were transfected with cDNA coding for either GFP alone, GFP-S-OGA (130 kDa) or GFP-L-OGA (160 kDa). (A) Total cell lysate (TCL), mitochondria (Mito) and cytosolic (Cyto) enriched fractions from these cells were submitted to western-blotting using an anti-GFP antibody. Mitochondrial and cytosol enrichment was controlled using anti-ATP5A and anti-α-tubulin antibodies. In mitochondria-enriched fractions, recovery of transfected GFP-S-OGA was higher than GFP-L-OGA, whereas GFP-L-OGA recovery was higher in the cytosolic enriched fraction. (E) Densitometric analysis of the 130 kDa and 160 kDa GFP-OGA bands in HEK-293T cells. The results are the mean ± SEM of the ratio of the 130 kDa to 160 kDa GFP-OGA signals detected in TCL, cytosol- and mitochondria-enriched fractions (n=3; *: p<0.05).

Journal: bioRxiv

Article Title: Short OGA is targeted to the mitochondria and regulates mitochondrial reactive oxygen species level

doi: 10.1101/2021.12.25.474160

Figure Lengend Snippet: (A) HEK-293T cells were transfected with pcDNA3 empty vector or plasmids coding for S-OGA or L-OGA. S-OGA and L-OGA in total cell lysates were detected as bands of apparent molecular weight of 95 kDa and 130 kDa by western-blotting using anti-OGA Novus antibody. (B) HEK-293T cells were transfected with plasmids coding for GFP-tagged S-OGA or L-OGA. GFP-S-OGA and GFP-L-OGA (left panel) were detected as bands of apparent molecular weights of 130 kDa and 160 kDa by western-blotting with an anti-GFP antibody. In mitochondria-enriched fractions from these cells, recovery of transfected GFP-S-OGA was higher than GFP-L-OGA, whereas GFP-L-OGA recovery was higher in the cytosolic enriched fraction. Mitochondrial and cytosol fractions were controlled using anti-ATP5A and anti-GAPDH antibodies. (C) Densitometric analysis of the 130 kDa and 160 kDa GFP-OGA bands in HEK-293T cells. The results are the mean ± SEM of the ratio of the 130 kDa to 160 kDa GFP-OGA bands detected in cytosol- and mitochondria-enriched fractions (n=3; *: p<0.05). (D) MEF were transfected with cDNA coding for either GFP alone, GFP-S-OGA (130 kDa) or GFP-L-OGA (160 kDa). (A) Total cell lysate (TCL), mitochondria (Mito) and cytosolic (Cyto) enriched fractions from these cells were submitted to western-blotting using an anti-GFP antibody. Mitochondrial and cytosol enrichment was controlled using anti-ATP5A and anti-α-tubulin antibodies. In mitochondria-enriched fractions, recovery of transfected GFP-S-OGA was higher than GFP-L-OGA, whereas GFP-L-OGA recovery was higher in the cytosolic enriched fraction. (E) Densitometric analysis of the 130 kDa and 160 kDa GFP-OGA bands in HEK-293T cells. The results are the mean ± SEM of the ratio of the 130 kDa to 160 kDa GFP-OGA signals detected in TCL, cytosol- and mitochondria-enriched fractions (n=3; *: p<0.05).

Article Snippet: HEK-293T cells plated on polylysine-coated coverslips were transfected with cDNA (100 ng/40000 cells) coding for mitochondrial-targeted mCherry (mCherry-Mito-7, a gift from Michael Davidson (Addgene plasmid # 55102), ( Olenych et al , 2007 )) and either GFP-tagged long or short OGA isoforms (gifts from John A. Hanover).

Techniques: Transfection, Plasmid Preparation, Molecular Weight, Western Blot

(A) HEK-293T cells were transfected with cDNA coding for either GFP or GFP fused at its C-terminus with OGA intron-derived peptide (GFP-IdP). Total cell lysate, mitochondrial and cytosol enriched fractions were analysed by western-blotting with anti-GFP antibody. GFP-IdP was more abundant in the mitochondrial-enriched fraction whereas GFP was more abundant in the cytosol enriched fraction. (B) Densitometric analysis of the GFP and GFP-IdP bands. The results are the mean ± SEM of the ratio of the GFP-IdP to GFP signals detected in cytosol- and mitochondria-enriched fractions (n=5; *: p<0.05). (C) HEK-293T cells and HeLa cells were transfected with cDNA coding GFP fused at its C-terminus with OGA intron-derived peptide (GFP-IdP), fixed and labelled with mitotracker. Confocal microscopy experiments confirmed co-localisation of GFP-IdP with mitotracker. GFP alone was widely distributed into the cells, including the nucleus (HeLa cells).

Journal: bioRxiv

Article Title: Short OGA is targeted to the mitochondria and regulates mitochondrial reactive oxygen species level

doi: 10.1101/2021.12.25.474160

Figure Lengend Snippet: (A) HEK-293T cells were transfected with cDNA coding for either GFP or GFP fused at its C-terminus with OGA intron-derived peptide (GFP-IdP). Total cell lysate, mitochondrial and cytosol enriched fractions were analysed by western-blotting with anti-GFP antibody. GFP-IdP was more abundant in the mitochondrial-enriched fraction whereas GFP was more abundant in the cytosol enriched fraction. (B) Densitometric analysis of the GFP and GFP-IdP bands. The results are the mean ± SEM of the ratio of the GFP-IdP to GFP signals detected in cytosol- and mitochondria-enriched fractions (n=5; *: p<0.05). (C) HEK-293T cells and HeLa cells were transfected with cDNA coding GFP fused at its C-terminus with OGA intron-derived peptide (GFP-IdP), fixed and labelled with mitotracker. Confocal microscopy experiments confirmed co-localisation of GFP-IdP with mitotracker. GFP alone was widely distributed into the cells, including the nucleus (HeLa cells).

Article Snippet: HEK-293T cells plated on polylysine-coated coverslips were transfected with cDNA (100 ng/40000 cells) coding for mitochondrial-targeted mCherry (mCherry-Mito-7, a gift from Michael Davidson (Addgene plasmid # 55102), ( Olenych et al , 2007 )) and either GFP-tagged long or short OGA isoforms (gifts from John A. Hanover).

Techniques: Transfection, Derivative Assay, Western Blot, Confocal Microscopy