American Society of Hematology

Figure 6.

$Figure 6. NOX5 is expressed on the outer membrane of the mitochondria and interacts with p22phox. (A) Confocal microscopy of Mo-DC, labeled with anti-NOX5 and Alexa 488, anti-LAMP-1 (lysosome marker), anti-BCL2 (outer membrane mitochondrial marker), or anti-COXIV (inner membrane mitochondrial marker) and Alexa 555, and DAPI (nuclear staining). 40× objective, zoom 6×. (B) Representative western blots of Cyto and Mito fractions isolated by differential centrifugation. Blots were incubated with anti-NOX5, anti-GAPDH (for cytosol), and anti-COXIV (for mitochondria) antibodies. (C) Mitochondria isolation in Mo-DC by magnetic anti-TOM22 separation. A representative western blot from 2 independent isolations is shown. Blots were incubated with anti-NOX5, anti-TOM22, and anti-COXIV antibodies. (D) Representative western blot of co-IP in isolated mitochondria. Immunoprecipitation was achieved with p22phox Ab, and NOX5 Ab was used for detection. Immunoprecipitate fractions with or without antibodies (−Ab and +Ab). Supernatant (SUP) after pull-down. For control, COXIV was detected in the mitochondrial fraction and total (Tot) lysate (bottom panel). Each experiment was repeated 3 times.$

NOX5 is expressed on the outer membrane of the mitochondria and interacts with p22phox. (A) Confocal microscopy of Mo-DC, labeled with anti-NOX5 and Alexa 488, anti-LAMP-1 (lysosome marker), anti-BCL2 (outer membrane mitochondrial marker), or anti-COXIV (inner membrane mitochondrial marker) and Alexa 555, and DAPI (nuclear staining). 40× objective, zoom 6×. (B) Representative western blots of Cyto and Mito fractions isolated by differential centrifugation. Blots were incubated with anti-NOX5, anti-GAPDH (for cytosol), and anti-COXIV (for mitochondria) antibodies. (C) Mitochondria isolation in Mo-DC by magnetic anti-TOM22 separation. A representative western blot from 2 independent isolations is shown. Blots were incubated with anti-NOX5, anti-TOM22, and anti-COXIV antibodies. (D) Representative western blot of co-IP in isolated mitochondria. Immunoprecipitation was achieved with p22phox Ab, and NOX5 Ab was used for detection. Immunoprecipitate fractions with or without antibodies (−Ab and +Ab). Supernatant (SUP) after pull-down. For control, COXIV was detected in the mitochondrial fraction and total (Tot) lysate (bottom panel). Each experiment was repeated 3 times.

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