bioimagesUnknownKe ZhangThe Journal of Cell Biologyhttps://www.ebi.ac.uk/biostudies/studies/S-JCBD-201208033JCB

Shown is an anti-HA Western blot for ND42-HA in whole body extract from FRT19A; da>ND42-HA/+ (wt) and sicily mutant flies expressing a genomic copy of ND42-HA. The overall level, including the level of ND42-HA preprotein, is down-regulated in sicily mutants compared to “wt” animals. The picture is upside down relative to the published figure.

Shown is an anti-CytC Western blot of lysates from flies with or without expression of da>Hsp90 RNAi-1. The picture is rotated 90 degrees counterclockwise and flipped vertically relative to the published figure.

Shown is an anti-V5 Western blot of cell lysates expressing Sicily-V5 and ND42-HA and treated with 0.1-2 μM geldanamycin, a specific inhibitor for Hsp90. The level of Sicily-V5 is decreased when Hsp90 activity is inhibited.

Shown is immunolabeling for Sicily(-MTS)-V5 (red) and the mitochondrial ATP synthase α subunit (Complex V or CV; green) in S2 cells expressing Ubi-Sicily-MTS. Compared to full-length Sicily that is mostly localized in mitochondria (part a), Sicily-MTS is mostly localized in cytoplasm.<br />

Shown is a transmission EM of a 24 day old XE07E; eyFLP fly retina. XE07E mutant rhabdomeres are severely dissociated after aging for 24 days.

Shown is immunolabeling for Sicily-mCherry (anti-DsRed, red) and the mitochondrial ATP synthase α subunit (Complex V or CV; green) in a third instar fly posterior ventral nerve cord of a sicily mutant rescued with a genomic Sicily-mCherry construct. Sicily is present in both the cytoplasm and mitochondria.

Shown is an anti-HA Western blot for of lysates from cells expressing ND42(-MTS)-HA and/or mCherry and expressing Sicily-V5 or Sicily(-MTS)-V5. See lanes 1, 2, and 3. The picture is rotated 90 degrees counterclockwise relative to the published figure.

Shown is an anti-porin Western blot (loading control) of lysates from cells expressing ND42(-MTS)-HA and/or mCherry and expressing Sicily-V5 or Sicily(-MTS)-V5. See lanes 1, 2, and 3.

Shown is a Western blot for ND42 in wild-type, siciliyC, and sicily E strains. See lanes 1, 2, and 3 labeled Ct, C, and E, respectively. The picture is upside down and flipped horizontally relative to the published figure.

Shown is an anti-HA Western blot of cell lysates expressing Sicily-V5 and ND42-HA and treated with 0.1-2 μM geldanamycin, a specific inhibitor for Hsp90. The level of ND42-HA is decreased when Hsp90 activity is inhibited.

Shown is an anti-porin Western blot (loading control) of lysates from flies with or without expression of da>Hsp90 RNAi-1. The picture is upside down relative to the published figure. Lane 3 shows Hsp90 RNAi-2 that is referred to as “data not shown” in the manuscript.

Shown is an anti-V5 Western blot for Sicily-V5 that shows that Sicily-V5 and ND42-HA co-immunoprecipitate in an anti-HA IP. S2 cells transfected with Ubi-GAL4, UAS-Sicily-V5 and/or UAS-ND42-HA were harvested and lysed 48h posttransfection. Both Sicily and ND42 exhibit two isoforms. The smaller isoforms are more predominant in the input and IP, but the larger ones are more predominant in the co-IP. The picture is upside down relative to the published figure.

Shown is an anti-Hsp60 Western blot of mitochondrial fractions purified from third instar fly larvae with and without da>ND42 RNAi expression. Hsp60 is up-regulated.

Shown is an anti-HA Western blot for ND42-HA in whole body extract from genomic construct-expressing gND42-HA/+, ubiquitously overexpressing da>ND42-HA, and sicily overexpressing da>ND42HA flies. The overall level, including the level of ND42-HA preprotein, is down-regulated in sicily mutants. The picture is rotated 90 degreed clockwise and flipped horizontally relative to the published figure.

Shown is a transmission EM of a 24 day old FRT19A; eyFLP fly retina.

Shown is an anti-V5 Western blot for Sicily-V5 following anti-V5 immunoprecipitation. S2 cells transfected with Ubi-GAL4, UAS-Sicily-V5 and/or UAS-ND42-HA were harvested and lysed 48h posttransfection. Both Sicily and ND42 exhibit two isoforms. The smaller isoforms are more predominant in the input and IP, but the larger ones are more predominant in the co-IP. The picture is upside down relative to the published figure.

Shown is an anti-HA Western blot for ND42-HA following anti-HA immunoprecipitation. S2 cells transfected with Ubi-GAL4, UAS-Sicily-V5 and/or UAS-ND42-HA were harvested and lysed 48h posttransfection. Both Sicily and ND42 exhibit two isoforms. The smaller isoforms are more predominant in the input and IP, but the larger ones are more predominant in the co-IP. The picture is upside down relative to the published figure.

Shown is immunolabeling for Sicily-mCherry (anti-DsRed, red) and the mitochondrial ATP synthase α subunit (Complex V or CV; green) in the eye imaginal disc of a sicily mutant rescued with a genomic Sicily-mCherry construct. Sicily is present in both the cytoplasm and mitochondria.

Shown is immunolabeling for ND42-HA (anti-DsRed, red) and the mitochondrial ATP synthase α subunit (Complex V or CV; green) in S2 cells expressing Ubi>ND42-HA.

Shown is an anti-HA Western blot for ND42(-MTS)-HA from cell treated with the proteasomal inhibitor MG132, with bafilomycin A1 (BFA), or DMSO (control). MG132 treatment leads to ubiquitinated ND42. The anti-porin and anti-GFP blots are shown in part g of this figure (lanes 4-6).

Shown is an anti-V5 Western blot following anti-HA IP for NDUFS2-HA in S2 cells expressing both Siciliy-V5 and NDUFS3-HA. Full length Sicily preferentially interacts with NDUFS3. The picture is flipped vertically relative to the published figure.

Shown is an anti-ND42 Western blot of wild-type, sicilyE, and sicilyE;da>sicility-MTS strains (top half of image). sicily mutant males expressing Sicily-MTS under the control of the da-GAL4 driver, a ubiquitous driver, have restored levels of ND42 and NDUFS3.

Shown is immunolabeling for ND42(-MTS)-HA (anti-DsRed, red) and the mitochondrial ATP synthase α subunit (Complex V or CV; green) in S2 cells expressing Ubi>ND42(-MTS)-HA and Siciliy-V5. When coexpressed, Sicily does not target ND42-MTS to mitochondria but rather leads to an increase in level of ND42-MTS (compared to part e).

Shown is a transmission EM of a 10 day old XE07E; eyFLP fly retina. XE07E mutant rhabdomeres are dissociated after aging for 10 days.

Shown is a transmission EM of a 24-day old siciliy mutant fly retina rescued by the genomic construct.

Shown is a transmission EM image of a 10 day old GMR>ND42 RNAi-1 fly retina. Newly eclosed flies with ND42 RNAi display a normal number and arrangement of rhabdomeres, but the pigment cells are expanded when compared to the control flies (part d). Upon aging for 10 days, the retinas of these flies degenerate, rhabdomeres are missing or dissociating, and the cell bodies of some photoreceptors are vacuolated.<br />

Shown is an anti-HA Western blot following anti-V5 IP for Sicily-V5 or Sicily(-MTS)-V5 in S2 cells expressing both Sicily-V5 and ND42-HA. Sicily-MTS shows similar binding affinity to ND42 as Sicily containing its MTS (mitochondrial targeting sequence). An unrelated protein, Marf (the fly homologue of Mitofusin), serves as a negative control. The picture is rotated 90 degrees counterclockwise relative to the published figure.

Shown is an anti-HA Western blot for ND42-HA that shows that Sicily-V5 and ND42-HA co-immunoprecipitate in an anti-V5 IP. S2 cells transfected with Ubi-GAL4, UAS-Sicily-V5 and/or UAS-ND42-HA were harvested and lysed 48h posttransfection. Both Sicily and ND42 exhibit two isoforms. The smaller isoforms are more predominant in the input and IP, but the larger ones are more predominant in the co-IP. The picture is upside down relative to the published figure.

Shown is immunolabeling for Sicily-V5 (red) and the mitochondrial ATP synthase α subunit (Complex V or CV; green) in S2 cells expressing Ubi-Sicily.

Shown is an anti-porin Western blot of wild-type, sicilyE, and sicilyE;da>sicility-MTS strains (top half of image).

Shown is an anti-porin Western blot of subcellular fractionations of S2 cells expressing Sicily-V5 and ND42-HA. The picture is flipped horizontally relative to the published figure.

Shown is a longer exposure of an anti-HA Western blot of in vitro translated ND42-HA either with or without co-translation of Sicily-V5 or Sicily(-MTS)-V5. ND42-HA is up-regulated when Sicily or Sicily-MTS is co-translated. The picture is flipped horizontally relative to the published figure.

Shown is a transmission EM of a 10 day old XE07E; eyFLP fly retina after rearing in the dark. XE07 mutants display progressive neurodegenerative features in photoreceptors.

Shown is a transmission EM image of a newly eclosed GMR>ND42 RNAi-2 fly retina. Newly eclosed flies with ND42 RNAi display a normal number and arrangement of rhabdomeres, but the pigment cells are expanded when compared to the control flies (part d).

Shown is a transmission EM of a 1 day old FRT19A; eyFLP fly retina.

Shown is a transmission EM image of a 10 day old GMR/+ (“wt”) fly retina.

Shown is a transmission EM image of a newly eclosed GMR>ND42 RNAi-1 fly retina. Newly eclosed flies with ND42 RNAi display a normal number and arrangement of rhabdomeres, but the pigment cells are expanded when compared to the control flies (part d).

Shown is an anti-ND42 Western blot of lysates from flies with or without expression of da>Hsp90 RNAi-1. Complex I proteins are down-regulated in flies expressing Hsp90 RNAi. The picture is rotated 180 degrees relative to the published figure. Lane 3 shows Hsp90 RNAi-2 that is referred to as “data not shown” in the manuscript.

Shown is an anti-NDUFS3 Western blot of lysates from flies with or without expression of da>Hsp90 RNAi-1. Complex I proteins are down-regulated in flies expressing Hsp90 RNAi. The picture is upside down relative to the published figure. Lane 3 shows Hsp90 RNAi-2 that is referred to as “data not shown” in the manuscript.

Shown is an anti-Porin Western blot in whole body extract from gND42-HA/+ (“wt”), da>ND42HA, and sicily mutant flies expressing da>ND42HA. The first two lanes from left are for 6a, and the other three are for b.

Shown is an anti- NDUFS3 Western blot of subcellular fractionations of S2 cells expressing Sicily-V5 and ND42-HA. The picture is upside down and flipped horizontally relative to the published figure.

Shown is immunolabeling for ND42(-MTS)-HA (anti-DsRed, red) and the mitochondrial ATP synthase α subunit (Complex V or CV; green) in S2 cells expressing Ubi>ND42(-MTS)-HA. MTS-less ND42 is more broadly localized than full-length ND42 (part c).

Shown is an anti-GFP Western blot (transfection control) of cell lysates expressing Sicily-V5 and ND42-HA and treated with 0.1-2 μM geldanamycin, a specific inhibitor for Hsp90.

Shown are Western blots for CytC, NDUFS3, and Porin in wild-type, siciliyC, and sicily E strains.

Shown is an anti-HA Western blot of subcellular fractionations of S2 cells expressing Sicily-V5 and ND42-HA. The larger isoform of ND42 is enriched in the cytoplasm (C), whereas the smaller isoform is enriched in mitochondria (M). The picture is upside down and flipped horizontally relative to the published figure.

Shown is a transmission EM image of a newly eclosed GMR/+ (“wt”) fly retina.

Shown is an anti-porin Western blot of mitochondrial fractions purified from third instar fly larvae with and without da>ND42 RNAi expression. The picture is upside down relative to the published figure.

Shown is immunolabeling for ND42-HA (anti-DsRed, red) and the mitochondrial ATP synthase α subunit (Complex V or CV; green) in S2 cells expressing Ubi>ND42-HA and Siciliy-V5.

Shown is an anti-HA Western blot of in vitro translated ND42-HA either with or without co-translation of Sicily-V5 or Sicily(-MTS)-V5. ND42-HA is up-regulated when Sicily or Sicily-MTS is co-translated. The picture is flipped horizontally relative to the published figure.

Shown is an anti-GFP Western blot for Hsp90-GFP that shows that Sicily-V5 and Hsp90-GFPco-immunoprecipitate in an anti-GFP IP from cultured cells. Two different exposures are shown.

Shown is an anti-tubulin Western blot of subcellular fractionations of S2 cells expressing Sicily-V5 and ND42-HA.

Shown is an anti-GFP Western blot for Hsp90-GFP that shows that Hsp90-GFP and ND42-HA only co-immunoprecipitate in an anti-GFP IP from cultured cells when Sicily-V5 is co-expressed. Two different exposures are shown.

Shown are two exposures of an anti-NDUFS3 Western blot of mitochondrial fractions purified from third instar fly larvae with and without da>ND42 RNAi expression. Complex I subunits are almost completely lost.

Shown is a transmission EM of a 1 day old XE07E; eyFLP fly retina. XE07E mutants display normal differentiation of seven rhabdomeres at day 1, but the pigment cells are largely expanded compared to wildtype.

Shown is a transmission EM image of a 10 day old GMR>ND42 RNAi-2 fly retina. Newly eclosed flies with ND42 RNAi display a normal number and arrangement of rhabdomeres, but the pigment cells are expanded when compared to the control flies (part d). Upon aging for 10 days, the retinas of these flies degenerate, rhabdomeres are missing or dissociating, and the cell bodies of some photoreceptors are vacuolated.

Shown is an anti-GFP Western blot (transfection control) of lysates from cells expressing ND42(-MTS)-HA and/or mCherry and expressing Sicily-V5 or Sicily(-MTS)-V5. The picture is rotated 180 degrees relative to the published figure. See lanes 1, 2, and 3.

Shown is an anti-porin Western blot (loading control) of cell lysates expressing Sicily-V5 and ND42-HA and treated with 0.1-2 μM geldanamycin, a specific inhibitor for Hsp90.

Shown is immunolabeling for Sicily-mCherry (anti-DsRed, red) and the mitochondrial ATP synthase α subunit (Complex V or CV; green) in third instar motor neuron axons of a sicily mutant rescued with a genomic Sicily-mCherry construct. Sicily is present in both the cytoplasm and mitochondria.

Shown is an anti-NDUFS3 Western blot of wild-type, sicilyE, and sicilyE;da>sicility-MTS strains (bottom half of image). sicily mutant males expressing Sicily-MTS under the control of the da-GAL4 driver, a ubiquitous driver, have restored levels of ND42 and NDUFS3. The picture is flipped horizontally relative to the published figure.

bioimagesImage 137142 (Figure 6 - g)Image 137152 (Figure 7 - e)Image 137148 (Figure 7 - a)Figure 1 - dFigure 1 - cImage 140948 (Figure 8 - f)Image 137112 (Figure 2 - e)Image 137123 (Figure 4 - a)Image 137135 (Figure 6 - c)Figure 7 - e'Image 137161 (Figure 8 - f)Image 137145 (Figure 6 - h)Figure 8 - eFigure 8 - fFigure 8 - aFigure 8 - bImage 137119 (Figure 3 - c)Image 137117 (Figure 3 - b)Image 137172 (Supplemental Figure 3 - a)Image 137132 (Figure 6 - a)Image 141937 (Figure 1 - d)Image 141935 (Figure 8 - e)Figure 5 - aImage 137121 (Figure 3 - c)Figure 5 - fFigure 5 - bFigure 5 - eImage 137109 (Figure 1 - d')Figure 7 - d''Figure 6 - abImage 137113 (Figure 2 - e)Figure 3 - fFigure 3 - gSupplemental Figure 1 - cSupplemental Figure 1 - bImage 137153 (Figure 7 - e')Image 137155 (Figure 8 - a)Image 137130 (Figure 5 - e)Image 141932 (Figure 8 - e)Figure 3 - bFigure 3 - cFigure 5Image 137147 (Figure 7 - a)Figure 4Image 137127 (Figure 5 - a)Figure 7Figure 6Figure 1 - d''Figure 8Image 137138 (Figure 6 - f)Figure 1Image 140942 (Figure 6 - i)Figure 3Figure 5 - cdFigure 2Image 140940 (Figure 6 - h)Supplemental Figure 3 - bImage 137136 (Figure 6 - d)Supplemental Figure 3 - aSupplemental Figure 3Supplemental Figure 1Image 137111 (Figure 2 - c)Image 140939 (Figure 6 - ab)Image 141934 (Figure 8 - e)Figure 2 - eFigure 1 - d'Image 137118 (Figure 3 - c)Image 137173 (Supplemental Figure 3 - b)Image 137115 (Figure 3 - b)Figure 7 - e''Image 137151 (Figure 7 - d'')Figure 2 - cImage 140938 (Figure 6 - b)Image 137124 (Figure 4 - a)Image 137140 (Figure 6 - g)Image 137150 (Figure 7 - d')Image 137149 (Figure 7 - d)Figure 7 - aFigure 7 - dFigure 7 - eImage 141931 (Figure 4 - a)Image 137141 (Figure 6 - g)Image 137122 (Figure 3 - f)Image 137110 (Figure 1 - d'')Figure 6 - iImage 137156 (Figure 8 - b)Image 137114 (Figure 3 - b)Image 137169 (Supplemental Figure 1 - b)Image 137131 (Figure 5 - f)Image 137116 (Figure 3 - b)Figure 6 - cFigure 6 - dImage 141933 (Figure 8 - e)Image 137126 (Figure 3 - g)Figure 6 - aFigure 6 - bFigure 6 - gFigure 6 - hFigure 6 - eFigure 6 - fImage 137146 (Figure 7 - a)Image 137128 (Figure 5 - b)Image 137107 (Figure 1 - c)Image 137154 (Figure 7 - e'')Figure 4 - aImage 137163 (Figure 8 - f)Image 137129 (Figure 5 - cd)Figure 7 - d'Image 137137 (Figure 6 - e)Image 137171 (Supplemental Figure 1 - c)Image 137120 (Figure 3 - c)Image 140947 (Figure 8 - f)Hector SandovalBrett H. GrahamBo XiongWu-Lin CharngHugo J. BellenVafa BayatKe ZhangZhihong LiManish JaiswalGabriela DavidClaire HaueterShinya YamamotofalseThe C8ORF38 homologue Sicily is a cytosolic chaperone for a mitochondrial complex I subunit2013-03-18T11:23:48Z2018-11-29T11:23:48Z2018-11-29T11:23:48ZS-JCBD-20120803310.1083/jcb.201208033