a5 na k atpase  (Developmental Studies Hybridoma Bank)

Journal: EMBO Reports

Article Title: Functional profiling and visualization of the sphingolipid metabolic network in vivo

doi: 10.1038/s44319-025-00632-0

Figure Lengend Snippet: ( A ) A schematic figure of the OlyA w biosensor for labeling SPL- and sterol-rich membrane rafts. ( B ) OlyA w expression in the L3 salivary glands of control ( tubulin -GAL4/ UAS-OlyA w ) and Cpes KO ( Cpes KO /Cpes KO ; tubulin -GAL4/ UAS-OlyA w ). Cnx99A is a marker of the ER membrane (magenta). ( C ) Quantification of Pearson’s coefficient of OlyA w and Cnx99A (ER membrane) channels. Data are represented as mean ± SEM of 3 independent experiments. Data points represent biological repeats. The P value ( P = 0.005) was calculated using two-tailed unpaired Student’s t test. ( D ) Quantification of mean intensities of the OlyA w channel colocalizing with the ER membrane. Data are represented as mean ± SEM of 3 independent experiments. Data points represent biological repeats. The P value ( P = 0.004) was calculated using two-tailed unpaired Student’s t test. ( E ) A Schematic of an ommatidium cross-section. Pigment glia occupy the interommatidial space. ( F ) OlyA w expression driven by a pigment glia driver ( 54C -GAL4) in the adult eye. Na + -K + -ATPase is the marker of the plasma membrane (PM; magenta). OlyA w puncta can be found in the cytosol of photoreceptors (arrowhead). ( G ) OlyA w expression driven by a photoreceptor driver ( rh1 -GAL4) in the adult eye. Na + -K + -ATPase is the marker of the plasma membrane (PM; magenta). OlyA w puncta can be found in the interommatidial space (arrowhead). ( H ) Confocal images of the adult brain for showing colocalization OlyA w with a membrane raft marker, flo2-RFP. OlyA w was co-expressed with flo2-RFP ( Actin -GAL4/ + ; UAS-OlyA w / UAS-flo2-RFP ). Arrowheads indicate OlyA w puncta colocalized with flo2-RFP. ( I ) Confocal images of the adult brain for showing colocalization OlyA w with a membrane raft marker, mCherry-GPI. OlyA w was co-expressed with mCherry-GPI ( Actin -GAL4/ UAS-HA-mCherry-GPI ; UAS-OlyA w /+). Arrowheads indicate OlyA w puncta colocalized with mCherry-GPI. ( J ) Quantification of the percentage of flo2-GFP and mCherry-GPI labeled by OlyA w . Data are represented as mean ± SEM. Data points represent biological repeats. .

Article Snippet: The brains were permeabilized with 2% PBST for 30 min, then blocked in 0.25% PBST with 1% BSA for 1 h. Next, the samples were incubated with primary antibodies, rat anti-Elav (1:200, Rat-Elav-7E8A10, DSHB), mouse anti-Repo (1:200, 8D12 anti-Repo, DSHB), mouse anti-lamin (1:200, ADL67.10, DSHB), mouse anti-ATP5A (1:500, ab14748, Abcam), mouse anti-Cathepsin L (1:500, MAB22591, R and D Systems), mouse anti-Na + /K + -ATPase (1:200, a5, DSHB), rabbit anti-Ref2P (1:200, ab178440, abcam), mouse anti-DLG (1:200, 4F3 anti-discs large, DSHB), rabbit anti-GM130 (1:200, ab30637, Abcam), goat anti-Golgin245 (1:500, Golgin245, DSHB), mouse anti-Cnx99A (1:200, Cnx99A 6-2-1, DSHB), mouse anti-Golgin84 (1:200, Golgin84 12-1, DSHB), chicken anti-GFP (1:500, ab13970, Abcam), rabbit anti-Ifc (1:200, Jung et al, ) rabbit anti-Calnexin (1:200, ab75801, Abcam), rabbit anti-rab5 (1:200, ab31261, Abcam), diluted in 0.25% PBST with 1% BSA overnight at 4 °C, washed in 1% PBST four times for 30 min, and incubated in secondary antibody (1:500, 111-605-003, 715-545-150, 711-545-152, 112-545-167, 111-165-003, 115-605-166, Jackson ImmunoResearch Laboratories) diluted in 0.25% PBST with 1% BSA overnight at 4 °C.

Techniques: Labeling, Membrane, Expressing, Control, Marker, Two Tailed Test, Clinical Proteomics

Journal: EMBO Reports

Article Title: Functional profiling and visualization of the sphingolipid metabolic network in vivo

doi: 10.1038/s44319-025-00632-0

Figure Lengend Snippet: ( A ) OlyA w expression and SMases RNAi knockdowns in neurons. UAS-OlyA w expression and SMases RNAi were driven by nSyb -GAL4 ( nSyb -GAL4> OlyA w + UAS-RNAi ). Confocal images were captured from the posterior view of the Calyx in the brain of young adult flies (1 week old). (Right) Quantitative analysis of OlyA w intensity in the cortical region. P values [dSMPD4-IR (BDSC 51682), P < 0.001; dSMPD4-IR (VDRC 110163), P = 0.03; nSMase-IR (BDSC 36759), P = 0.09; nSMase-IR (VDRC 107062), P = 0.25; aSMase-IR (VDRC 12227), P = 0.46] were calculated using one-way ANOVA with Dunnett’s multiple comparisons. Data are represented as mean ± SEM ( n ≥ 4, biological repeats). Data are representative of at least two independent experiments. ( B ) OlyA w expression in neurons and SMases RNAi knockdowns in glia. LexAop-OlyA w expression is driven by nSyb -LexA, while SMases RNAi were driven by repo -GAL4 ( nSyb -LexA> LexAop-OlyA w ; repo -GAL4 > UAS-RNAi ). Confocal images were captured from the posterior view of the Calyx in the adult brain of young adult flies (1 week old). (Right) Quantifications of OlyA w intensity in the cortical region. P values [dSMPD4-IR (BDSC 51682), P = 0.10; dSMPD4-IR (VDRC 110163), P = 0.49; nSMase-IR (BDSC 36759), P = 0.90; nSMase-IR (VDRC 107062), P = 0.41; aSMase-IR (VDRC 12227), P < 0.001] were calculated using one-way ANOVA with Tukey’s multiple comparisons. Data are represented as mean ± SEM ( n ≥ 4, biological repeats). Data are representative of at least two independent experiments. ( C ) Quantification of the fold change in total lipid levels of dSMPD4 KO and nSMase KO compared to w 1118 brains (day 3 females). P values ( nSMase KO vs. w 1118 : Total lipids, P = 0.973; PS, P = 0.955; PE, P = 0.995; PC, P = 0.294; CerPE, P < 0.001; Cer, P < 0.001. dSMPD4 KO vs. w 1118 : Total lipids, P = 0.946; PS, P = 0.988; PE, P = 0.938; PC, P = 0.779; CerPE, P = 0.101; Cer, P = 0.768.) were calculated using one-way ANOVA with Dunnett’s multiple comparisons. Data are represented as mean ± SEM ( n = 4, biological repeats). ( D ) Quantification of the fold change in CerPE of dSMPD4 -knockout compared to w 1118 brains (day 3 females). P values (d16:2/22:0, P = 0.345; d16:2/20:0, P = 0.832; d16:2/18:0, P = 0.428; d16:1/22:0, P = 0.842; d16:0/22:0, P = 0.122; d14:2/24:0, P = 0.932; d14:2/22:0, P = 0.227; d14:2/20:0, P = 0.096; d14:2/18:1, P = 0.005; d14:2/18:0, P = 0.027; d14:1/24:1, P = 0.171; d14:1/24:0, P = 0.766; d14:1/22:0, P = 0.379; d14:1/20:0, P = 0.024; d14:1/18:0, P = 0.001; d14:0/22:1, P = 0.152; d14:0/20:1, P = 0.061) were calculated using two-tailed unpaired Student’s t test. Data are represented as mean ± SEM ( n = 4, biological repeats). ( E ) Quantification of the fold change in CerPE of nSMase -knockout compared to w 1118 brains (day 3 females). P values (d16:2/22:0, P = 0.081; d16:2/20:0, P < 0.001; d16:2/18:0, P < 0.001; d16:1/22:0, P < 0.001; d16:1/20:0, P < 0.001; d16:1/18:0, P < 0.001; d14:2/24:0, P = 0.610; d14:2/22:0, P = 0.663; d14:2/20:0, P < 0.001; d14:2/18:1, P < 0.001; d14:2/18:0, P < 0.001; d14:1/24:0, P = 0.203; d14:1/22:0, P = 0.016; d14:1/20:0, P < 0.001; d14:1/18:0, P < 0.001; d14:1/16:0, P < 0.001; d14:0/22:1, P = 0.001; d14:0/20:1, P < 0.001; d14:0/18:1, P < 0.001) were calculated using two-tailed unpaired Student’s t-test. Data are represented as mean ± SEM ( n = 4, biological repeats). ( F ) Anti-HA and anti-Lamin co-staining of the adult brain from dSMPD4 -HG line. Lamin is used as a marker of the nuclear envelope (NE). ( G ) Quantification of dSMPD4-3XHA subcellular localization. Box plot shows the Pearson’s coefficient of anti-HA and organellar markers, including anti-Lamin (nuclear envelope), anti-ATP5A (mitochondria), anti-Na + -K + -ATPase (Plasma membrane), and anti-Cathepsin L (Lysosome). Box plots illustrate the data distribution. The center line within the box represents the median, which is the 50th percentile. The lower and upper bounds of the box correspond to the 25th and 75th percentiles, respectively. The ends of the whiskers indicate the minima and maxima. Data points represent biological repeats. P values (Nuclear envelope vs. Mitochondria, P < 0.001; Nuclear envelope vs. Plasma membrane, P < 0.001; Nuclear envelope vs. Lysosome, P < 0.001) were calculated using one-way ANOVA with Tukey’s multiple comparisons. Representative images of anti-HA and organellar markers co-stainings are presented in Appendix Fig. . ( H ) Morphology of the nuclear membrane (anti-Lamin, magenta) in adult brains (1 week old) of indicated genotypes. ( I ) Quantification of the percentage of nuclei with blebs. Data are represented as mean ± SEM ( n = 3, biological repeats). P values ( w 1118 vs. dSMPD4 KO , P < 0.001; w 1118 vs. dSMPD4 KO ; actin > dSMPD4-myc , P = 0.999; w 1118 vs. nSMase KO , P = 0.547) were calculated using one-way ANOVA with Dunnett’s multiple comparisons. Data are representative of at least two independent experiments. ( J ) Olfactory associative memory assay in dSMPD4 -knockout, nSMase -knockout, and w 1118 control flies (1-week-old). Data are represented as mean ± SEM. P values ( w 1118 vs. dSMPD4 KO , P = 0.018; w 1118 vs. nSMase KO , P = 0.915) were calculated using one-way ANOVA with Dunnett’s multiple comparisons. Data are representative of four independent experiments of biological repeats. .

Article Snippet: The brains were permeabilized with 2% PBST for 30 min, then blocked in 0.25% PBST with 1% BSA for 1 h. Next, the samples were incubated with primary antibodies, rat anti-Elav (1:200, Rat-Elav-7E8A10, DSHB), mouse anti-Repo (1:200, 8D12 anti-Repo, DSHB), mouse anti-lamin (1:200, ADL67.10, DSHB), mouse anti-ATP5A (1:500, ab14748, Abcam), mouse anti-Cathepsin L (1:500, MAB22591, R and D Systems), mouse anti-Na + /K + -ATPase (1:200, a5, DSHB), rabbit anti-Ref2P (1:200, ab178440, abcam), mouse anti-DLG (1:200, 4F3 anti-discs large, DSHB), rabbit anti-GM130 (1:200, ab30637, Abcam), goat anti-Golgin245 (1:500, Golgin245, DSHB), mouse anti-Cnx99A (1:200, Cnx99A 6-2-1, DSHB), mouse anti-Golgin84 (1:200, Golgin84 12-1, DSHB), chicken anti-GFP (1:500, ab13970, Abcam), rabbit anti-Ifc (1:200, Jung et al, ) rabbit anti-Calnexin (1:200, ab75801, Abcam), rabbit anti-rab5 (1:200, ab31261, Abcam), diluted in 0.25% PBST with 1% BSA overnight at 4 °C, washed in 1% PBST four times for 30 min, and incubated in secondary antibody (1:500, 111-605-003, 715-545-150, 711-545-152, 112-545-167, 111-165-003, 115-605-166, Jackson ImmunoResearch Laboratories) diluted in 0.25% PBST with 1% BSA overnight at 4 °C.

Techniques: Expressing, Knock-Out, Two Tailed Test, Staining, Marker, Clinical Proteomics, Membrane, Control

Journal: EMBO Reports

Article Title: Functional profiling and visualization of the sphingolipid metabolic network in vivo

doi: 10.1038/s44319-025-00632-0

Figure Lengend Snippet: ( A ) A schematic figure of the OlyA w biosensor for labeling SPL- and sterol-rich membrane rafts. ( B ) OlyA w expression in the L3 salivary glands of control ( tubulin -GAL4/ UAS-OlyA w ) and Cpes KO ( Cpes KO /Cpes KO ; tubulin -GAL4/ UAS-OlyA w ). Cnx99A is a marker of the ER membrane (magenta). ( C ) Quantification of Pearson’s coefficient of OlyA w and Cnx99A (ER membrane) channels. Data are represented as mean ± SEM of 3 independent experiments. Data points represent biological repeats. The P value ( P = 0.005) was calculated using two-tailed unpaired Student’s t test. ( D ) Quantification of mean intensities of the OlyA w channel colocalizing with the ER membrane. Data are represented as mean ± SEM of 3 independent experiments. Data points represent biological repeats. The P value ( P = 0.004) was calculated using two-tailed unpaired Student’s t test. ( E ) A Schematic of an ommatidium cross-section. Pigment glia occupy the interommatidial space. ( F ) OlyA w expression driven by a pigment glia driver ( 54C -GAL4) in the adult eye. Na + -K + -ATPase is the marker of the plasma membrane (PM; magenta). OlyA w puncta can be found in the cytosol of photoreceptors (arrowhead). ( G ) OlyA w expression driven by a photoreceptor driver ( rh1 -GAL4) in the adult eye. Na + -K + -ATPase is the marker of the plasma membrane (PM; magenta). OlyA w puncta can be found in the interommatidial space (arrowhead). ( H ) Confocal images of the adult brain for showing colocalization OlyA w with a membrane raft marker, flo2-RFP. OlyA w was co-expressed with flo2-RFP ( Actin -GAL4/ + ; UAS-OlyA w / UAS-flo2-RFP ). Arrowheads indicate OlyA w puncta colocalized with flo2-RFP. ( I ) Confocal images of the adult brain for showing colocalization OlyA w with a membrane raft marker, mCherry-GPI. OlyA w was co-expressed with mCherry-GPI ( Actin -GAL4/ UAS-HA-mCherry-GPI ; UAS-OlyA w /+). Arrowheads indicate OlyA w puncta colocalized with mCherry-GPI. ( J ) Quantification of the percentage of flo2-GFP and mCherry-GPI labeled by OlyA w . Data are represented as mean ± SEM. Data points represent biological repeats. .

Article Snippet: Anti-Na + /K + -ATPase , Developmental Studies Hybridoma Bank , Cat# a5.

Techniques: Labeling, Membrane, Expressing, Control, Marker, Two Tailed Test, Clinical Proteomics

Journal: EMBO Reports

Article Title: Functional profiling and visualization of the sphingolipid metabolic network in vivo

doi: 10.1038/s44319-025-00632-0

Figure Lengend Snippet: ( A ) OlyA w expression and SMases RNAi knockdowns in neurons. UAS-OlyA w expression and SMases RNAi were driven by nSyb -GAL4 ( nSyb -GAL4> OlyA w + UAS-RNAi ). Confocal images were captured from the posterior view of the Calyx in the brain of young adult flies (1 week old). (Right) Quantitative analysis of OlyA w intensity in the cortical region. P values [dSMPD4-IR (BDSC 51682), P < 0.001; dSMPD4-IR (VDRC 110163), P = 0.03; nSMase-IR (BDSC 36759), P = 0.09; nSMase-IR (VDRC 107062), P = 0.25; aSMase-IR (VDRC 12227), P = 0.46] were calculated using one-way ANOVA with Dunnett’s multiple comparisons. Data are represented as mean ± SEM ( n ≥ 4, biological repeats). Data are representative of at least two independent experiments. ( B ) OlyA w expression in neurons and SMases RNAi knockdowns in glia. LexAop-OlyA w expression is driven by nSyb -LexA, while SMases RNAi were driven by repo -GAL4 ( nSyb -LexA> LexAop-OlyA w ; repo -GAL4 > UAS-RNAi ). Confocal images were captured from the posterior view of the Calyx in the adult brain of young adult flies (1 week old). (Right) Quantifications of OlyA w intensity in the cortical region. P values [dSMPD4-IR (BDSC 51682), P = 0.10; dSMPD4-IR (VDRC 110163), P = 0.49; nSMase-IR (BDSC 36759), P = 0.90; nSMase-IR (VDRC 107062), P = 0.41; aSMase-IR (VDRC 12227), P < 0.001] were calculated using one-way ANOVA with Tukey’s multiple comparisons. Data are represented as mean ± SEM ( n ≥ 4, biological repeats). Data are representative of at least two independent experiments. ( C ) Quantification of the fold change in total lipid levels of dSMPD4 KO and nSMase KO compared to w 1118 brains (day 3 females). P values ( nSMase KO vs. w 1118 : Total lipids, P = 0.973; PS, P = 0.955; PE, P = 0.995; PC, P = 0.294; CerPE, P < 0.001; Cer, P < 0.001. dSMPD4 KO vs. w 1118 : Total lipids, P = 0.946; PS, P = 0.988; PE, P = 0.938; PC, P = 0.779; CerPE, P = 0.101; Cer, P = 0.768.) were calculated using one-way ANOVA with Dunnett’s multiple comparisons. Data are represented as mean ± SEM ( n = 4, biological repeats). ( D ) Quantification of the fold change in CerPE of dSMPD4 -knockout compared to w 1118 brains (day 3 females). P values (d16:2/22:0, P = 0.345; d16:2/20:0, P = 0.832; d16:2/18:0, P = 0.428; d16:1/22:0, P = 0.842; d16:0/22:0, P = 0.122; d14:2/24:0, P = 0.932; d14:2/22:0, P = 0.227; d14:2/20:0, P = 0.096; d14:2/18:1, P = 0.005; d14:2/18:0, P = 0.027; d14:1/24:1, P = 0.171; d14:1/24:0, P = 0.766; d14:1/22:0, P = 0.379; d14:1/20:0, P = 0.024; d14:1/18:0, P = 0.001; d14:0/22:1, P = 0.152; d14:0/20:1, P = 0.061) were calculated using two-tailed unpaired Student’s t test. Data are represented as mean ± SEM ( n = 4, biological repeats). ( E ) Quantification of the fold change in CerPE of nSMase -knockout compared to w 1118 brains (day 3 females). P values (d16:2/22:0, P = 0.081; d16:2/20:0, P < 0.001; d16:2/18:0, P < 0.001; d16:1/22:0, P < 0.001; d16:1/20:0, P < 0.001; d16:1/18:0, P < 0.001; d14:2/24:0, P = 0.610; d14:2/22:0, P = 0.663; d14:2/20:0, P < 0.001; d14:2/18:1, P < 0.001; d14:2/18:0, P < 0.001; d14:1/24:0, P = 0.203; d14:1/22:0, P = 0.016; d14:1/20:0, P < 0.001; d14:1/18:0, P < 0.001; d14:1/16:0, P < 0.001; d14:0/22:1, P = 0.001; d14:0/20:1, P < 0.001; d14:0/18:1, P < 0.001) were calculated using two-tailed unpaired Student’s t-test. Data are represented as mean ± SEM ( n = 4, biological repeats). ( F ) Anti-HA and anti-Lamin co-staining of the adult brain from dSMPD4 -HG line. Lamin is used as a marker of the nuclear envelope (NE). ( G ) Quantification of dSMPD4-3XHA subcellular localization. Box plot shows the Pearson’s coefficient of anti-HA and organellar markers, including anti-Lamin (nuclear envelope), anti-ATP5A (mitochondria), anti-Na + -K + -ATPase (Plasma membrane), and anti-Cathepsin L (Lysosome). Box plots illustrate the data distribution. The center line within the box represents the median, which is the 50th percentile. The lower and upper bounds of the box correspond to the 25th and 75th percentiles, respectively. The ends of the whiskers indicate the minima and maxima. Data points represent biological repeats. P values (Nuclear envelope vs. Mitochondria, P < 0.001; Nuclear envelope vs. Plasma membrane, P < 0.001; Nuclear envelope vs. Lysosome, P < 0.001) were calculated using one-way ANOVA with Tukey’s multiple comparisons. Representative images of anti-HA and organellar markers co-stainings are presented in Appendix Fig. . ( H ) Morphology of the nuclear membrane (anti-Lamin, magenta) in adult brains (1 week old) of indicated genotypes. ( I ) Quantification of the percentage of nuclei with blebs. Data are represented as mean ± SEM ( n = 3, biological repeats). P values ( w 1118 vs. dSMPD4 KO , P < 0.001; w 1118 vs. dSMPD4 KO ; actin > dSMPD4-myc , P = 0.999; w 1118 vs. nSMase KO , P = 0.547) were calculated using one-way ANOVA with Dunnett’s multiple comparisons. Data are representative of at least two independent experiments. ( J ) Olfactory associative memory assay in dSMPD4 -knockout, nSMase -knockout, and w 1118 control flies (1-week-old). Data are represented as mean ± SEM. P values ( w 1118 vs. dSMPD4 KO , P = 0.018; w 1118 vs. nSMase KO , P = 0.915) were calculated using one-way ANOVA with Dunnett’s multiple comparisons. Data are representative of four independent experiments of biological repeats. .

Article Snippet: Anti-Na + /K + -ATPase , Developmental Studies Hybridoma Bank , Cat# a5.

Techniques: Expressing, Knock-Out, Two Tailed Test, Staining, Marker, Clinical Proteomics, Membrane, Control

Journal: Nature Communications

Article Title: Choroid plexus-mediated CSF secretion remains stable in aging rats via high and age-resistant metabolic activity

doi: 10.1038/s41467-025-61889-6

Figure Lengend Snippet: a GO enrichment analysis of the differentially expressed genes in choroid plexus obtained from the different age groups into protein classes. b All differentially expressed proteins within the ‘transporter’ category with a mean of trimmed mean of M values (TMM) above 100. Listed are the gene names together with their percentage fluctuations from the mean in the 1-, 3-, 6-, 12-, 18-, and 24-months-old groups, with the average expression in TMM. c Schematic of choroid plexus illustrating the polarized expression of transporters implicated in CSF secretion. NCBE and NBCe2; Na + -coupled HCO 3 - transporters, AE2; Cl - /HCO 3 - exchanger, AQP1; aquaporin 1, NKCC1; Na + ,K + ,2Cl - cotransporter 1, NKA; Na + /K + -ATPase. Tight junctions are marked with black bars. d Expression levels as a function of age illustrated as z-score of the mean (circles) with the 95% confidence interval indicated as a shaded area. The expression level of each transporter at each age is indicated in transcripts per million (TPM) below each graph, n = 3. e Quantification of Western blots (from Supplementary Fig. ) of Na + /K + -ATPase (NKA), NKCC1, and AQP1, n = 3, data are presented as mean ± SEM and statistical significance was tested with one-way ANOVA followed by Tukey’s multiple comparisons test. Asterisks denote statistical significance (* P < 0.05), non-significance is not illustrated for clarity. 1 M; turquoise, 3 M; green, 6 M; orange, 12 M; red, 18 M; purple, 24 M; grey. Source data are provided as a Source Data file and exact P -values in Supplementary Data .

Article Snippet: The membranes were blocked in Odyssey blocking buffer PBS (LI-COR) and probed with primary antibodies: mouse anti-Na + /K + -ATPase α1 a6F (1:60, a6F, DSHB), rabbit anti-claudin-1 (1:1000, ab307692, Abcam), rabbit anti-AQP1-1 (1:1000, ab307692, Alomone), sheep anti-NKCC1 (2 μg/ml, S022D, Dundee University, and as a loading control; chicken anti-GAPDH (1:800, AB2302, Millipore), all diluted in blocking buffer:PBS-T 1:1.

Techniques: Expressing, Western Blot