Structured Review

GE Healthcare anti rabbit igg
Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . <t>IgG</t> influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers <t>Occludin</t> (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p
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Images

1) Product Images from "Decreased Plasma Aβ in Hyperlipidemic APPSL Transgenic Mice Is Associated with BBB Dysfunction"

Article Title: Decreased Plasma Aβ in Hyperlipidemic APPSL Transgenic Mice Is Associated with BBB Dysfunction

Journal: Frontiers in Neuroscience

doi: 10.3389/fnins.2016.00232

Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p
Figure Legend Snippet: Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p

Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Marker

Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p
Figure Legend Snippet: Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p

Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Marker

Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p
Figure Legend Snippet: Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p

Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Marker

Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p
Figure Legend Snippet: Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p

Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Marker

2) Product Images from "JMJD1C Exhibits Multiple Functions in Epigenetic Regulation during Spermatogenesis"

Article Title: JMJD1C Exhibits Multiple Functions in Epigenetic Regulation during Spermatogenesis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0163466

Detection of hypo-acetylated K16 on histone H4 in the Jmjd1C gt/gt testis. Approximately 15 μg protein extracts from +/+ and gt/gt testes (postnatal day 56 littermates) were applied on SDS-PAGE (10–20% gradient gel) and blotted onto membranes. A) The membranes were reacted with a mixture of three antibodies against HSC70t, histone H3 (H3) and acetylated H4K16 (H4K16Ac) (left) or four antibodies against VASA, MOF, histone H3 and acetylated H4K16 (right) and then detected with HRP-conjugated anti-IgG and an ECL detection reagent. Specific reactant of each antibody used in this mixed immunoblotting is shown in S5 Fig . B) Comparison of the detected protein bands between the +/+ and gt/gt extracts. C) Semi-quantitative comparison of the acetylated H4K16 bands between the +/+ and gt/gt testes extracts. Values obtained from the +/+ and gt/gt testes were normalized with those of the histone H3, VASA and HSC70t protein bands, which roughly corresponded to the numbers of total cells, germ cells at stages from spermatogonium to late spermatid, and spermatids, respectively. Bars in the graph show relative ratios (%) of values of the H4K16ac band in gt/gt against those in +/+ after normalizing with values of standard protein bands as indicated below. Error bars indicate the SEM (n = 4–6).
Figure Legend Snippet: Detection of hypo-acetylated K16 on histone H4 in the Jmjd1C gt/gt testis. Approximately 15 μg protein extracts from +/+ and gt/gt testes (postnatal day 56 littermates) were applied on SDS-PAGE (10–20% gradient gel) and blotted onto membranes. A) The membranes were reacted with a mixture of three antibodies against HSC70t, histone H3 (H3) and acetylated H4K16 (H4K16Ac) (left) or four antibodies against VASA, MOF, histone H3 and acetylated H4K16 (right) and then detected with HRP-conjugated anti-IgG and an ECL detection reagent. Specific reactant of each antibody used in this mixed immunoblotting is shown in S5 Fig . B) Comparison of the detected protein bands between the +/+ and gt/gt extracts. C) Semi-quantitative comparison of the acetylated H4K16 bands between the +/+ and gt/gt testes extracts. Values obtained from the +/+ and gt/gt testes were normalized with those of the histone H3, VASA and HSC70t protein bands, which roughly corresponded to the numbers of total cells, germ cells at stages from spermatogonium to late spermatid, and spermatids, respectively. Bars in the graph show relative ratios (%) of values of the H4K16ac band in gt/gt against those in +/+ after normalizing with values of standard protein bands as indicated below. Error bars indicate the SEM (n = 4–6).

Techniques Used: SDS Page

Detection of methylated non-histone proteins is affected by Jmjd1C deficiency. A) Total extracts of 15 μg were prepared from +/+ and gt/gt testes of littermate mice. IP samples obtained using combinations of anti-MDC1 and +/+ or gt/gt testis lysates were applied for SDS-PAGE (10%), blotted and then detected with anti-methylated-Lys in conjugation with HRP-labeled anti-rabbit IgG. An arrow indicates a 90 kDa protein band. B) MDC1-IP samples from +/+ and gt/gt testes (A), control IgG-IP and the input lysates from the +/+ and gt/gt testes were immunoblotted. The membrane was reacted with anti-HSP90α and then visualized with HRP-anti-rabbit IgG and the detection kit.
Figure Legend Snippet: Detection of methylated non-histone proteins is affected by Jmjd1C deficiency. A) Total extracts of 15 μg were prepared from +/+ and gt/gt testes of littermate mice. IP samples obtained using combinations of anti-MDC1 and +/+ or gt/gt testis lysates were applied for SDS-PAGE (10%), blotted and then detected with anti-methylated-Lys in conjugation with HRP-labeled anti-rabbit IgG. An arrow indicates a 90 kDa protein band. B) MDC1-IP samples from +/+ and gt/gt testes (A), control IgG-IP and the input lysates from the +/+ and gt/gt testes were immunoblotted. The membrane was reacted with anti-HSP90α and then visualized with HRP-anti-rabbit IgG and the detection kit.

Techniques Used: Methylation, Mouse Assay, SDS Page, Conjugation Assay, Labeling

3) Product Images from "WIPI3 and WIPI4 β-propellers are scaffolds for LKB1-AMPK-TSC signalling circuits in the control of autophagy"

Article Title: WIPI3 and WIPI4 β-propellers are scaffolds for LKB1-AMPK-TSC signalling circuits in the control of autophagy

Journal: Nature Communications

doi: 10.1038/ncomms15637

WIPI3 interacts with the TSC complex. ( a ) Stable GFP-WIPI1, GFP-WIPI2B, GFP-WIPI3 or GFP-WIPI4 U2OS cells were starved (3 h), and subjected to anti-GFP immunoprecipitation and anti-TSC2, anti-phospho-TSC2 (S1387), anti-TSC1 or anti-GFP immunoblotting. ( b ) U2OS cells were analysed by anti-TSC1 immunoprecipitation (TSC1-IP), anti-TSC1, anti-TSC2 and anti-WIPI1 (right panel), anti-WIPI2 (right panel), anti-WIPI3 (left panel) or anti-WIPI4 (right panel) immunoblotting. ( c ) ATG5 WT or KO MEFs expressing GFP-WIPI3 (W3) or GFP were subjected to anti-GFP immunoprecipitation and anti-TSC1, anti-TSC2 and anti-GFP immunoblotting. Conditions (3 h): fed (F) or starved (S). ( d ) Stable GFP-WIPI3 or GFP U2OS cells were analysed by anti-GFP immunoprecipitation and anti-TSC2 and anti-GFP immunoblotting. Conditions (15 min. to 3 h): fed (F), starved (S), starved with LY294002 (S+LY). ( e ) Myc-tagged TSC1 fragments (TSC1-M, TSC1-C; full length: TSC-FL) were expressed in stable GFP-WIPI3 U2OS cells and subjected to anti-GFP immunoprecipitation and anti-GFP or anti-myc immunoblotting. ( f ) Lysosomal fractions (no. 1–7; total protein control: TP) from stable GFP-WIPI3 U2OS cells (BafA1, 3 h) were immunoblotted using anti-GFP, anti-LAMP2, anti-TSC2, anti-WIPI2 or anti-Rab4 antibodies. ( g ) Stable GFP-WIPI3 U2OS cells were starved (3 h) and immunostained with anti-TSC2/IgG-Alexa Fluor 546 and anti-LAMP2/IgG-Alexa Fluor 633 antibodies for confocal LSM. ( h ) Stable GFP-WIPI3 U2OS cells were immunostained with anti-Lamp2/IgG-Alexa Fluor 633 and anti-WIPI2/IgG-Alexa Fluor 546 for confocal LSM. Conditions (3 h): fed (F), starved (S). ( i ) Stable GFP-WIPI3 U2OS cells with siControl or siTSC2 were fed (F) or starved (S) with or without BafA1 (3 h). Upper panel: anti-TSC2 immunoblotting, lower panel: GFP-WIPI3 puncta assessment (up to 493 cells per condition, n =4). ( j ) U2OS cells were subjected to immunoblotting using anti-phospho-mTOR (S2481), anti-mTOR, anti-phospho-ULK1 (S757), anti-ULK1 and anti-tubulin antibodies. Treatments: fed (F, 4 h), starved (S, 4 h), starved (3 h) and fed (1 h) (S→F). ( k ) Stable GFP-WIPI3 U2OS cells were immunostained with anti-mTOR/IgG-Alexa Fluor 546 and anti-LAMP2/IgG-Alexa Fluor 633 antibodies. Treatments: starved (S, 2 h), starved (2 h) and fed (1 h) (S→F). Co-localizations are indicated with arrows ( g , h , k ). Supplementary Material is available: Supplementary Fig. 5 . Statistics and source data: Supplementary Data 1 . Mean±s.d.; heteroscedastic t -testing; P values: * P
Figure Legend Snippet: WIPI3 interacts with the TSC complex. ( a ) Stable GFP-WIPI1, GFP-WIPI2B, GFP-WIPI3 or GFP-WIPI4 U2OS cells were starved (3 h), and subjected to anti-GFP immunoprecipitation and anti-TSC2, anti-phospho-TSC2 (S1387), anti-TSC1 or anti-GFP immunoblotting. ( b ) U2OS cells were analysed by anti-TSC1 immunoprecipitation (TSC1-IP), anti-TSC1, anti-TSC2 and anti-WIPI1 (right panel), anti-WIPI2 (right panel), anti-WIPI3 (left panel) or anti-WIPI4 (right panel) immunoblotting. ( c ) ATG5 WT or KO MEFs expressing GFP-WIPI3 (W3) or GFP were subjected to anti-GFP immunoprecipitation and anti-TSC1, anti-TSC2 and anti-GFP immunoblotting. Conditions (3 h): fed (F) or starved (S). ( d ) Stable GFP-WIPI3 or GFP U2OS cells were analysed by anti-GFP immunoprecipitation and anti-TSC2 and anti-GFP immunoblotting. Conditions (15 min. to 3 h): fed (F), starved (S), starved with LY294002 (S+LY). ( e ) Myc-tagged TSC1 fragments (TSC1-M, TSC1-C; full length: TSC-FL) were expressed in stable GFP-WIPI3 U2OS cells and subjected to anti-GFP immunoprecipitation and anti-GFP or anti-myc immunoblotting. ( f ) Lysosomal fractions (no. 1–7; total protein control: TP) from stable GFP-WIPI3 U2OS cells (BafA1, 3 h) were immunoblotted using anti-GFP, anti-LAMP2, anti-TSC2, anti-WIPI2 or anti-Rab4 antibodies. ( g ) Stable GFP-WIPI3 U2OS cells were starved (3 h) and immunostained with anti-TSC2/IgG-Alexa Fluor 546 and anti-LAMP2/IgG-Alexa Fluor 633 antibodies for confocal LSM. ( h ) Stable GFP-WIPI3 U2OS cells were immunostained with anti-Lamp2/IgG-Alexa Fluor 633 and anti-WIPI2/IgG-Alexa Fluor 546 for confocal LSM. Conditions (3 h): fed (F), starved (S). ( i ) Stable GFP-WIPI3 U2OS cells with siControl or siTSC2 were fed (F) or starved (S) with or without BafA1 (3 h). Upper panel: anti-TSC2 immunoblotting, lower panel: GFP-WIPI3 puncta assessment (up to 493 cells per condition, n =4). ( j ) U2OS cells were subjected to immunoblotting using anti-phospho-mTOR (S2481), anti-mTOR, anti-phospho-ULK1 (S757), anti-ULK1 and anti-tubulin antibodies. Treatments: fed (F, 4 h), starved (S, 4 h), starved (3 h) and fed (1 h) (S→F). ( k ) Stable GFP-WIPI3 U2OS cells were immunostained with anti-mTOR/IgG-Alexa Fluor 546 and anti-LAMP2/IgG-Alexa Fluor 633 antibodies. Treatments: starved (S, 2 h), starved (2 h) and fed (1 h) (S→F). Co-localizations are indicated with arrows ( g , h , k ). Supplementary Material is available: Supplementary Fig. 5 . Statistics and source data: Supplementary Data 1 . Mean±s.d.; heteroscedastic t -testing; P values: * P

Techniques Used: Immunoprecipitation, Expressing

WIPI1 assists WIPI2 in recruiting ATG16L for LC3 lipidation. ( a ) U2OS cells transiently expressing GFP-WIPI1, GFP-WIPI2B, GFP-WIPI2D, GFP-WIPI3, GFP-WIPI4 or GFP alone were starved for 3 h and analysed by anti-GFP immunoprecipitation followed by anti-GFP or anti-ATG16L immunoblotting. The asterisk in the right panel (GFP-IP) represents a nonspecific band. Endogenous ATG16L isoforms co-precipitated with GFP-WIPI1, GFP-WIPI2B and GFP-WIPI2D as indicated in the right panel. ( b , c ) G361 cells stably expressing shRNA targeting WIPI2 ( b , shWIPI2), WIPI1 ( c , shWIPI1) or the non-targeting control (shControl) were fed (F) or starved (S) for 3 h, and endogenous ATG16L was detected using anti-ATG16L/IgG-Alexa Fluor 488 antibodies and fluorescence microscopy. Mean percentages of ATG16L-puncta-positive cells (up to 369 individual cells per condition, n =3) are presented. ( d ) Monoclonal U2OS cells stably expressing GFP-WIPI1 and shWIPI2 or shControl were starved (S) for 3 h, and the percentage of cells displaying an accumulation of perinuclear GFP-WIPI1 was calculated using fluorescence microscopy. Mean values (400 cells per condition, n =4) are presented. ( e , f ) G361 cells stably expressing shRNAs targeting WIPI1 ( e , shWIPI1) or WIPI2 ( f , shWIPI2) were fed (F) or starved (S) for 3 h and immunostained using anti-WIPI4/IgG-Alexa Fluor 488 antibodies. Mean percentages of WIPI4-puncta-positive cells (up to 340 cells per condition, n =3) are presented. ( g ) G361 cells stably expressing shRNAs targeting WIPI3 (shWIPI3), WIPI4 (shWIPI4) or the non-targeting shRNA control (shControl) were fed (F) or starved (S) for 3 h and immunostained using anti-ATG16L/IgG-Alexa Fluor 488 antibodies for analysis by fluorescence microscopy (left panel). Mean percentages of ATG16L-puncta-positive cells (up to 387 cells per condition, n =3) are presented (right panel). ( h , i ) ATG5 wild-type (WT) or knockout (KO) mouse embryonic fibroblasts (MEFs) transiently expressing GFP-WIPI1, GFP-WIPI2B, GFP-WIPI3 or GFP-WIPI4 were analysed by confocal LSM, and images from ATG5 WT MEFs ( h , upper panel) were processed using Volocity to generate 3D-reconstruction fly-through movies ( Supplementary Movies 5–8 ), from which still images are presented ( h , lower panel). Mean percentages of GFP-WIPI puncta-positive cells (300 cells, n =3). Mean±s.d.; heteroscedastic t -testing; P values: * P
Figure Legend Snippet: WIPI1 assists WIPI2 in recruiting ATG16L for LC3 lipidation. ( a ) U2OS cells transiently expressing GFP-WIPI1, GFP-WIPI2B, GFP-WIPI2D, GFP-WIPI3, GFP-WIPI4 or GFP alone were starved for 3 h and analysed by anti-GFP immunoprecipitation followed by anti-GFP or anti-ATG16L immunoblotting. The asterisk in the right panel (GFP-IP) represents a nonspecific band. Endogenous ATG16L isoforms co-precipitated with GFP-WIPI1, GFP-WIPI2B and GFP-WIPI2D as indicated in the right panel. ( b , c ) G361 cells stably expressing shRNA targeting WIPI2 ( b , shWIPI2), WIPI1 ( c , shWIPI1) or the non-targeting control (shControl) were fed (F) or starved (S) for 3 h, and endogenous ATG16L was detected using anti-ATG16L/IgG-Alexa Fluor 488 antibodies and fluorescence microscopy. Mean percentages of ATG16L-puncta-positive cells (up to 369 individual cells per condition, n =3) are presented. ( d ) Monoclonal U2OS cells stably expressing GFP-WIPI1 and shWIPI2 or shControl were starved (S) for 3 h, and the percentage of cells displaying an accumulation of perinuclear GFP-WIPI1 was calculated using fluorescence microscopy. Mean values (400 cells per condition, n =4) are presented. ( e , f ) G361 cells stably expressing shRNAs targeting WIPI1 ( e , shWIPI1) or WIPI2 ( f , shWIPI2) were fed (F) or starved (S) for 3 h and immunostained using anti-WIPI4/IgG-Alexa Fluor 488 antibodies. Mean percentages of WIPI4-puncta-positive cells (up to 340 cells per condition, n =3) are presented. ( g ) G361 cells stably expressing shRNAs targeting WIPI3 (shWIPI3), WIPI4 (shWIPI4) or the non-targeting shRNA control (shControl) were fed (F) or starved (S) for 3 h and immunostained using anti-ATG16L/IgG-Alexa Fluor 488 antibodies for analysis by fluorescence microscopy (left panel). Mean percentages of ATG16L-puncta-positive cells (up to 387 cells per condition, n =3) are presented (right panel). ( h , i ) ATG5 wild-type (WT) or knockout (KO) mouse embryonic fibroblasts (MEFs) transiently expressing GFP-WIPI1, GFP-WIPI2B, GFP-WIPI3 or GFP-WIPI4 were analysed by confocal LSM, and images from ATG5 WT MEFs ( h , upper panel) were processed using Volocity to generate 3D-reconstruction fly-through movies ( Supplementary Movies 5–8 ), from which still images are presented ( h , lower panel). Mean percentages of GFP-WIPI puncta-positive cells (300 cells, n =3). Mean±s.d.; heteroscedastic t -testing; P values: * P

Techniques Used: Expressing, Immunoprecipitation, Stable Transfection, shRNA, Fluorescence, Microscopy, Knock-Out

WIPI3 interacts with FIP200 at nascent autophagosomes and at LAMP2-positive lysosomes/late endosomes. ( a ) Mouse embryonic fibroblasts transiently expressing GFP-WIPI3 or GFP alone were fed (F) or starved (S) for 3 h and analysed by immunoprecipitation using anti-GFP antibodies and anti-FIP200 or anti-GFP immunoblotting. ( b ) U2OS cells stably expressing GFP-WIPI3 were transfected with myc-WIPI2B and starved (S) in the absence or presence of BafA1 for 3 h. Cells were immunostained with anti-myc/IgG-Alexa Fluor 546 or anti-FIP200/IgG-Alexa Fluor 633 antibodies and visualized by confocal LSM. ( c ) U2OS cells stably expressing GFP-WIPI3 were starved (S) in the absence or presence of BafA1 for 3 h, immunostained with anti-FIP200/IgG-Alexa Fluor 546 and anti-p62/IgG-Alexa Fluor 633 antibodies and visualized by confocal LSM. ( d , e ) U2OS cells stably overexpressing GFP-WIPI3 were transfected with control siRNA or FIP200 siRNA. Subsequently, the cells were starved for 3 h in the absence or presence BafA1. FIP200 downregulation was confirmed by immunoblotting using anti-FIP200 and anti-tubulin antibodies ( d ). Mean percentages of GFP-WIPI3-puncta-positive cells (up to 481 cells per condition, n =4) are presented ( e ). ( f ) U2OS cells stably overexpressing GFP-WIPI1 were transfected with control siRNA or FIP200 siRNA. Subsequently, the cells were starved for 3 h with nutrient-free medium in the absence or presence BafA1. Using automated image acquisition and analysis, the mean percentages of GFP-WIPI1-puncta-positive cells (up to 5,856 cells per condition, n =4) were calculated. ( g ) In parallel, this experiment was conducted using GFP-LC3-expressing U2OS cells and mean numbers of GFP-LC3 puncta per cell (up to 4,798 cells per condition, n =4) calculated. ( h ) U2OS cells stably expressing GFP-WIPI3 were starved (S) for 2 h or starved and replenished with amino acids for 1 h (S→AAs). Subsequently, cells were immunostained with anti-FIP200/IgG-Alexa Fluor 546 and anti-LAMP2/IgG-Alexa Fluor 633 antibodies and visualized by confocal LSM. Image magnifications are presented ( b , c , h ) and co-localizations indicated with white arrows. Supplementary Material is available: Supplementary Fig. 6 . Statistics and source data can be found in Supplementary Data 1 . Mean±s.d.; heteroscedastic t -testing; P values: * P
Figure Legend Snippet: WIPI3 interacts with FIP200 at nascent autophagosomes and at LAMP2-positive lysosomes/late endosomes. ( a ) Mouse embryonic fibroblasts transiently expressing GFP-WIPI3 or GFP alone were fed (F) or starved (S) for 3 h and analysed by immunoprecipitation using anti-GFP antibodies and anti-FIP200 or anti-GFP immunoblotting. ( b ) U2OS cells stably expressing GFP-WIPI3 were transfected with myc-WIPI2B and starved (S) in the absence or presence of BafA1 for 3 h. Cells were immunostained with anti-myc/IgG-Alexa Fluor 546 or anti-FIP200/IgG-Alexa Fluor 633 antibodies and visualized by confocal LSM. ( c ) U2OS cells stably expressing GFP-WIPI3 were starved (S) in the absence or presence of BafA1 for 3 h, immunostained with anti-FIP200/IgG-Alexa Fluor 546 and anti-p62/IgG-Alexa Fluor 633 antibodies and visualized by confocal LSM. ( d , e ) U2OS cells stably overexpressing GFP-WIPI3 were transfected with control siRNA or FIP200 siRNA. Subsequently, the cells were starved for 3 h in the absence or presence BafA1. FIP200 downregulation was confirmed by immunoblotting using anti-FIP200 and anti-tubulin antibodies ( d ). Mean percentages of GFP-WIPI3-puncta-positive cells (up to 481 cells per condition, n =4) are presented ( e ). ( f ) U2OS cells stably overexpressing GFP-WIPI1 were transfected with control siRNA or FIP200 siRNA. Subsequently, the cells were starved for 3 h with nutrient-free medium in the absence or presence BafA1. Using automated image acquisition and analysis, the mean percentages of GFP-WIPI1-puncta-positive cells (up to 5,856 cells per condition, n =4) were calculated. ( g ) In parallel, this experiment was conducted using GFP-LC3-expressing U2OS cells and mean numbers of GFP-LC3 puncta per cell (up to 4,798 cells per condition, n =4) calculated. ( h ) U2OS cells stably expressing GFP-WIPI3 were starved (S) for 2 h or starved and replenished with amino acids for 1 h (S→AAs). Subsequently, cells were immunostained with anti-FIP200/IgG-Alexa Fluor 546 and anti-LAMP2/IgG-Alexa Fluor 633 antibodies and visualized by confocal LSM. Image magnifications are presented ( b , c , h ) and co-localizations indicated with white arrows. Supplementary Material is available: Supplementary Fig. 6 . Statistics and source data can be found in Supplementary Data 1 . Mean±s.d.; heteroscedastic t -testing; P values: * P

Techniques Used: Expressing, Immunoprecipitation, Stable Transfection, Transfection

4) Product Images from "Determination of the CD148-Interacting Region in Thrombospondin-1"

Article Title: Determination of the CD148-Interacting Region in Thrombospondin-1

Journal: PLoS ONE

doi: 10.1371/journal.pone.0154916

Trimeric TSP1 fragments that contain the 1 st type 1 repeat increase CD148 catalytic activity and reduce tyrosine phosphorylation of EGFR and ERK1/2 in A431D/CD148wt cells. (A) Left: A431D/CD148wt cells were treated with the indicated trimeric TSP1 fragments (12 nM) or whole TSP1 protein (12 nM) for 15 min. CD148 was immunoprecipitated using anti-CD148 antibody or class-matched control IgG. The washed immunocomplexes were subjected to a PTP activity assay with or without 1 mM sodium orthovanadate (VO 4 ). The amount of CD148 in the immunocomplexes was evaluated by immunoblotting using anti-CD148 antibody (lower panel). The data show mean ± SEM of quadruplicate determinations. Representative data of five independent experiments is shown. ** P
Figure Legend Snippet: Trimeric TSP1 fragments that contain the 1 st type 1 repeat increase CD148 catalytic activity and reduce tyrosine phosphorylation of EGFR and ERK1/2 in A431D/CD148wt cells. (A) Left: A431D/CD148wt cells were treated with the indicated trimeric TSP1 fragments (12 nM) or whole TSP1 protein (12 nM) for 15 min. CD148 was immunoprecipitated using anti-CD148 antibody or class-matched control IgG. The washed immunocomplexes were subjected to a PTP activity assay with or without 1 mM sodium orthovanadate (VO 4 ). The amount of CD148 in the immunocomplexes was evaluated by immunoblotting using anti-CD148 antibody (lower panel). The data show mean ± SEM of quadruplicate determinations. Representative data of five independent experiments is shown. ** P

Techniques Used: Activity Assay, Immunoprecipitation

5) Product Images from "HTLV-1 bZIP factor enhances TGF-? signaling through p300 coactivator"

Article Title: HTLV-1 bZIP factor enhances TGF-? signaling through p300 coactivator

Journal: Blood

doi: 10.1182/blood-2010-12-326199

Physiologic level of HBZ overcame the Tax-mediated suppression of TGF-β signaling. (A) Comparing the level of HBZ protein in HBZ-transfected HepG2 cells with the level of HBZ in ATL and HTLV-1–immortalized cell lines. Total protein was extracted from sHBZ-transfected HepG2 cells (samples from supplemental Figure 1) and the indicated cell lines, and subjected to immunoblotting using HBZ antibody. (B) Endogenous HBZ interacted with Smad3. ATL-55T cells were treated with 10 ng/mL TGF-β. After 10 hours, whole cell lysate was subjected to immunoprecipitation with anti-Smad3 or control IgG, and immunoprecipitates were probed with anti-HBZ antibody. (C) HBZ overcame the repression of TGF-β signaling induced by Tax. In 12-well plates, HepG2 cells were cotransfected with 3TP-Lux (0.5 μg), phRL-TK (2 ng), pCG-Tax (0, 0.2 μg), and pcDNA3.1-mycHis-sHBZ (0, 0.2 μg). At 24 hours after transfection, the cells were treated with or without 10 ng/mL TGF-β. After 24 hours, the cells were harvested and analyzed for luciferase activity. sHBZ and Tax were detected by Western blot (middle panel). CBB staining was shown as the loading control (bottom panel).
Figure Legend Snippet: Physiologic level of HBZ overcame the Tax-mediated suppression of TGF-β signaling. (A) Comparing the level of HBZ protein in HBZ-transfected HepG2 cells with the level of HBZ in ATL and HTLV-1–immortalized cell lines. Total protein was extracted from sHBZ-transfected HepG2 cells (samples from supplemental Figure 1) and the indicated cell lines, and subjected to immunoblotting using HBZ antibody. (B) Endogenous HBZ interacted with Smad3. ATL-55T cells were treated with 10 ng/mL TGF-β. After 10 hours, whole cell lysate was subjected to immunoprecipitation with anti-Smad3 or control IgG, and immunoprecipitates were probed with anti-HBZ antibody. (C) HBZ overcame the repression of TGF-β signaling induced by Tax. In 12-well plates, HepG2 cells were cotransfected with 3TP-Lux (0.5 μg), phRL-TK (2 ng), pCG-Tax (0, 0.2 μg), and pcDNA3.1-mycHis-sHBZ (0, 0.2 μg). At 24 hours after transfection, the cells were treated with or without 10 ng/mL TGF-β. After 24 hours, the cells were harvested and analyzed for luciferase activity. sHBZ and Tax were detected by Western blot (middle panel). CBB staining was shown as the loading control (bottom panel).

Techniques Used: Transfection, Immunoprecipitation, Luciferase, Activity Assay, Western Blot, Staining

6) Product Images from "Decreased Plasma Aβ in Hyperlipidemic APPSL Transgenic Mice Is Associated with BBB Dysfunction"

Article Title: Decreased Plasma Aβ in Hyperlipidemic APPSL Transgenic Mice Is Associated with BBB Dysfunction

Journal: Frontiers in Neuroscience

doi: 10.3389/fnins.2016.00232

Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p
Figure Legend Snippet: Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p

Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Marker

Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p
Figure Legend Snippet: Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p

Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Marker

Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p
Figure Legend Snippet: Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p

Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Marker

Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p
Figure Legend Snippet: Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p

Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Marker

7) Product Images from "Subtype-Specific Translocation of the ? Subtype of Protein Kinase C and Its Activation by Tyrosine Phosphorylation Induced by Ceramide in HeLa Cells"

Article Title: Subtype-Specific Translocation of the ? Subtype of Protein Kinase C and Its Activation by Tyrosine Phosphorylation Induced by Ceramide in HeLa Cells

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.21.5.1769-1783.2001

Localization of fluorescent ceramide (C 6 -NBD-ceramide) in HeLa cells. (A) C 6 -NBD-ceramide (NBD-C 6 -Cer) at 10 μM rapidly accumulated on the plasma membrane and perinuclear region of HeLa cells 1 min after the treatment, and the fluorescence in the perinuclear region increased significantly until 10 min (top row). C 6 -ceramide (C 6 -Cer)-induced translocation of δPKC-GFP showed a similar time course to that of C 6 -NBD-ceramide (bottom row). (B) HeLa cells transfected with δPKC were fixed after treatment with 10 μM C 6 -NBD-ceramide for 20 min. Cells were immunostained with anti-δPKC monoclonal antibody and with Cy3-labeled IgG as secondary antibody to make the expressed δPKC visible. The localization of C 6 -NBD-ceramide (NBD) is shown in green (left) and of δPKC is shown in red (center). On the merged image, the overlapped signals of C 6 -NBD-ceramide and Cy3 appear in yellow (right). The results shown are representative of three independent experiments. Bars, 10 μm (A, top row) and 20 μm (A, bottom row, and B).
Figure Legend Snippet: Localization of fluorescent ceramide (C 6 -NBD-ceramide) in HeLa cells. (A) C 6 -NBD-ceramide (NBD-C 6 -Cer) at 10 μM rapidly accumulated on the plasma membrane and perinuclear region of HeLa cells 1 min after the treatment, and the fluorescence in the perinuclear region increased significantly until 10 min (top row). C 6 -ceramide (C 6 -Cer)-induced translocation of δPKC-GFP showed a similar time course to that of C 6 -NBD-ceramide (bottom row). (B) HeLa cells transfected with δPKC were fixed after treatment with 10 μM C 6 -NBD-ceramide for 20 min. Cells were immunostained with anti-δPKC monoclonal antibody and with Cy3-labeled IgG as secondary antibody to make the expressed δPKC visible. The localization of C 6 -NBD-ceramide (NBD) is shown in green (left) and of δPKC is shown in red (center). On the merged image, the overlapped signals of C 6 -NBD-ceramide and Cy3 appear in yellow (right). The results shown are representative of three independent experiments. Bars, 10 μm (A, top row) and 20 μm (A, bottom row, and B).

Techniques Used: Fluorescence, Translocation Assay, Transfection, Labeling

8) Product Images from "Mutant huntingtin impairs Ku70-mediated DNA repair"

Article Title: Mutant huntingtin impairs Ku70-mediated DNA repair

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200905138

Protein–protein interaction between Ku70 and mutant Htt. (A) IP assay with mouse brain samples. The nuclear extract from brain samples (cerebral cortex and striatum) of R6/2 or nontransgenic littermate (wt) mice at 9 wk were precipitated with anti-Ku70, -1C2, or -HD1 antibody and blotted with the other antibody. In the case of HD1 antibody, wild-type full-length Htt and aggregation of mutant Htt at the gel top are shown in input (arrow). In output, only the mutant Htt was coprecipitated with Ku70. As negative controls, mouse and rabbit IgG were used for precipitation. (B) IP assay showing interaction between endogenous Ku70 and overexpressed mutant Htt88Q exon 1 in HeLa cells. Interaction between normal Htt and endogenous Ku70 was not clear in this condition. (C) Deletion analysis demonstrated that mutant Htt interacts with the N-terminal domain of Ku70. (top) Diagram shows the constructs for the deletion analysis. (bottom) Coprecipitation pattern of GFP-HttQ88 with various Flag-Ku70 deletion mutants. NLS, nuclear localization signal. (D) The amount of mutant Htt necessary for interaction with endogenous Ku70 was titrated with Tet-on stable cells in which mutant Htt expression can be regulated by the concentration of tetracyclin in the medium. (top) Lanes 1–5 indicate a semiquantitative RT-PCR experiment with Htt-specific primers using cDNAs obtained from the Tet-on EGFP-HttQ88 stable cells treated with tetracyclin at different concentrations (0–100,00 pg/ml) for 48 h. The Htt-specific primers are designed to amplify the CAG repeat region to distinguish the endogenous Htt and induced EGFP-HttQ88 genes. The single asterisk denotes induced GFP-HttQ88 transcripts, the double asterisk denotes endogenous Htt transcripts (lanes 1–5). As a control, corresponding PCR with GAPDH-specific primers was performed. As a semiquantitative PCR calibration, PCR was performed with the Htt primers using plasmid DNAs, pEGFP-HttQ17 (lanes 6–11), and pEGFP-HttQ88 (lanes 12–17) as PCR templates with indicated amounts to evaluate the RT-PCR. (middle) Equal amounts of cell lysate from the Tet-on stable cell lines treated with the indicated concentration of tetracyclin for 48 h were blotted with anti-GFP antibody. (bottom) The lysates were also subjected to IP with anti-GFP antibody, and the precipitants were blotted with anti-Ku70 and GFP antibodies. IB, immunoblot.
Figure Legend Snippet: Protein–protein interaction between Ku70 and mutant Htt. (A) IP assay with mouse brain samples. The nuclear extract from brain samples (cerebral cortex and striatum) of R6/2 or nontransgenic littermate (wt) mice at 9 wk were precipitated with anti-Ku70, -1C2, or -HD1 antibody and blotted with the other antibody. In the case of HD1 antibody, wild-type full-length Htt and aggregation of mutant Htt at the gel top are shown in input (arrow). In output, only the mutant Htt was coprecipitated with Ku70. As negative controls, mouse and rabbit IgG were used for precipitation. (B) IP assay showing interaction between endogenous Ku70 and overexpressed mutant Htt88Q exon 1 in HeLa cells. Interaction between normal Htt and endogenous Ku70 was not clear in this condition. (C) Deletion analysis demonstrated that mutant Htt interacts with the N-terminal domain of Ku70. (top) Diagram shows the constructs for the deletion analysis. (bottom) Coprecipitation pattern of GFP-HttQ88 with various Flag-Ku70 deletion mutants. NLS, nuclear localization signal. (D) The amount of mutant Htt necessary for interaction with endogenous Ku70 was titrated with Tet-on stable cells in which mutant Htt expression can be regulated by the concentration of tetracyclin in the medium. (top) Lanes 1–5 indicate a semiquantitative RT-PCR experiment with Htt-specific primers using cDNAs obtained from the Tet-on EGFP-HttQ88 stable cells treated with tetracyclin at different concentrations (0–100,00 pg/ml) for 48 h. The Htt-specific primers are designed to amplify the CAG repeat region to distinguish the endogenous Htt and induced EGFP-HttQ88 genes. The single asterisk denotes induced GFP-HttQ88 transcripts, the double asterisk denotes endogenous Htt transcripts (lanes 1–5). As a control, corresponding PCR with GAPDH-specific primers was performed. As a semiquantitative PCR calibration, PCR was performed with the Htt primers using plasmid DNAs, pEGFP-HttQ17 (lanes 6–11), and pEGFP-HttQ88 (lanes 12–17) as PCR templates with indicated amounts to evaluate the RT-PCR. (middle) Equal amounts of cell lysate from the Tet-on stable cell lines treated with the indicated concentration of tetracyclin for 48 h were blotted with anti-GFP antibody. (bottom) The lysates were also subjected to IP with anti-GFP antibody, and the precipitants were blotted with anti-Ku70 and GFP antibodies. IB, immunoblot.

Techniques Used: Mutagenesis, Mouse Assay, Construct, Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Plasmid Preparation, Stable Transfection

9) Product Images from "A signaling organelle containing the nerve growth factor-activated receptor tyrosine kinase, TrkA"

Article Title: A signaling organelle containing the nerve growth factor-activated receptor tyrosine kinase, TrkA

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

( A ) TrkA was crosslinked to NGF in small endocytic vesicles. Cells were bound to 125 I-NGF, washed, warmed 10 min, chilled and analyzed after an in vitro reaction with ATP. The membrane permeable crosslinking reagent disuccinimidyl suberate was added (2 mM, 4°C, 30 min) to the permeabilized cell suspension before fractionation of membranes. One-fifth of the cell ghost membranes (1,000 × g pellet, P1), one-half of the 8,000 × g pellet (P2) one-half of the 100,000 × g pellet (P3), and one-tenth of the 100,000 × g ) and analyzed by SDS/PAGE and autoradiography (22 day exposure). The position of molecular weight markers (kDa) is indicated. ( B ) TrkA is still activated in intracellular organelles after in vitro reactions. Untreated PC12 cells or PC12 cells bound to NGF (1 nM, 4°C) were warmed 10 min and fractionated and immunoprecipitated as in A after in vitro ) was used for immunoprecipitations. Western blots were probed with RTA followed by horseradish peroxidase-conjugated anti-rabbit IgG and ECL ( Left , 1 min exposure). Two proteins were identified, gp140 TrkA and gp110 TrkA (indicated). The latter is a precursor to gp140 TrkA ), and remained mostly with the cell ghosts after in vitro reactions with ATP. TrkA immunoprecipitates were also probed with anti-phosphotyrosine (4G10, a gift of S. Robbins and M. Bishop, University of California, San Francisco) followed by 125 I-goat anti-mouse IgG ( Right , 34 day exposure). Mature 140-kDa TrkA was tyrosine phosphorylated, while the 110-kDa immature glycosylated TrkA was not. Equal amounts of cells were used to compare conditions. The top and bottom edges of the panels mark the position of the 200- and 97.4-kDa molecular weight markers, respectively. ( C–E ) Quantification of TrkA and tyrosine phosphorylated TrkA in intracellular organelles. Data for gp140 TrkA ( C ) and gp110 TrkA ( D ) from 4 experiments as in B ( Left ) were quantified by densitometry and plotted as a percent of the total in all cell fractions with error bars showing standard deviations. The conditions and fractions are labeled at the left. Data for tyrosine phosphorylated TrkA ( E ) from four experiments as in ( B Right ) were quantified by phosphorimaging or densitometry and plotted in reference to total tyrosine phosphorylated TrkA following NGF treatment with error bars as in C . ( F ) Sodium orthovanadate was added to in vitro incubations with ATP. Untreated cells ( Left ) and NGF-treated cells ( Right ) were permeabilized and incubated in vitro with ATP in the presence of 1 mM sodium orthovanadate. TrkA was immunoprecipitated as in A ). In the P3 fraction, PLC-γ, then TrkA was immunoprecipitated from the P3 fractions (indicated by IP). Proteins were Western blotted with antiphosphotyrosine followed by horseradish peroxidase-conjugated antimouse and detected using ECL (20 sec exposure). Equal amounts of cells were used to compare conditions. The top and bottom edges of the panels mark the position of the 200- and 97.4-kDa molecular weight markers, respectively.
Figure Legend Snippet: ( A ) TrkA was crosslinked to NGF in small endocytic vesicles. Cells were bound to 125 I-NGF, washed, warmed 10 min, chilled and analyzed after an in vitro reaction with ATP. The membrane permeable crosslinking reagent disuccinimidyl suberate was added (2 mM, 4°C, 30 min) to the permeabilized cell suspension before fractionation of membranes. One-fifth of the cell ghost membranes (1,000 × g pellet, P1), one-half of the 8,000 × g pellet (P2) one-half of the 100,000 × g pellet (P3), and one-tenth of the 100,000 × g ) and analyzed by SDS/PAGE and autoradiography (22 day exposure). The position of molecular weight markers (kDa) is indicated. ( B ) TrkA is still activated in intracellular organelles after in vitro reactions. Untreated PC12 cells or PC12 cells bound to NGF (1 nM, 4°C) were warmed 10 min and fractionated and immunoprecipitated as in A after in vitro ) was used for immunoprecipitations. Western blots were probed with RTA followed by horseradish peroxidase-conjugated anti-rabbit IgG and ECL ( Left , 1 min exposure). Two proteins were identified, gp140 TrkA and gp110 TrkA (indicated). The latter is a precursor to gp140 TrkA ), and remained mostly with the cell ghosts after in vitro reactions with ATP. TrkA immunoprecipitates were also probed with anti-phosphotyrosine (4G10, a gift of S. Robbins and M. Bishop, University of California, San Francisco) followed by 125 I-goat anti-mouse IgG ( Right , 34 day exposure). Mature 140-kDa TrkA was tyrosine phosphorylated, while the 110-kDa immature glycosylated TrkA was not. Equal amounts of cells were used to compare conditions. The top and bottom edges of the panels mark the position of the 200- and 97.4-kDa molecular weight markers, respectively. ( C–E ) Quantification of TrkA and tyrosine phosphorylated TrkA in intracellular organelles. Data for gp140 TrkA ( C ) and gp110 TrkA ( D ) from 4 experiments as in B ( Left ) were quantified by densitometry and plotted as a percent of the total in all cell fractions with error bars showing standard deviations. The conditions and fractions are labeled at the left. Data for tyrosine phosphorylated TrkA ( E ) from four experiments as in ( B Right ) were quantified by phosphorimaging or densitometry and plotted in reference to total tyrosine phosphorylated TrkA following NGF treatment with error bars as in C . ( F ) Sodium orthovanadate was added to in vitro incubations with ATP. Untreated cells ( Left ) and NGF-treated cells ( Right ) were permeabilized and incubated in vitro with ATP in the presence of 1 mM sodium orthovanadate. TrkA was immunoprecipitated as in A ). In the P3 fraction, PLC-γ, then TrkA was immunoprecipitated from the P3 fractions (indicated by IP). Proteins were Western blotted with antiphosphotyrosine followed by horseradish peroxidase-conjugated antimouse and detected using ECL (20 sec exposure). Equal amounts of cells were used to compare conditions. The top and bottom edges of the panels mark the position of the 200- and 97.4-kDa molecular weight markers, respectively.

Techniques Used: In Vitro, Fractionation, SDS Page, Autoradiography, Molecular Weight, Immunoprecipitation, Western Blot, Labeling, Incubation, Planar Chromatography, Size-exclusion Chromatography

10) Product Images from "Decreased Plasma Aβ in Hyperlipidemic APPSL Transgenic Mice Is Associated with BBB Dysfunction"

Article Title: Decreased Plasma Aβ in Hyperlipidemic APPSL Transgenic Mice Is Associated with BBB Dysfunction

Journal: Frontiers in Neuroscience

doi: 10.3389/fnins.2016.00232

Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p
Figure Legend Snippet: Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p

Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Marker

Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p
Figure Legend Snippet: Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p

Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Marker

Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p
Figure Legend Snippet: Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p

Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Marker

Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p
Figure Legend Snippet: Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p

Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Marker

11) Product Images from "Decreased Plasma Aβ in Hyperlipidemic APPSL Transgenic Mice Is Associated with BBB Dysfunction"

Article Title: Decreased Plasma Aβ in Hyperlipidemic APPSL Transgenic Mice Is Associated with BBB Dysfunction

Journal: Frontiers in Neuroscience

doi: 10.3389/fnins.2016.00232

Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p
Figure Legend Snippet: Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p

Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Marker

Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p
Figure Legend Snippet: Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p

Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Marker

Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p
Figure Legend Snippet: Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p

Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Marker

Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p
Figure Legend Snippet: Changes in protein levels of EC and BBB integrity associated markers due to HFD and genotype measured with ELISA . IgG influx into the cortex in APP SL and WT mice on HFD and ND (A) . Cortical levels of the tight junction markers Occludin (B) and ZO1 (C) in APP SL and WT mice on both diets. LRP1 (D) as well as EC marker CD31 (E) and VCAM1 (F) levels in the cortex of APP SL and WT mice on ND and HFD. Relative differences between groups were expressed as x-fold change to WT on ND. N = 5–8 animals per group; Statistical analyses: Two-way-ANOVA with Bonferroni's post-test, * p

Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Marker

12) Product Images from "SILAC-Based Proteomic Profiling of the Human MDA-MB-231 Metastatic Breast Cancer Cell Line in Response to the Two Antitumoral Lactoferrin Isoforms: The Secreted Lactoferrin and the Intracellular Delta-Lactoferrin"

Article Title: SILAC-Based Proteomic Profiling of the Human MDA-MB-231 Metastatic Breast Cancer Cell Line in Response to the Two Antitumoral Lactoferrin Isoforms: The Secreted Lactoferrin and the Intracellular Delta-Lactoferrin

Journal: PLoS ONE

doi: 10.1371/journal.pone.0104563

In vivo recruitment of ΔLf on new target genes, GTF2F2 and UBE2E1 . Panel A. Differential protein expression was confirmed using Western blotting. MDA-MB-231 dox- cells were lysed 24 h after treatment and samples (20 µg of protein) were subjected to SDS-PAGE and immunoblotted with antibodies specific to GTF2F2 and UBE2E1. Histone H2B served as loading control. Panel B. GTF2F2 and UBE2E1 mRNA are upregulated in ΔLf-expressing MDA-MB-231 dox- cells. Cells were either untreated or induced with doxycycline (2 µg/mL) or transfected with pcDNA-ΔLf or treated with exogenous hLf (50 or 500 µg/mL). mRNA content was determined by qRT-PCR. Panel C. HEK 293 cells were transfected with the pCMV-3XFLAG-ΔLf or pcDNA-hLf. 24 h post transfection, a ChIP assay was performed. ChIP product was then amplified by real time PCR using specific primer pairs targeting the ΔLfRE containing fragment of the each targeted promoter. ChIP assays were performed using an anti-FLAG (M2), an anti-hLf (α-Lf) and anti-rabbit IgG as nonspecific antibody control (IR). As a further control, the assay was performed without binding of an antibody to the protein G plus Sepharose (NIP). The isolated genomic DNA was analysed by real time PCR using primers specific for ΔLfRE putative binding site on GTF2F2 and UBE2E1 promoters. The results were normalized with the levels of ΔLfRE present in the samples (input). Data are expressed as fold enrichment related to null-transfected cells, and are the mean ±SD of triplicates from three independent assays. *p
Figure Legend Snippet: In vivo recruitment of ΔLf on new target genes, GTF2F2 and UBE2E1 . Panel A. Differential protein expression was confirmed using Western blotting. MDA-MB-231 dox- cells were lysed 24 h after treatment and samples (20 µg of protein) were subjected to SDS-PAGE and immunoblotted with antibodies specific to GTF2F2 and UBE2E1. Histone H2B served as loading control. Panel B. GTF2F2 and UBE2E1 mRNA are upregulated in ΔLf-expressing MDA-MB-231 dox- cells. Cells were either untreated or induced with doxycycline (2 µg/mL) or transfected with pcDNA-ΔLf or treated with exogenous hLf (50 or 500 µg/mL). mRNA content was determined by qRT-PCR. Panel C. HEK 293 cells were transfected with the pCMV-3XFLAG-ΔLf or pcDNA-hLf. 24 h post transfection, a ChIP assay was performed. ChIP product was then amplified by real time PCR using specific primer pairs targeting the ΔLfRE containing fragment of the each targeted promoter. ChIP assays were performed using an anti-FLAG (M2), an anti-hLf (α-Lf) and anti-rabbit IgG as nonspecific antibody control (IR). As a further control, the assay was performed without binding of an antibody to the protein G plus Sepharose (NIP). The isolated genomic DNA was analysed by real time PCR using primers specific for ΔLfRE putative binding site on GTF2F2 and UBE2E1 promoters. The results were normalized with the levels of ΔLfRE present in the samples (input). Data are expressed as fold enrichment related to null-transfected cells, and are the mean ±SD of triplicates from three independent assays. *p

Techniques Used: In Vivo, Expressing, Western Blot, Multiple Displacement Amplification, SDS Page, Transfection, Quantitative RT-PCR, Chromatin Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Binding Assay, Isolation

Both ΔLf and hLf act as transcription factors and target the SelH promoter. Panels A and B. SelH mRNA overexpression is not cell specific. MDA-MB 231, HeLa, MCF7 and HEK 293 cells were induced with doxycycline (2 µg/mL) or transfected with pcDNA-ΔLf (A). MDA-MB 231, HeLa, MCF7 and HEK 293 cells were transfected with pcDNA-hLf whereas only MDA-MB 231 cells were treated with exogenous hLf (50 µg/mL) (B). The expression pattern of SelH transcripts in MDA-MB-231, HeLa, MCF-7 and HEK-293 cells after treatment was followed by qRT-PCR. Data are normalized to HPRT and are expressed as the fold increase (2 −ΔΔCt ) under ΔLf (A) or hLf (B) treatment (n = 3). Panel C. HEK 293 cells were cotransfected with pGL3-SelH-Luc construct (50 ng/well) and pcDNA-hLf expression vector (200 ng/well) encoding intracellular hLf or pcDNA-ΔLf expression vector (200 ng/well) encoding ΔLf. 24 h after transfection, cells were lysed and samples were assayed for protein content and luciferase activity. The relative luciferase activity reported is expressed as the fold increase of the ratio of the pGL3 reporter activity to protein content. Values represent the mean ±SD of triplicates from three independent measurements. Panel D. ΔLf and hLf are recruited in vivo on the SelH promoter. HEK 293 cells were transfected with the pcDNA-hLf or the pCMV-3XFLAG-ΔLf. 24 h post transfection, ChIP assays were performed, using an anti-FLAG (M2), an anti-hLf (α-Lf) and anti-rabbit IgG as nonspecific antibody control (IR). As a further control, the assay was performed without binding of an antibody to the protein G plus Sepharose (NIP). The isolated genomic DNA was analyzed by real time PCR using primers that link the ΔLfRE binding site on the SelH promoter. The results were normalized with the levels of ΔLfRE present in the samples (input). Data are expressed as fold enrichment related to null-transfected cells, and are the mean ±SD of triplicates from three independent assays. *p
Figure Legend Snippet: Both ΔLf and hLf act as transcription factors and target the SelH promoter. Panels A and B. SelH mRNA overexpression is not cell specific. MDA-MB 231, HeLa, MCF7 and HEK 293 cells were induced with doxycycline (2 µg/mL) or transfected with pcDNA-ΔLf (A). MDA-MB 231, HeLa, MCF7 and HEK 293 cells were transfected with pcDNA-hLf whereas only MDA-MB 231 cells were treated with exogenous hLf (50 µg/mL) (B). The expression pattern of SelH transcripts in MDA-MB-231, HeLa, MCF-7 and HEK-293 cells after treatment was followed by qRT-PCR. Data are normalized to HPRT and are expressed as the fold increase (2 −ΔΔCt ) under ΔLf (A) or hLf (B) treatment (n = 3). Panel C. HEK 293 cells were cotransfected with pGL3-SelH-Luc construct (50 ng/well) and pcDNA-hLf expression vector (200 ng/well) encoding intracellular hLf or pcDNA-ΔLf expression vector (200 ng/well) encoding ΔLf. 24 h after transfection, cells were lysed and samples were assayed for protein content and luciferase activity. The relative luciferase activity reported is expressed as the fold increase of the ratio of the pGL3 reporter activity to protein content. Values represent the mean ±SD of triplicates from three independent measurements. Panel D. ΔLf and hLf are recruited in vivo on the SelH promoter. HEK 293 cells were transfected with the pcDNA-hLf or the pCMV-3XFLAG-ΔLf. 24 h post transfection, ChIP assays were performed, using an anti-FLAG (M2), an anti-hLf (α-Lf) and anti-rabbit IgG as nonspecific antibody control (IR). As a further control, the assay was performed without binding of an antibody to the protein G plus Sepharose (NIP). The isolated genomic DNA was analyzed by real time PCR using primers that link the ΔLfRE binding site on the SelH promoter. The results were normalized with the levels of ΔLfRE present in the samples (input). Data are expressed as fold enrichment related to null-transfected cells, and are the mean ±SD of triplicates from three independent assays. *p

Techniques Used: Activated Clotting Time Assay, Over Expression, Multiple Displacement Amplification, Transfection, Expressing, Quantitative RT-PCR, Construct, Plasmid Preparation, Luciferase, Activity Assay, In Vivo, Chromatin Immunoprecipitation, Binding Assay, Isolation, Real-time Polymerase Chain Reaction

13) Product Images from "Label-Free Proteomic Identification of Endogenous, Insulin-Stimulated Interaction Partners of Insulin Receptor Substrate-1"

Article Title: Label-Free Proteomic Identification of Endogenous, Insulin-Stimulated Interaction Partners of Insulin Receptor Substrate-1

Journal: Journal of the American Society for Mass Spectrometry

doi: 10.1007/s13361-010-0051-2

PPP1R12A and the p85 regulatory subunit of PI 3-K interact with IRS-1. L6 myotubes were serum-starved for 4 h and either left untreated or stimulated with insulin (100 nM) for 15 min at 37 °C. One mg of lysate proteins were immunoprecipitated with normal IgG or IRS-1 antibodies and Western blotted with antibodies specific to either IRS-1, PPP1R12A, or p85; each graph is representative of data obtained from three independent experiments
Figure Legend Snippet: PPP1R12A and the p85 regulatory subunit of PI 3-K interact with IRS-1. L6 myotubes were serum-starved for 4 h and either left untreated or stimulated with insulin (100 nM) for 15 min at 37 °C. One mg of lysate proteins were immunoprecipitated with normal IgG or IRS-1 antibodies and Western blotted with antibodies specific to either IRS-1, PPP1R12A, or p85; each graph is representative of data obtained from three independent experiments

Techniques Used: Immunoprecipitation, Western Blot

14) Product Images from "Retinoid and thiazolidinedione therapies in melanoma: an analysis of differential response based on nuclear hormone receptor expression"

Article Title: Retinoid and thiazolidinedione therapies in melanoma: an analysis of differential response based on nuclear hormone receptor expression

Journal: Molecular Cancer

doi: 10.1186/1476-4598-8-16

Western blot of nuclear hormone receptors in A375(DRO) resistant cell lines . 3a : 60 μg of nuclear protein extract from the resistant A375(DRO) sublines was size-separated on a 10% SDS-PAGE gel and transferred to nitrocellulose. The blot was blocked with 10% nonfat milk and incubated with RXRγ (MS-1343-P NeoMarkers) and RXRα (sc D-20) antibodies at a concentration of 1:500 and PPARγ (H-100) rabbit polyclonal ab (sc-7196, Santa Cruz Biotechnology, Santa Cruz, CA) at 1:500. Secondary antibodies were anti-rabbit IgG conjugated to horse-radish peroxidase at a 1:5000 dilution for RXRs and 1:1000 for PPARγ (GE Healthcare UK). β-Actin was measured as a loading control. 3b: PPARγ receptor to β-Actin ratio was calculated using an Alpha Innotech alpha imager.
Figure Legend Snippet: Western blot of nuclear hormone receptors in A375(DRO) resistant cell lines . 3a : 60 μg of nuclear protein extract from the resistant A375(DRO) sublines was size-separated on a 10% SDS-PAGE gel and transferred to nitrocellulose. The blot was blocked with 10% nonfat milk and incubated with RXRγ (MS-1343-P NeoMarkers) and RXRα (sc D-20) antibodies at a concentration of 1:500 and PPARγ (H-100) rabbit polyclonal ab (sc-7196, Santa Cruz Biotechnology, Santa Cruz, CA) at 1:500. Secondary antibodies were anti-rabbit IgG conjugated to horse-radish peroxidase at a 1:5000 dilution for RXRs and 1:1000 for PPARγ (GE Healthcare UK). β-Actin was measured as a loading control. 3b: PPARγ receptor to β-Actin ratio was calculated using an Alpha Innotech alpha imager.

Techniques Used: Western Blot, SDS Page, Incubation, Mass Spectrometry, Concentration Assay

15) Product Images from "Loss of the SxxSS Motif in a Human T-Cell Factor-4 Isoform Confers Hypoxia Resistance to Liver Cancer: An Oncogenic Switch in Wnt Signaling"

Article Title: Loss of the SxxSS Motif in a Human T-Cell Factor-4 Isoform Confers Hypoxia Resistance to Liver Cancer: An Oncogenic Switch in Wnt Signaling

Journal: PLoS ONE

doi: 10.1371/journal.pone.0039981

Lack of SxxSS motif contributes to protein stability of HIF-αs in J cells. (A) Cells were treated with 150 µM CoCl 2 for 36 hr followed by incubation with or without MG-132 (10 µM) for 2 hr. Total cell lysates were employed for immunoblot analysis. Relative expression of HIF-αs was normalized to actin. (B) Protein stability of HIF-αs was evaluated by immunoblot analysis, where cells were exposed to 5 µg/mL cycloheximide (CHX) to inhibit protein synthesis for 0 or 60 min at the last phase of the 48 hr CoCl 2 treatment period. (C) Expression of VHL in TCF-4J and K cells. Cells were treated 0 or 150 µM CoCl 2 and nuclear and cytoplamic fractions was prepared for immunoblot analysis. Expression level of HIF-2α and VHL was normalized by lamin (bottom panel); VHL-C, cytoplasmic VHL. (D) Interaction between VHL and HIF-2α in nucleus of TCF-4J and K cells. (E) Polyubiquitination of HIF-2α in cytoplasmic and nuclear fractions of TCF-4J and K cells. Immunoprecipitation (IP)/immunoblot (IB) analysis was performed by using antibodies for HIF-2α and ubiquitin (Ub); *, IgG heavy chain. Note the decreased ubiquitination of HIF-2α and weak VHL-HIF-2α interaction in J cells, which was in contrast to the observations in K cells.
Figure Legend Snippet: Lack of SxxSS motif contributes to protein stability of HIF-αs in J cells. (A) Cells were treated with 150 µM CoCl 2 for 36 hr followed by incubation with or without MG-132 (10 µM) for 2 hr. Total cell lysates were employed for immunoblot analysis. Relative expression of HIF-αs was normalized to actin. (B) Protein stability of HIF-αs was evaluated by immunoblot analysis, where cells were exposed to 5 µg/mL cycloheximide (CHX) to inhibit protein synthesis for 0 or 60 min at the last phase of the 48 hr CoCl 2 treatment period. (C) Expression of VHL in TCF-4J and K cells. Cells were treated 0 or 150 µM CoCl 2 and nuclear and cytoplamic fractions was prepared for immunoblot analysis. Expression level of HIF-2α and VHL was normalized by lamin (bottom panel); VHL-C, cytoplasmic VHL. (D) Interaction between VHL and HIF-2α in nucleus of TCF-4J and K cells. (E) Polyubiquitination of HIF-2α in cytoplasmic and nuclear fractions of TCF-4J and K cells. Immunoprecipitation (IP)/immunoblot (IB) analysis was performed by using antibodies for HIF-2α and ubiquitin (Ub); *, IgG heavy chain. Note the decreased ubiquitination of HIF-2α and weak VHL-HIF-2α interaction in J cells, which was in contrast to the observations in K cells.

Techniques Used: Incubation, Expressing, Immunoprecipitation

16) Product Images from "WIPI3 and WIPI4 β-propellers are scaffolds for LKB1-AMPK-TSC signalling circuits in the control of autophagy"

Article Title: WIPI3 and WIPI4 β-propellers are scaffolds for LKB1-AMPK-TSC signalling circuits in the control of autophagy

Journal: Nature Communications

doi: 10.1038/ncomms15637

WIPI3 interacts with the TSC complex. ( a ) Stable GFP-WIPI1, GFP-WIPI2B, GFP-WIPI3 or GFP-WIPI4 U2OS cells were starved (3 h), and subjected to anti-GFP immunoprecipitation and anti-TSC2, anti-phospho-TSC2 (S1387), anti-TSC1 or anti-GFP immunoblotting. ( b ) U2OS cells were analysed by anti-TSC1 immunoprecipitation (TSC1-IP), anti-TSC1, anti-TSC2 and anti-WIPI1 (right panel), anti-WIPI2 (right panel), anti-WIPI3 (left panel) or anti-WIPI4 (right panel) immunoblotting. ( c ) ATG5 WT or KO MEFs expressing GFP-WIPI3 (W3) or GFP were subjected to anti-GFP immunoprecipitation and anti-TSC1, anti-TSC2 and anti-GFP immunoblotting. Conditions (3 h): fed (F) or starved (S). ( d ) Stable GFP-WIPI3 or GFP U2OS cells were analysed by anti-GFP immunoprecipitation and anti-TSC2 and anti-GFP immunoblotting. Conditions (15 min. to 3 h): fed (F), starved (S), starved with LY294002 (S+LY). ( e ) Myc-tagged TSC1 fragments (TSC1-M, TSC1-C; full length: TSC-FL) were expressed in stable GFP-WIPI3 U2OS cells and subjected to anti-GFP immunoprecipitation and anti-GFP or anti-myc immunoblotting. ( f ) Lysosomal fractions (no. 1–7; total protein control: TP) from stable GFP-WIPI3 U2OS cells (BafA1, 3 h) were immunoblotted using anti-GFP, anti-LAMP2, anti-TSC2, anti-WIPI2 or anti-Rab4 antibodies. ( g ) Stable GFP-WIPI3 U2OS cells were starved (3 h) and immunostained with anti-TSC2/IgG-Alexa Fluor 546 and anti-LAMP2/IgG-Alexa Fluor 633 antibodies for confocal LSM. ( h ) Stable GFP-WIPI3 U2OS cells were immunostained with anti-Lamp2/IgG-Alexa Fluor 633 and anti-WIPI2/IgG-Alexa Fluor 546 for confocal LSM. Conditions (3 h): fed (F), starved (S). ( i ) Stable GFP-WIPI3 U2OS cells with siControl or siTSC2 were fed (F) or starved (S) with or without BafA1 (3 h). Upper panel: anti-TSC2 immunoblotting, lower panel: GFP-WIPI3 puncta assessment (up to 493 cells per condition, n =4). ( j ) U2OS cells were subjected to immunoblotting using anti-phospho-mTOR (S2481), anti-mTOR, anti-phospho-ULK1 (S757), anti-ULK1 and anti-tubulin antibodies. Treatments: fed (F, 4 h), starved (S, 4 h), starved (3 h) and fed (1 h) (S→F). ( k ) Stable GFP-WIPI3 U2OS cells were immunostained with anti-mTOR/IgG-Alexa Fluor 546 and anti-LAMP2/IgG-Alexa Fluor 633 antibodies. Treatments: starved (S, 2 h), starved (2 h) and fed (1 h) (S→F). Co-localizations are indicated with arrows ( g , h , k ). Supplementary Material is available: Supplementary Fig. 5 . Statistics and source data: Supplementary Data 1 . Mean±s.d.; heteroscedastic t -testing; P values: * P
Figure Legend Snippet: WIPI3 interacts with the TSC complex. ( a ) Stable GFP-WIPI1, GFP-WIPI2B, GFP-WIPI3 or GFP-WIPI4 U2OS cells were starved (3 h), and subjected to anti-GFP immunoprecipitation and anti-TSC2, anti-phospho-TSC2 (S1387), anti-TSC1 or anti-GFP immunoblotting. ( b ) U2OS cells were analysed by anti-TSC1 immunoprecipitation (TSC1-IP), anti-TSC1, anti-TSC2 and anti-WIPI1 (right panel), anti-WIPI2 (right panel), anti-WIPI3 (left panel) or anti-WIPI4 (right panel) immunoblotting. ( c ) ATG5 WT or KO MEFs expressing GFP-WIPI3 (W3) or GFP were subjected to anti-GFP immunoprecipitation and anti-TSC1, anti-TSC2 and anti-GFP immunoblotting. Conditions (3 h): fed (F) or starved (S). ( d ) Stable GFP-WIPI3 or GFP U2OS cells were analysed by anti-GFP immunoprecipitation and anti-TSC2 and anti-GFP immunoblotting. Conditions (15 min. to 3 h): fed (F), starved (S), starved with LY294002 (S+LY). ( e ) Myc-tagged TSC1 fragments (TSC1-M, TSC1-C; full length: TSC-FL) were expressed in stable GFP-WIPI3 U2OS cells and subjected to anti-GFP immunoprecipitation and anti-GFP or anti-myc immunoblotting. ( f ) Lysosomal fractions (no. 1–7; total protein control: TP) from stable GFP-WIPI3 U2OS cells (BafA1, 3 h) were immunoblotted using anti-GFP, anti-LAMP2, anti-TSC2, anti-WIPI2 or anti-Rab4 antibodies. ( g ) Stable GFP-WIPI3 U2OS cells were starved (3 h) and immunostained with anti-TSC2/IgG-Alexa Fluor 546 and anti-LAMP2/IgG-Alexa Fluor 633 antibodies for confocal LSM. ( h ) Stable GFP-WIPI3 U2OS cells were immunostained with anti-Lamp2/IgG-Alexa Fluor 633 and anti-WIPI2/IgG-Alexa Fluor 546 for confocal LSM. Conditions (3 h): fed (F), starved (S). ( i ) Stable GFP-WIPI3 U2OS cells with siControl or siTSC2 were fed (F) or starved (S) with or without BafA1 (3 h). Upper panel: anti-TSC2 immunoblotting, lower panel: GFP-WIPI3 puncta assessment (up to 493 cells per condition, n =4). ( j ) U2OS cells were subjected to immunoblotting using anti-phospho-mTOR (S2481), anti-mTOR, anti-phospho-ULK1 (S757), anti-ULK1 and anti-tubulin antibodies. Treatments: fed (F, 4 h), starved (S, 4 h), starved (3 h) and fed (1 h) (S→F). ( k ) Stable GFP-WIPI3 U2OS cells were immunostained with anti-mTOR/IgG-Alexa Fluor 546 and anti-LAMP2/IgG-Alexa Fluor 633 antibodies. Treatments: starved (S, 2 h), starved (2 h) and fed (1 h) (S→F). Co-localizations are indicated with arrows ( g , h , k ). Supplementary Material is available: Supplementary Fig. 5 . Statistics and source data: Supplementary Data 1 . Mean±s.d.; heteroscedastic t -testing; P values: * P

Techniques Used: Immunoprecipitation, Expressing

WIPI1 assists WIPI2 in recruiting ATG16L for LC3 lipidation. ( a ) U2OS cells transiently expressing GFP-WIPI1, GFP-WIPI2B, GFP-WIPI2D, GFP-WIPI3, GFP-WIPI4 or GFP alone were starved for 3 h and analysed by anti-GFP immunoprecipitation followed by anti-GFP or anti-ATG16L immunoblotting. The asterisk in the right panel (GFP-IP) represents a nonspecific band. Endogenous ATG16L isoforms co-precipitated with GFP-WIPI1, GFP-WIPI2B and GFP-WIPI2D as indicated in the right panel. ( b , c ) G361 cells stably expressing shRNA targeting WIPI2 ( b , shWIPI2), WIPI1 ( c , shWIPI1) or the non-targeting control (shControl) were fed (F) or starved (S) for 3 h, and endogenous ATG16L was detected using anti-ATG16L/IgG-Alexa Fluor 488 antibodies and fluorescence microscopy. Mean percentages of ATG16L-puncta-positive cells (up to 369 individual cells per condition, n =3) are presented. ( d ) Monoclonal U2OS cells stably expressing GFP-WIPI1 and shWIPI2 or shControl were starved (S) for 3 h, and the percentage of cells displaying an accumulation of perinuclear GFP-WIPI1 was calculated using fluorescence microscopy. Mean values (400 cells per condition, n =4) are presented. ( e , f ) G361 cells stably expressing shRNAs targeting WIPI1 ( e , shWIPI1) or WIPI2 ( f , shWIPI2) were fed (F) or starved (S) for 3 h and immunostained using anti-WIPI4/IgG-Alexa Fluor 488 antibodies. Mean percentages of WIPI4-puncta-positive cells (up to 340 cells per condition, n =3) are presented. ( g ) G361 cells stably expressing shRNAs targeting WIPI3 (shWIPI3), WIPI4 (shWIPI4) or the non-targeting shRNA control (shControl) were fed (F) or starved (S) for 3 h and immunostained using anti-ATG16L/IgG-Alexa Fluor 488 antibodies for analysis by fluorescence microscopy (left panel). Mean percentages of ATG16L-puncta-positive cells (up to 387 cells per condition, n =3) are presented (right panel). ( h , i ) ATG5 wild-type (WT) or knockout (KO) mouse embryonic fibroblasts (MEFs) transiently expressing GFP-WIPI1, GFP-WIPI2B, GFP-WIPI3 or GFP-WIPI4 were analysed by confocal LSM, and images from ATG5 WT MEFs ( h , upper panel) were processed using Volocity to generate 3D-reconstruction fly-through movies ( Supplementary Movies 5–8 ), from which still images are presented ( h , lower panel). Mean percentages of GFP-WIPI puncta-positive cells (300 cells, n =3). Mean±s.d.; heteroscedastic t -testing; P values: * P
Figure Legend Snippet: WIPI1 assists WIPI2 in recruiting ATG16L for LC3 lipidation. ( a ) U2OS cells transiently expressing GFP-WIPI1, GFP-WIPI2B, GFP-WIPI2D, GFP-WIPI3, GFP-WIPI4 or GFP alone were starved for 3 h and analysed by anti-GFP immunoprecipitation followed by anti-GFP or anti-ATG16L immunoblotting. The asterisk in the right panel (GFP-IP) represents a nonspecific band. Endogenous ATG16L isoforms co-precipitated with GFP-WIPI1, GFP-WIPI2B and GFP-WIPI2D as indicated in the right panel. ( b , c ) G361 cells stably expressing shRNA targeting WIPI2 ( b , shWIPI2), WIPI1 ( c , shWIPI1) or the non-targeting control (shControl) were fed (F) or starved (S) for 3 h, and endogenous ATG16L was detected using anti-ATG16L/IgG-Alexa Fluor 488 antibodies and fluorescence microscopy. Mean percentages of ATG16L-puncta-positive cells (up to 369 individual cells per condition, n =3) are presented. ( d ) Monoclonal U2OS cells stably expressing GFP-WIPI1 and shWIPI2 or shControl were starved (S) for 3 h, and the percentage of cells displaying an accumulation of perinuclear GFP-WIPI1 was calculated using fluorescence microscopy. Mean values (400 cells per condition, n =4) are presented. ( e , f ) G361 cells stably expressing shRNAs targeting WIPI1 ( e , shWIPI1) or WIPI2 ( f , shWIPI2) were fed (F) or starved (S) for 3 h and immunostained using anti-WIPI4/IgG-Alexa Fluor 488 antibodies. Mean percentages of WIPI4-puncta-positive cells (up to 340 cells per condition, n =3) are presented. ( g ) G361 cells stably expressing shRNAs targeting WIPI3 (shWIPI3), WIPI4 (shWIPI4) or the non-targeting shRNA control (shControl) were fed (F) or starved (S) for 3 h and immunostained using anti-ATG16L/IgG-Alexa Fluor 488 antibodies for analysis by fluorescence microscopy (left panel). Mean percentages of ATG16L-puncta-positive cells (up to 387 cells per condition, n =3) are presented (right panel). ( h , i ) ATG5 wild-type (WT) or knockout (KO) mouse embryonic fibroblasts (MEFs) transiently expressing GFP-WIPI1, GFP-WIPI2B, GFP-WIPI3 or GFP-WIPI4 were analysed by confocal LSM, and images from ATG5 WT MEFs ( h , upper panel) were processed using Volocity to generate 3D-reconstruction fly-through movies ( Supplementary Movies 5–8 ), from which still images are presented ( h , lower panel). Mean percentages of GFP-WIPI puncta-positive cells (300 cells, n =3). Mean±s.d.; heteroscedastic t -testing; P values: * P

Techniques Used: Expressing, Immunoprecipitation, Stable Transfection, shRNA, Fluorescence, Microscopy, Knock-Out

WIPI3 interacts with FIP200 at nascent autophagosomes and at LAMP2-positive lysosomes/late endosomes. ( a ) Mouse embryonic fibroblasts transiently expressing GFP-WIPI3 or GFP alone were fed (F) or starved (S) for 3 h and analysed by immunoprecipitation using anti-GFP antibodies and anti-FIP200 or anti-GFP immunoblotting. ( b ) U2OS cells stably expressing GFP-WIPI3 were transfected with myc-WIPI2B and starved (S) in the absence or presence of BafA1 for 3 h. Cells were immunostained with anti-myc/IgG-Alexa Fluor 546 or anti-FIP200/IgG-Alexa Fluor 633 antibodies and visualized by confocal LSM. ( c ) U2OS cells stably expressing GFP-WIPI3 were starved (S) in the absence or presence of BafA1 for 3 h, immunostained with anti-FIP200/IgG-Alexa Fluor 546 and anti-p62/IgG-Alexa Fluor 633 antibodies and visualized by confocal LSM. ( d , e ) U2OS cells stably overexpressing GFP-WIPI3 were transfected with control siRNA or FIP200 siRNA. Subsequently, the cells were starved for 3 h in the absence or presence BafA1. FIP200 downregulation was confirmed by immunoblotting using anti-FIP200 and anti-tubulin antibodies ( d ). Mean percentages of GFP-WIPI3-puncta-positive cells (up to 481 cells per condition, n =4) are presented ( e ). ( f ) U2OS cells stably overexpressing GFP-WIPI1 were transfected with control siRNA or FIP200 siRNA. Subsequently, the cells were starved for 3 h with nutrient-free medium in the absence or presence BafA1. Using automated image acquisition and analysis, the mean percentages of GFP-WIPI1-puncta-positive cells (up to 5,856 cells per condition, n =4) were calculated. ( g ) In parallel, this experiment was conducted using GFP-LC3-expressing U2OS cells and mean numbers of GFP-LC3 puncta per cell (up to 4,798 cells per condition, n =4) calculated. ( h ) U2OS cells stably expressing GFP-WIPI3 were starved (S) for 2 h or starved and replenished with amino acids for 1 h (S→AAs). Subsequently, cells were immunostained with anti-FIP200/IgG-Alexa Fluor 546 and anti-LAMP2/IgG-Alexa Fluor 633 antibodies and visualized by confocal LSM. Image magnifications are presented ( b , c , h ) and co-localizations indicated with white arrows. Supplementary Material is available: Supplementary Fig. 6 . Statistics and source data can be found in Supplementary Data 1 . Mean±s.d.; heteroscedastic t -testing; P values: * P
Figure Legend Snippet: WIPI3 interacts with FIP200 at nascent autophagosomes and at LAMP2-positive lysosomes/late endosomes. ( a ) Mouse embryonic fibroblasts transiently expressing GFP-WIPI3 or GFP alone were fed (F) or starved (S) for 3 h and analysed by immunoprecipitation using anti-GFP antibodies and anti-FIP200 or anti-GFP immunoblotting. ( b ) U2OS cells stably expressing GFP-WIPI3 were transfected with myc-WIPI2B and starved (S) in the absence or presence of BafA1 for 3 h. Cells were immunostained with anti-myc/IgG-Alexa Fluor 546 or anti-FIP200/IgG-Alexa Fluor 633 antibodies and visualized by confocal LSM. ( c ) U2OS cells stably expressing GFP-WIPI3 were starved (S) in the absence or presence of BafA1 for 3 h, immunostained with anti-FIP200/IgG-Alexa Fluor 546 and anti-p62/IgG-Alexa Fluor 633 antibodies and visualized by confocal LSM. ( d , e ) U2OS cells stably overexpressing GFP-WIPI3 were transfected with control siRNA or FIP200 siRNA. Subsequently, the cells were starved for 3 h in the absence or presence BafA1. FIP200 downregulation was confirmed by immunoblotting using anti-FIP200 and anti-tubulin antibodies ( d ). Mean percentages of GFP-WIPI3-puncta-positive cells (up to 481 cells per condition, n =4) are presented ( e ). ( f ) U2OS cells stably overexpressing GFP-WIPI1 were transfected with control siRNA or FIP200 siRNA. Subsequently, the cells were starved for 3 h with nutrient-free medium in the absence or presence BafA1. Using automated image acquisition and analysis, the mean percentages of GFP-WIPI1-puncta-positive cells (up to 5,856 cells per condition, n =4) were calculated. ( g ) In parallel, this experiment was conducted using GFP-LC3-expressing U2OS cells and mean numbers of GFP-LC3 puncta per cell (up to 4,798 cells per condition, n =4) calculated. ( h ) U2OS cells stably expressing GFP-WIPI3 were starved (S) for 2 h or starved and replenished with amino acids for 1 h (S→AAs). Subsequently, cells were immunostained with anti-FIP200/IgG-Alexa Fluor 546 and anti-LAMP2/IgG-Alexa Fluor 633 antibodies and visualized by confocal LSM. Image magnifications are presented ( b , c , h ) and co-localizations indicated with white arrows. Supplementary Material is available: Supplementary Fig. 6 . Statistics and source data can be found in Supplementary Data 1 . Mean±s.d.; heteroscedastic t -testing; P values: * P

Techniques Used: Expressing, Immunoprecipitation, Stable Transfection, Transfection

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Western Blot:

Article Title: JMJD1C Exhibits Multiple Functions in Epigenetic Regulation during Spermatogenesis
Article Snippet: .. The blotted membranes (Immobilon-XP) were immunoreacted with primary antibodies and visualized with the ECL Prime Western blotting Detection kit (GE Healthcare) containing secondary antibodies, HRP (horseradish peroxidase)-labeled anti-mouse IgG and anti-rabbit IgG, and the Quant LAS4000-mini (GE Healthcare) in accordance with the manufacturer’s instruction. .. The antibodies used for immunofluorescence and immunoblotting detection are listed in .

Article Title: WIPI3 and WIPI4 β-propellers are scaffolds for LKB1-AMPK-TSC signalling circuits in the control of autophagy
Article Snippet: .. For immunoblotting the following secondary antibodies were used in this study: anti-rabbit IgG, horseradish peroxidase (HRP)-linked (GE Healthcare; NA934V; WB 1:10,000), anti-mouse IgG, HRP-linked (GE Healthcare; NA931V; WB 1:10,000), anti-rabbit IgG, HRP-linked (Cell Signaling; #7,074; WB 1:10,000), anti-mouse IgG, HRP-linked (Cell Signaling; #7,076; WB 1:10,000). .. Short interfering RNAs The following siRNAs were used in this study: WIPI2 (EHU095551), WIPI3/WDR45L (EHU109971), WIPI4/WDR45 (EHU023161) and the control esiRNA targeting FLUC (EHUFLUC) were purchased from Sigma-Aldrich.

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    GE Healthcare horseradish peroxidase conjugated anti rabbit igg
    Internalisation of Ecrg4(71–132) and activation of NF-κB by Ecrg4(71–132) via several scavenger receptors. ( A ) HEK293 T cells were transfected with several Flag-tagged scavenger receptors. Two days after transfection, the cells were treated with Ecrg4(71–132) for 2 h. Ecrg4(71–132)–Fc was detected with a FITC-labelled anti-human <t>IgG</t> Fc antibody (green) and the transiently expressed Flag-tagged receptors were detected with an anti-Flag antibody (red). Nuclei were stained with Hoechst (blue). ( B ) Fluorescence intensities of Ecrg4(71–132) bound to primary microglia. Primary microglia were incubated with Fc(N293A)-fused Ecrg4(71–132) for 1 h in the presence of PBS, polyI, or polyC. ( C ) Immunoblotting for <t>p65</t> in microglial cells treated with Ecrg4(71–132) in the presence of PBS, polyI, or polyC. The band intensity was quantified by a ChemiDoc™ MP Imaging system (Bio-Rad), and the relative band intensity ratio NF-κB p65(pSer536)/NF-κB p65 is shown (pSer536/cont). Representative data from three independent experiments are indicated.
    Horseradish Peroxidase Conjugated Anti Rabbit Igg, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 97 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    GE Healthcare horse radish peroxidase conjugated goat anti rabbit igg
    A human mAb generated against a non-amyloid target binds aggregated <t>Aβ.</t> ( A ) Left panel: SEC chromatograms for ~15 mg/mL of mAb Avastin (anti-VEGF) and IVIg. SEC was carried out using a Superdex 200 Increase 10/300 GL column (GE Healthcare) equilibrated in PBS, pH 7.4. Right panel: Antibody binding curves against plate-immobilized PFs for unfractionated (Unfrac) IVIg, and for Avastin used unfractionated or as SEC-isolated monomers and dimers. ( B ) The top Western blots show immunoprecipitation (IP) of synthetic Aβ conformers (monomers (Mon), dimers, and PFs) by 100 μg/mL of Avastin and IVIg, and by 200 μg/mL of a pan-Aβ reactive polyclonal antibody, AW8. The blots were probed for Aβ using an Aβ N-terminal reactive mAb, 6E10 (Signet Laboratories). The lower Western blots show 20 μg/mL mAb Avastin's ability to IP 5 μg/mL of Aβ dimers and PFs in the presence of a 5-molar excess (with respect to Avastin) of a N-terminal 165-amino acid fragment of its immunogen VEGF (VEGF-165). Control IP experiments (Ctls) were carried out using 5 μg/mL mAb 6E10 and a mixture of Aβ dimers and PFs, or with 20 μg/mL Avastin and 1 μg/mL VEGF-165. The blots were probed for Aβ and VEGF-165 using mAb 6E10 and Avastin, respectively. In IPs carried out in the absence of VEGF-165, cross-reactivity of the secondary antibody, goat anti-human <t>IgG</t> (heavy and light, Jackson Immunoresearch Laboratories Inc), with Avastin’s Ig light chain caused a faint band that migrated near VEGF-165. ( C ) Avastin IgG conformers binding curves against plate-immobilized PFs in the presence or absence of a 1:10 dilution of IgG-depleted normal human sera. ( D ) Bar charts for solution-phase PF's, Aβ monomers, and non-amyloid native and aggregated molecule's inhibition of Avastin monomers an dimers binding to plate-immobilized PFs. Competition studies were carried out using 0.1 mg/mL competitors and concentrations of Avastin conformers that were equivalent to their EC 50 values for PFs: 500 nM IgG Mon, and 200 nM IgG dimer. Each competition curve was carried out in duplicate, and bars represent the standard error.
    Horse Radish Peroxidase Conjugated Goat Anti Rabbit Igg, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    GE Healthcare donkey anti rabbit immunoglobulin g igg conjugated
    FFSS rapidly stimulates ERK1/2 activity, nuclear translocation, chromatin binding, and RUNX2 phosphorylation. ( A ) FFSS stimulates ERK/MAPK and RUNX2-S319 phosphorylation. Cell lysates were analyzed by probing Western blots with the indicated antibodies, except in the case of P-RUNX2 for which P-RUNX2 antibody was used for immunoprecipitation followed by Western blot detection of total RUNX2. ( B , C ) FFSS increases nuclear localization of P-ERK, Runx2, and P-Runx2. Immunofluorescence confocal microscopy was used to localize the indicated factors. FFSS stimulated the translocation of P-ERK from a perinuclear cytoplasmic compartment to the nucleus without affecting the nuclear localization of RUNX2 in B , and increased nuclear RUNX2-S319-P in C . Note colocalization of P-ERK and RUNX2 (in B , merged image) and P-RUNX2 with total RUNX2 (in C . ( D , E ) FFSS stimulates binding of P-ERK and P-RUNX2 to proximal promoter regions of Bglap2 and Ibsp chromatin. ChIP assays were used to measure chromatin-bound RUNX2, P-RUNX2, and P-ERK1/2. Nonspecifically precipitated chromatin was measured using isotype-matched <t>IgG.</t> PCR primers were designed to detect OSE2a and OSE2b regions of the Bglap2 gene in D , and proximal and distal (nonfunctional) RUNX2 binding sites in Ibsp in E .
    Donkey Anti Rabbit Immunoglobulin G Igg Conjugated, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    GE Healthcare hrp horseradish peroxidase labelled anti rabbit igg immunoglobulin g
    DHODH is associated with complexes II and III ( A ) DHODH associates with respiratory complexes II and III. DHODH–HA-transfected HeLa cells were treated with DOX for 48 h. The mitochondrial fraction was purified on a Percoll density gradient and lysed with TNE buffer. After cross-linking, immunoprecipitates obtained with anti-HA and mouse <t>IgG</t> antibodies were separated by SDS/PAGE and immunoblotted with anti-HA, NDUFA9 (complex I), SDHA (complex II), UQCRFS1 (complex III) and COXVa (complex IV) antibodies. IP, immunoprecipitate; α, antibody. ( B ) BN/SDS/PAGE analysis. Isolated mitochondria before and after induction by DOX were solubilized by n - D -maltoside and protein complexes were first resolved by BN-PAGE (polyacrylamide concentration gradient, 3–12%) and resolved in a second dimension by SDS/PAGE. The transferred proteins were immunoblotted with the indicated antibodies. SC I–III 2 , supercomplex formed from complexes I and III 2 ; CIII 2 , dimeric complex III; CIV, complex IV; II, succinate dehydrogenase complex II; III 2 –DHODH, association with complex III and DHODH; II–DHODH, association with complex II and DHODH. DHODH is shown as monomers. The molecular masses (in kDa) of the molecular mass standards are shown.
    Hrp Horseradish Peroxidase Labelled Anti Rabbit Igg Immunoglobulin G, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Internalisation of Ecrg4(71–132) and activation of NF-κB by Ecrg4(71–132) via several scavenger receptors. ( A ) HEK293 T cells were transfected with several Flag-tagged scavenger receptors. Two days after transfection, the cells were treated with Ecrg4(71–132) for 2 h. Ecrg4(71–132)–Fc was detected with a FITC-labelled anti-human IgG Fc antibody (green) and the transiently expressed Flag-tagged receptors were detected with an anti-Flag antibody (red). Nuclei were stained with Hoechst (blue). ( B ) Fluorescence intensities of Ecrg4(71–132) bound to primary microglia. Primary microglia were incubated with Fc(N293A)-fused Ecrg4(71–132) for 1 h in the presence of PBS, polyI, or polyC. ( C ) Immunoblotting for p65 in microglial cells treated with Ecrg4(71–132) in the presence of PBS, polyI, or polyC. The band intensity was quantified by a ChemiDoc™ MP Imaging system (Bio-Rad), and the relative band intensity ratio NF-κB p65(pSer536)/NF-κB p65 is shown (pSer536/cont). Representative data from three independent experiments are indicated.

    Journal: Scientific Reports

    Article Title: Ecrg4 peptide is the ligand of multiple scavenger receptors

    doi: 10.1038/s41598-018-22440-4

    Figure Lengend Snippet: Internalisation of Ecrg4(71–132) and activation of NF-κB by Ecrg4(71–132) via several scavenger receptors. ( A ) HEK293 T cells were transfected with several Flag-tagged scavenger receptors. Two days after transfection, the cells were treated with Ecrg4(71–132) for 2 h. Ecrg4(71–132)–Fc was detected with a FITC-labelled anti-human IgG Fc antibody (green) and the transiently expressed Flag-tagged receptors were detected with an anti-Flag antibody (red). Nuclei were stained with Hoechst (blue). ( B ) Fluorescence intensities of Ecrg4(71–132) bound to primary microglia. Primary microglia were incubated with Fc(N293A)-fused Ecrg4(71–132) for 1 h in the presence of PBS, polyI, or polyC. ( C ) Immunoblotting for p65 in microglial cells treated with Ecrg4(71–132) in the presence of PBS, polyI, or polyC. The band intensity was quantified by a ChemiDoc™ MP Imaging system (Bio-Rad), and the relative band intensity ratio NF-κB p65(pSer536)/NF-κB p65 is shown (pSer536/cont). Representative data from three independent experiments are indicated.

    Article Snippet: The membranes were probed with antibodies against phospho-p65 (pSer536) and p65 (Cell Signaling Technology) overnight at 4 °C and then with horseradish-peroxidase-conjugated anti-rabbit IgG (GE Healthcare).

    Techniques: Activation Assay, Transfection, Staining, Fluorescence, Incubation, Imaging

    A human mAb generated against a non-amyloid target binds aggregated Aβ. ( A ) Left panel: SEC chromatograms for ~15 mg/mL of mAb Avastin (anti-VEGF) and IVIg. SEC was carried out using a Superdex 200 Increase 10/300 GL column (GE Healthcare) equilibrated in PBS, pH 7.4. Right panel: Antibody binding curves against plate-immobilized PFs for unfractionated (Unfrac) IVIg, and for Avastin used unfractionated or as SEC-isolated monomers and dimers. ( B ) The top Western blots show immunoprecipitation (IP) of synthetic Aβ conformers (monomers (Mon), dimers, and PFs) by 100 μg/mL of Avastin and IVIg, and by 200 μg/mL of a pan-Aβ reactive polyclonal antibody, AW8. The blots were probed for Aβ using an Aβ N-terminal reactive mAb, 6E10 (Signet Laboratories). The lower Western blots show 20 μg/mL mAb Avastin's ability to IP 5 μg/mL of Aβ dimers and PFs in the presence of a 5-molar excess (with respect to Avastin) of a N-terminal 165-amino acid fragment of its immunogen VEGF (VEGF-165). Control IP experiments (Ctls) were carried out using 5 μg/mL mAb 6E10 and a mixture of Aβ dimers and PFs, or with 20 μg/mL Avastin and 1 μg/mL VEGF-165. The blots were probed for Aβ and VEGF-165 using mAb 6E10 and Avastin, respectively. In IPs carried out in the absence of VEGF-165, cross-reactivity of the secondary antibody, goat anti-human IgG (heavy and light, Jackson Immunoresearch Laboratories Inc), with Avastin’s Ig light chain caused a faint band that migrated near VEGF-165. ( C ) Avastin IgG conformers binding curves against plate-immobilized PFs in the presence or absence of a 1:10 dilution of IgG-depleted normal human sera. ( D ) Bar charts for solution-phase PF's, Aβ monomers, and non-amyloid native and aggregated molecule's inhibition of Avastin monomers an dimers binding to plate-immobilized PFs. Competition studies were carried out using 0.1 mg/mL competitors and concentrations of Avastin conformers that were equivalent to their EC 50 values for PFs: 500 nM IgG Mon, and 200 nM IgG dimer. Each competition curve was carried out in duplicate, and bars represent the standard error.

    Journal: PLoS ONE

    Article Title: IgG Conformer's Binding to Amyloidogenic Aggregates

    doi: 10.1371/journal.pone.0137344

    Figure Lengend Snippet: A human mAb generated against a non-amyloid target binds aggregated Aβ. ( A ) Left panel: SEC chromatograms for ~15 mg/mL of mAb Avastin (anti-VEGF) and IVIg. SEC was carried out using a Superdex 200 Increase 10/300 GL column (GE Healthcare) equilibrated in PBS, pH 7.4. Right panel: Antibody binding curves against plate-immobilized PFs for unfractionated (Unfrac) IVIg, and for Avastin used unfractionated or as SEC-isolated monomers and dimers. ( B ) The top Western blots show immunoprecipitation (IP) of synthetic Aβ conformers (monomers (Mon), dimers, and PFs) by 100 μg/mL of Avastin and IVIg, and by 200 μg/mL of a pan-Aβ reactive polyclonal antibody, AW8. The blots were probed for Aβ using an Aβ N-terminal reactive mAb, 6E10 (Signet Laboratories). The lower Western blots show 20 μg/mL mAb Avastin's ability to IP 5 μg/mL of Aβ dimers and PFs in the presence of a 5-molar excess (with respect to Avastin) of a N-terminal 165-amino acid fragment of its immunogen VEGF (VEGF-165). Control IP experiments (Ctls) were carried out using 5 μg/mL mAb 6E10 and a mixture of Aβ dimers and PFs, or with 20 μg/mL Avastin and 1 μg/mL VEGF-165. The blots were probed for Aβ and VEGF-165 using mAb 6E10 and Avastin, respectively. In IPs carried out in the absence of VEGF-165, cross-reactivity of the secondary antibody, goat anti-human IgG (heavy and light, Jackson Immunoresearch Laboratories Inc), with Avastin’s Ig light chain caused a faint band that migrated near VEGF-165. ( C ) Avastin IgG conformers binding curves against plate-immobilized PFs in the presence or absence of a 1:10 dilution of IgG-depleted normal human sera. ( D ) Bar charts for solution-phase PF's, Aβ monomers, and non-amyloid native and aggregated molecule's inhibition of Avastin monomers an dimers binding to plate-immobilized PFs. Competition studies were carried out using 0.1 mg/mL competitors and concentrations of Avastin conformers that were equivalent to their EC 50 values for PFs: 500 nM IgG Mon, and 200 nM IgG dimer. Each competition curve was carried out in duplicate, and bars represent the standard error.

    Article Snippet: The detection system consisted of an in house pan-Aβ reactive polyclonal rabbit antibody, AW7 [ ], a horse radish peroxidase-conjugated goat anti-rabbit IgG (GE healthcare), and TMB substrate (SureBlue ReserveTM; KPL).

    Techniques: Generated, Size-exclusion Chromatography, Binding Assay, Isolation, Western Blot, Immunoprecipitation, Inhibition

    PAb aggregates have diverse avidities for Aβ. ( A ) Left panel: SEC chromatograms for untreated IVIg and for supernatants of ~4 mg/mL of SEC-isolated monomeric IVIg in PBS, pH 7.4, which was heated at 71°C until light scattering at A 400nm was 0.6 or 3.9, respectively. Right panel: Antibody binding curves against plate-immobilized PFs for untreated IVIg, Aβ-isolated IVIg IgGs, and for IgG supernatants (sup) and PBS resuspendend pellets (pellet) of heat-treated IVIg. (B ) Left panel: SEC chromatograms for ~4 mg/mL of untreated IVIg and for IVIg that was buffer exchanged at room temperature from gentle elution buffer (Pierce), pH 6.6, into PBS, pH 7.4. Right panel: Antibody binding curves against plate-immobilized PFs for untreated IVIg, buffered exchanged IVIg that was used unfractionated or as SEC-isolated monomers, and for Aβ-isolated IVIg IgGs.

    Journal: PLoS ONE

    Article Title: IgG Conformer's Binding to Amyloidogenic Aggregates

    doi: 10.1371/journal.pone.0137344

    Figure Lengend Snippet: PAb aggregates have diverse avidities for Aβ. ( A ) Left panel: SEC chromatograms for untreated IVIg and for supernatants of ~4 mg/mL of SEC-isolated monomeric IVIg in PBS, pH 7.4, which was heated at 71°C until light scattering at A 400nm was 0.6 or 3.9, respectively. Right panel: Antibody binding curves against plate-immobilized PFs for untreated IVIg, Aβ-isolated IVIg IgGs, and for IgG supernatants (sup) and PBS resuspendend pellets (pellet) of heat-treated IVIg. (B ) Left panel: SEC chromatograms for ~4 mg/mL of untreated IVIg and for IVIg that was buffer exchanged at room temperature from gentle elution buffer (Pierce), pH 6.6, into PBS, pH 7.4. Right panel: Antibody binding curves against plate-immobilized PFs for untreated IVIg, buffered exchanged IVIg that was used unfractionated or as SEC-isolated monomers, and for Aβ-isolated IVIg IgGs.

    Article Snippet: The detection system consisted of an in house pan-Aβ reactive polyclonal rabbit antibody, AW7 [ ], a horse radish peroxidase-conjugated goat anti-rabbit IgG (GE healthcare), and TMB substrate (SureBlue ReserveTM; KPL).

    Techniques: Size-exclusion Chromatography, Isolation, Binding Assay

    IVIg and Protein A-purified human and murine pAbs have similar anti-amyloid activities. ( A ) IgG binding curves against plate-immobilized PFs for IVIg and protein A-purified human (Hu) and murine (Mu) pAbs from pooled normal plasmas. Antibody binding curves are also shown for pAbs present in or dosed back into plasma. ( B ) Hybrid capture/competition ELISA curves for pAb's and IVIg's dose-dependent inhibition of PFs binding by plate-immobilized IVIg F(ab') fragments. The assay was carried out using 8 μg/ml solution-phase PFs. ( C ) Antibody binding curves for Hu and Mu pAb's nM cross-reactivity with plate-immobilized Aβ and TTR fibrils. ( D ) Left panel: Competition curves for solution-phase PF's and Aβ monomer's (Mon) inhibition of pAbs and IVIg binding to plate-immobilized PFs. Right panel: SAgg's and native TTR's (Nat) inhibition of pAbs and IVIg binding to plate-immobilized TTR fibrils. Competition studies were carried out using IgG concentrations, ~500 nM, which were equivalent to their EC 50 values for binding to PFs or TTR fibrils. Each binding or competition curve was carried out in duplicate, and bars represent the standard errors.

    Journal: PLoS ONE

    Article Title: IgG Conformer's Binding to Amyloidogenic Aggregates

    doi: 10.1371/journal.pone.0137344

    Figure Lengend Snippet: IVIg and Protein A-purified human and murine pAbs have similar anti-amyloid activities. ( A ) IgG binding curves against plate-immobilized PFs for IVIg and protein A-purified human (Hu) and murine (Mu) pAbs from pooled normal plasmas. Antibody binding curves are also shown for pAbs present in or dosed back into plasma. ( B ) Hybrid capture/competition ELISA curves for pAb's and IVIg's dose-dependent inhibition of PFs binding by plate-immobilized IVIg F(ab') fragments. The assay was carried out using 8 μg/ml solution-phase PFs. ( C ) Antibody binding curves for Hu and Mu pAb's nM cross-reactivity with plate-immobilized Aβ and TTR fibrils. ( D ) Left panel: Competition curves for solution-phase PF's and Aβ monomer's (Mon) inhibition of pAbs and IVIg binding to plate-immobilized PFs. Right panel: SAgg's and native TTR's (Nat) inhibition of pAbs and IVIg binding to plate-immobilized TTR fibrils. Competition studies were carried out using IgG concentrations, ~500 nM, which were equivalent to their EC 50 values for binding to PFs or TTR fibrils. Each binding or competition curve was carried out in duplicate, and bars represent the standard errors.

    Article Snippet: The detection system consisted of an in house pan-Aβ reactive polyclonal rabbit antibody, AW7 [ ], a horse radish peroxidase-conjugated goat anti-rabbit IgG (GE healthcare), and TMB substrate (SureBlue ReserveTM; KPL).

    Techniques: Purification, Binding Assay, Enzyme-linked Immunosorbent Assay, Inhibition

    IVIg IgG conformers retain binding to Aβ in the presence of normal human sera and non-amyloid molecules. ( A ) Antibody binding curves against plate-immobilized PFs for SEC-isolated IVIg monomers (SEC Mon), dimers (SEC Dimer), and for Aβ-isolated IVIg IgGs with or without a 1:10 dilution of IgG-depleted normal human sera. Preparations of Aβ-isolated IVIg IgGs contained HMW, dimeric, and monomeric species (see Fig 3A ). ( B ) Bar charts for solution-phase PFs, monomeric Aβ and for non-amyloid molecules inhibition of IVIg IgG conformers binding to plate-immobilized PFs. Non-amyloid molecules were chosen based on their: 1) Abundance in vivo (extracellular matrix and elastin fibrils), 2) Association with polyreactive autoantibodies (DNA), 3) High hydrophobicity (maize protein zein), and 4) Non-amyloid aggregate state [amorphous aggregated carboxymethylated ovalbumin (CM-Oval)]. Competition studies were carried out using 0.1 mg/mL competitors and concentrations of IgG conformers that were equivalent to their EC 50 values for PFs: 400 nM IgG Monomers; 50 nM IgG dimers, and 20 nM Aβ-isolated IgGs. Each competition curve was carried out in duplicate, and bars represent the standard error. ( C ) IVIg IgG conformer binding curves against PFs and non-amyloid molecules, murine extracellular matrix gel (ECM) and human DNA.

    Journal: PLoS ONE

    Article Title: IgG Conformer's Binding to Amyloidogenic Aggregates

    doi: 10.1371/journal.pone.0137344

    Figure Lengend Snippet: IVIg IgG conformers retain binding to Aβ in the presence of normal human sera and non-amyloid molecules. ( A ) Antibody binding curves against plate-immobilized PFs for SEC-isolated IVIg monomers (SEC Mon), dimers (SEC Dimer), and for Aβ-isolated IVIg IgGs with or without a 1:10 dilution of IgG-depleted normal human sera. Preparations of Aβ-isolated IVIg IgGs contained HMW, dimeric, and monomeric species (see Fig 3A ). ( B ) Bar charts for solution-phase PFs, monomeric Aβ and for non-amyloid molecules inhibition of IVIg IgG conformers binding to plate-immobilized PFs. Non-amyloid molecules were chosen based on their: 1) Abundance in vivo (extracellular matrix and elastin fibrils), 2) Association with polyreactive autoantibodies (DNA), 3) High hydrophobicity (maize protein zein), and 4) Non-amyloid aggregate state [amorphous aggregated carboxymethylated ovalbumin (CM-Oval)]. Competition studies were carried out using 0.1 mg/mL competitors and concentrations of IgG conformers that were equivalent to their EC 50 values for PFs: 400 nM IgG Monomers; 50 nM IgG dimers, and 20 nM Aβ-isolated IgGs. Each competition curve was carried out in duplicate, and bars represent the standard error. ( C ) IVIg IgG conformer binding curves against PFs and non-amyloid molecules, murine extracellular matrix gel (ECM) and human DNA.

    Article Snippet: The detection system consisted of an in house pan-Aβ reactive polyclonal rabbit antibody, AW7 [ ], a horse radish peroxidase-conjugated goat anti-rabbit IgG (GE healthcare), and TMB substrate (SureBlue ReserveTM; KPL).

    Techniques: Binding Assay, Size-exclusion Chromatography, Isolation, Inhibition, In Vivo

    Aβ-isolated but not heat-induced Avastin aggregates have enhanced avidity for PFs. ( A ) Left panel: SEC chromatograms for 0.3 mg/mL of Aβ-isolated Avastin IgGs, untreated Avastin, and for the antibody diluted into elution buffer (0.1 M glycine, pH 2.7) that was used to elute Aβ-bound Avastin IgGs. SEC was carried out using a Superdex 200 increase 10/300 GL column (GE Healthcare) that was equilibrated with PBS, pH 7.4. Right panel: Antibody binding curves against PFs for unfractionated IVIg and Avastin, and for Aβ-isolated Avastin IgGs. ( B ) Left panel: SEC chromatograms for ~5 mg/mL of unfractionated Avastin in PBS, pH 7.4, and for IgG conformers contained in supernatant of 71°C heated Avastin monomers (A 400nm 0.5 sup) in PBS, pH 7.4. Right panel: Antibody binding curves against PFs for soluble (A 400nm 0.5 sup) and insoluble (A 400nm 0.5 pellet) IgG conformers of heat-treated Avastin monomers, and for untreated Avastin and IVIg.

    Journal: PLoS ONE

    Article Title: IgG Conformer's Binding to Amyloidogenic Aggregates

    doi: 10.1371/journal.pone.0137344

    Figure Lengend Snippet: Aβ-isolated but not heat-induced Avastin aggregates have enhanced avidity for PFs. ( A ) Left panel: SEC chromatograms for 0.3 mg/mL of Aβ-isolated Avastin IgGs, untreated Avastin, and for the antibody diluted into elution buffer (0.1 M glycine, pH 2.7) that was used to elute Aβ-bound Avastin IgGs. SEC was carried out using a Superdex 200 increase 10/300 GL column (GE Healthcare) that was equilibrated with PBS, pH 7.4. Right panel: Antibody binding curves against PFs for unfractionated IVIg and Avastin, and for Aβ-isolated Avastin IgGs. ( B ) Left panel: SEC chromatograms for ~5 mg/mL of unfractionated Avastin in PBS, pH 7.4, and for IgG conformers contained in supernatant of 71°C heated Avastin monomers (A 400nm 0.5 sup) in PBS, pH 7.4. Right panel: Antibody binding curves against PFs for soluble (A 400nm 0.5 sup) and insoluble (A 400nm 0.5 pellet) IgG conformers of heat-treated Avastin monomers, and for untreated Avastin and IVIg.

    Article Snippet: The detection system consisted of an in house pan-Aβ reactive polyclonal rabbit antibody, AW7 [ ], a horse radish peroxidase-conjugated goat anti-rabbit IgG (GE healthcare), and TMB substrate (SureBlue ReserveTM; KPL).

    Techniques: Isolation, Size-exclusion Chromatography, Binding Assay

    IgG aggregates are primarily responsible for the enhanced anti-amyloid activities of Aβ- and Cibacron blue-isolated pAb IgGs. ( A ) Left panel: SEC chromatograms for ~0.5 mg/mL of Aβ-isolated IVIg IgGs, and for IVIg, untreated, or diluted into column elution buffer (0.1 M glycine, pH 2.7) that was used to elute Aβ-bound IVIg IgGs. SEC was carried out using a Superdex 200 increase 10/300 GL column (GE Healthcare) that was equilibrated with PBS, pH 7.4. Right panel: IgG binding curves against plate-immobilized PFs for untreated IVIg, and for Aβ column-isolated IVIg IgGs that were used unfractionated (Unfrac) or as SEC-isolated monomers (SEC Mon) or aggregates (SEC Aggs). SEC Aggs consisted of a pool of IgG conformers (dimers and HMW species) that eluted before the monomeric antibody. ( B ) Left panel: SEC chromatograms for ~0.5 mg/mL dye-isolated IVIg IgGs, and for unfractionated IVIg that was untreated or diluted into column elution buffer (PBS containing 1.5 M NaCl, pH 7.4) that was used to elute dye-bound IVIg IgGs. Right panel: IgG binding curves against plate-immobilized PFs for unfractionated and SEC-isolated conformers of dye-isolated IVIg IgGs, and for untreated IVIg.

    Journal: PLoS ONE

    Article Title: IgG Conformer's Binding to Amyloidogenic Aggregates

    doi: 10.1371/journal.pone.0137344

    Figure Lengend Snippet: IgG aggregates are primarily responsible for the enhanced anti-amyloid activities of Aβ- and Cibacron blue-isolated pAb IgGs. ( A ) Left panel: SEC chromatograms for ~0.5 mg/mL of Aβ-isolated IVIg IgGs, and for IVIg, untreated, or diluted into column elution buffer (0.1 M glycine, pH 2.7) that was used to elute Aβ-bound IVIg IgGs. SEC was carried out using a Superdex 200 increase 10/300 GL column (GE Healthcare) that was equilibrated with PBS, pH 7.4. Right panel: IgG binding curves against plate-immobilized PFs for untreated IVIg, and for Aβ column-isolated IVIg IgGs that were used unfractionated (Unfrac) or as SEC-isolated monomers (SEC Mon) or aggregates (SEC Aggs). SEC Aggs consisted of a pool of IgG conformers (dimers and HMW species) that eluted before the monomeric antibody. ( B ) Left panel: SEC chromatograms for ~0.5 mg/mL dye-isolated IVIg IgGs, and for unfractionated IVIg that was untreated or diluted into column elution buffer (PBS containing 1.5 M NaCl, pH 7.4) that was used to elute dye-bound IVIg IgGs. Right panel: IgG binding curves against plate-immobilized PFs for unfractionated and SEC-isolated conformers of dye-isolated IVIg IgGs, and for untreated IVIg.

    Article Snippet: The detection system consisted of an in house pan-Aβ reactive polyclonal rabbit antibody, AW7 [ ], a horse radish peroxidase-conjugated goat anti-rabbit IgG (GE healthcare), and TMB substrate (SureBlue ReserveTM; KPL).

    Techniques: Isolation, Size-exclusion Chromatography, Binding Assay

    FFSS rapidly stimulates ERK1/2 activity, nuclear translocation, chromatin binding, and RUNX2 phosphorylation. ( A ) FFSS stimulates ERK/MAPK and RUNX2-S319 phosphorylation. Cell lysates were analyzed by probing Western blots with the indicated antibodies, except in the case of P-RUNX2 for which P-RUNX2 antibody was used for immunoprecipitation followed by Western blot detection of total RUNX2. ( B , C ) FFSS increases nuclear localization of P-ERK, Runx2, and P-Runx2. Immunofluorescence confocal microscopy was used to localize the indicated factors. FFSS stimulated the translocation of P-ERK from a perinuclear cytoplasmic compartment to the nucleus without affecting the nuclear localization of RUNX2 in B , and increased nuclear RUNX2-S319-P in C . Note colocalization of P-ERK and RUNX2 (in B , merged image) and P-RUNX2 with total RUNX2 (in C . ( D , E ) FFSS stimulates binding of P-ERK and P-RUNX2 to proximal promoter regions of Bglap2 and Ibsp chromatin. ChIP assays were used to measure chromatin-bound RUNX2, P-RUNX2, and P-ERK1/2. Nonspecifically precipitated chromatin was measured using isotype-matched IgG. PCR primers were designed to detect OSE2a and OSE2b regions of the Bglap2 gene in D , and proximal and distal (nonfunctional) RUNX2 binding sites in Ibsp in E .

    Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

    Article Title: Biomechanical Stimulation of Osteoblast Gene Expression Requires Phosphorylation of the RUNX2 Transcription Factor

    doi: 10.1002/jbmr.1574

    Figure Lengend Snippet: FFSS rapidly stimulates ERK1/2 activity, nuclear translocation, chromatin binding, and RUNX2 phosphorylation. ( A ) FFSS stimulates ERK/MAPK and RUNX2-S319 phosphorylation. Cell lysates were analyzed by probing Western blots with the indicated antibodies, except in the case of P-RUNX2 for which P-RUNX2 antibody was used for immunoprecipitation followed by Western blot detection of total RUNX2. ( B , C ) FFSS increases nuclear localization of P-ERK, Runx2, and P-Runx2. Immunofluorescence confocal microscopy was used to localize the indicated factors. FFSS stimulated the translocation of P-ERK from a perinuclear cytoplasmic compartment to the nucleus without affecting the nuclear localization of RUNX2 in B , and increased nuclear RUNX2-S319-P in C . Note colocalization of P-ERK and RUNX2 (in B , merged image) and P-RUNX2 with total RUNX2 (in C . ( D , E ) FFSS stimulates binding of P-ERK and P-RUNX2 to proximal promoter regions of Bglap2 and Ibsp chromatin. ChIP assays were used to measure chromatin-bound RUNX2, P-RUNX2, and P-ERK1/2. Nonspecifically precipitated chromatin was measured using isotype-matched IgG. PCR primers were designed to detect OSE2a and OSE2b regions of the Bglap2 gene in D , and proximal and distal (nonfunctional) RUNX2 binding sites in Ibsp in E .

    Article Snippet: The reagents used in this study were from the following sources: tissue culture medium and fetal bovine serum from Hyclone Laboratories (Logan, UT, USA) and Invitrogen (Carlsbad, CA, USA), RUNX2 antibody from Medical & Biological Laboratories (Nagoya, Japan; catalog number D130-3), P-ERK and total ERK antibodies from Cell Signaling Technology (Beverly, MA, USA; catalog numbers 9101 and 9102), acetylated histone H3 and H4 and histone H3 phosphorylated at serine 10 from Millipore (Billerica, MA, USA; catalog numbers 17–615, 17–630, and 06–570), and sheep anti-mouse or donkey anti-rabbit immunoglobulin G (IgG) conjugated with horseradish peroxidase (HRP) from GE Healthcare (Piscataway, NJ, USA).

    Techniques: Activity Assay, Translocation Assay, Binding Assay, Western Blot, Immunoprecipitation, Immunofluorescence, Confocal Microscopy, Chromatin Immunoprecipitation, Polymerase Chain Reaction

    DHODH is associated with complexes II and III ( A ) DHODH associates with respiratory complexes II and III. DHODH–HA-transfected HeLa cells were treated with DOX for 48 h. The mitochondrial fraction was purified on a Percoll density gradient and lysed with TNE buffer. After cross-linking, immunoprecipitates obtained with anti-HA and mouse IgG antibodies were separated by SDS/PAGE and immunoblotted with anti-HA, NDUFA9 (complex I), SDHA (complex II), UQCRFS1 (complex III) and COXVa (complex IV) antibodies. IP, immunoprecipitate; α, antibody. ( B ) BN/SDS/PAGE analysis. Isolated mitochondria before and after induction by DOX were solubilized by n - D -maltoside and protein complexes were first resolved by BN-PAGE (polyacrylamide concentration gradient, 3–12%) and resolved in a second dimension by SDS/PAGE. The transferred proteins were immunoblotted with the indicated antibodies. SC I–III 2 , supercomplex formed from complexes I and III 2 ; CIII 2 , dimeric complex III; CIV, complex IV; II, succinate dehydrogenase complex II; III 2 –DHODH, association with complex III and DHODH; II–DHODH, association with complex II and DHODH. DHODH is shown as monomers. The molecular masses (in kDa) of the molecular mass standards are shown.

    Journal: Bioscience Reports

    Article Title: Dihydro-orotate dehydrogenase is physically associated with the respiratory complex and its loss leads to mitochondrial dysfunction

    doi: 10.1042/BSR20120097

    Figure Lengend Snippet: DHODH is associated with complexes II and III ( A ) DHODH associates with respiratory complexes II and III. DHODH–HA-transfected HeLa cells were treated with DOX for 48 h. The mitochondrial fraction was purified on a Percoll density gradient and lysed with TNE buffer. After cross-linking, immunoprecipitates obtained with anti-HA and mouse IgG antibodies were separated by SDS/PAGE and immunoblotted with anti-HA, NDUFA9 (complex I), SDHA (complex II), UQCRFS1 (complex III) and COXVa (complex IV) antibodies. IP, immunoprecipitate; α, antibody. ( B ) BN/SDS/PAGE analysis. Isolated mitochondria before and after induction by DOX were solubilized by n - D -maltoside and protein complexes were first resolved by BN-PAGE (polyacrylamide concentration gradient, 3–12%) and resolved in a second dimension by SDS/PAGE. The transferred proteins were immunoblotted with the indicated antibodies. SC I–III 2 , supercomplex formed from complexes I and III 2 ; CIII 2 , dimeric complex III; CIV, complex IV; II, succinate dehydrogenase complex II; III 2 –DHODH, association with complex III and DHODH; II–DHODH, association with complex II and DHODH. DHODH is shown as monomers. The molecular masses (in kDa) of the molecular mass standards are shown.

    Article Snippet: The signals were visualized with HRP (horseradish peroxidase)-labelled anti-rabbit IgG (immunoglobulin G) and an ECL® (enhanced chemiluminescence) reagent (GE Healthcare).

    Techniques: Transfection, Purification, SDS Page, Isolation, Polyacrylamide Gel Electrophoresis, Concentration Assay