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Santa Cruz Biotechnology goat anti synaptopodin
Aggravation of podocyte injury upon autophagy inhibition. (A–B).The Western blot results confirmed that the expression of <t>synaptopodin</t> and LC3 II was lower in ATG7 (a key protein of autophagy) RNAi-mediated knock down differentiated MPCs (siRNA+R+P) than in the siRNA negative control group (siCon+R+P). Both groups were pretreated with rapamycin and treated with PAN,* P
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1) Product Images from "Rapamycin Upregulates Autophagy by Inhibiting the mTOR-ULK1 Pathway, Resulting in Reduced Podocyte Injury"

Article Title: Rapamycin Upregulates Autophagy by Inhibiting the mTOR-ULK1 Pathway, Resulting in Reduced Podocyte Injury

Journal: PLoS ONE

doi: 10.1371/journal.pone.0063799

Aggravation of podocyte injury upon autophagy inhibition. (A–B).The Western blot results confirmed that the expression of synaptopodin and LC3 II was lower in ATG7 (a key protein of autophagy) RNAi-mediated knock down differentiated MPCs (siRNA+R+P) than in the siRNA negative control group (siCon+R+P). Both groups were pretreated with rapamycin and treated with PAN,* P
Figure Legend Snippet: Aggravation of podocyte injury upon autophagy inhibition. (A–B).The Western blot results confirmed that the expression of synaptopodin and LC3 II was lower in ATG7 (a key protein of autophagy) RNAi-mediated knock down differentiated MPCs (siRNA+R+P) than in the siRNA negative control group (siCon+R+P). Both groups were pretreated with rapamycin and treated with PAN,* P

Techniques Used: Inhibition, Western Blot, Expressing, Negative Control

Rapamycin reduced podocyte injury by inhibiting the mTOR-ULK1 signaling pathway. (A–B).Western blot analysis of cellular proteins showed recovery of synaptopodin and LC3 II expression, a decrease in mTOR activity, and a decrease in ULK1 phosphorylation in rapamycin-pretreated cells (P+R) compared to PAN-treated cells in the absence of rapamycin (PAN), * P
Figure Legend Snippet: Rapamycin reduced podocyte injury by inhibiting the mTOR-ULK1 signaling pathway. (A–B).Western blot analysis of cellular proteins showed recovery of synaptopodin and LC3 II expression, a decrease in mTOR activity, and a decrease in ULK1 phosphorylation in rapamycin-pretreated cells (P+R) compared to PAN-treated cells in the absence of rapamycin (PAN), * P

Techniques Used: Western Blot, Expressing, Activity Assay

Preparation of passive Heymann nephritis rat model. (A).At day 1, 7, 14, 21, and 28 after antiserum injection, rats were sacrificed and the renal cortex was removed. Cryosections of the renal cortex were stained with goat anti-rat IgG-fluorescein isothiocyanate (FITC). Microscopic examination revealed that there were IgG depositions in the glomeruli of PHN rats compared to those of control rats. Depositions gradually increased over time, peaking on day 14. Magnification = × 400 (B). 24-hr urine was collected on days 0 (control), 1, 7, 14, 21, and 28, and analyzed for urinary albumin content using Coomassie Brilliant Blue G-250. (C). Electrophoresis followed by Coomassie blue staining. Each group urine sample was subjected to SDS-PAGE. Obvious albuminuria was apparent from day 7. (D-E).Western blot analysis of isolated glomerular protein on day 14 showed significantly reduced expression of synaptopodin in PHN rats compared to that in controls, * P
Figure Legend Snippet: Preparation of passive Heymann nephritis rat model. (A).At day 1, 7, 14, 21, and 28 after antiserum injection, rats were sacrificed and the renal cortex was removed. Cryosections of the renal cortex were stained with goat anti-rat IgG-fluorescein isothiocyanate (FITC). Microscopic examination revealed that there were IgG depositions in the glomeruli of PHN rats compared to those of control rats. Depositions gradually increased over time, peaking on day 14. Magnification = × 400 (B). 24-hr urine was collected on days 0 (control), 1, 7, 14, 21, and 28, and analyzed for urinary albumin content using Coomassie Brilliant Blue G-250. (C). Electrophoresis followed by Coomassie blue staining. Each group urine sample was subjected to SDS-PAGE. Obvious albuminuria was apparent from day 7. (D-E).Western blot analysis of isolated glomerular protein on day 14 showed significantly reduced expression of synaptopodin in PHN rats compared to that in controls, * P

Techniques Used: Injection, Staining, Electrophoresis, SDS Page, Western Blot, Isolation, Expressing

2) Product Images from "Rapamycin Upregulates Autophagy by Inhibiting the mTOR-ULK1 Pathway, Resulting in Reduced Podocyte Injury"

Article Title: Rapamycin Upregulates Autophagy by Inhibiting the mTOR-ULK1 Pathway, Resulting in Reduced Podocyte Injury

Journal: PLoS ONE

doi: 10.1371/journal.pone.0063799

Aggravation of podocyte injury upon autophagy inhibition. (A–B).The Western blot results confirmed that the expression of synaptopodin and LC3 II was lower in ATG7 (a key protein of autophagy) RNAi-mediated knock down differentiated MPCs (siRNA+R+P) than in the siRNA negative control group (siCon+R+P). Both groups were pretreated with rapamycin and treated with PAN,* P
Figure Legend Snippet: Aggravation of podocyte injury upon autophagy inhibition. (A–B).The Western blot results confirmed that the expression of synaptopodin and LC3 II was lower in ATG7 (a key protein of autophagy) RNAi-mediated knock down differentiated MPCs (siRNA+R+P) than in the siRNA negative control group (siCon+R+P). Both groups were pretreated with rapamycin and treated with PAN,* P

Techniques Used: Inhibition, Western Blot, Expressing, Negative Control

Rapamycin reduced podocyte injury by inhibiting the mTOR-ULK1 signaling pathway. (A–B).Western blot analysis of cellular proteins showed recovery of synaptopodin and LC3 II expression, a decrease in mTOR activity, and a decrease in ULK1 phosphorylation in rapamycin-pretreated cells (P+R) compared to PAN-treated cells in the absence of rapamycin (PAN), * P
Figure Legend Snippet: Rapamycin reduced podocyte injury by inhibiting the mTOR-ULK1 signaling pathway. (A–B).Western blot analysis of cellular proteins showed recovery of synaptopodin and LC3 II expression, a decrease in mTOR activity, and a decrease in ULK1 phosphorylation in rapamycin-pretreated cells (P+R) compared to PAN-treated cells in the absence of rapamycin (PAN), * P

Techniques Used: Western Blot, Expressing, Activity Assay

Preparation of passive Heymann nephritis rat model. (A).At day 1, 7, 14, 21, and 28 after antiserum injection, rats were sacrificed and the renal cortex was removed. Cryosections of the renal cortex were stained with goat anti-rat IgG-fluorescein isothiocyanate (FITC). Microscopic examination revealed that there were IgG depositions in the glomeruli of PHN rats compared to those of control rats. Depositions gradually increased over time, peaking on day 14. Magnification = × 400 (B). 24-hr urine was collected on days 0 (control), 1, 7, 14, 21, and 28, and analyzed for urinary albumin content using Coomassie Brilliant Blue G-250. (C). Electrophoresis followed by Coomassie blue staining. Each group urine sample was subjected to SDS-PAGE. Obvious albuminuria was apparent from day 7. (D-E).Western blot analysis of isolated glomerular protein on day 14 showed significantly reduced expression of synaptopodin in PHN rats compared to that in controls, * P
Figure Legend Snippet: Preparation of passive Heymann nephritis rat model. (A).At day 1, 7, 14, 21, and 28 after antiserum injection, rats were sacrificed and the renal cortex was removed. Cryosections of the renal cortex were stained with goat anti-rat IgG-fluorescein isothiocyanate (FITC). Microscopic examination revealed that there were IgG depositions in the glomeruli of PHN rats compared to those of control rats. Depositions gradually increased over time, peaking on day 14. Magnification = × 400 (B). 24-hr urine was collected on days 0 (control), 1, 7, 14, 21, and 28, and analyzed for urinary albumin content using Coomassie Brilliant Blue G-250. (C). Electrophoresis followed by Coomassie blue staining. Each group urine sample was subjected to SDS-PAGE. Obvious albuminuria was apparent from day 7. (D-E).Western blot analysis of isolated glomerular protein on day 14 showed significantly reduced expression of synaptopodin in PHN rats compared to that in controls, * P

Techniques Used: Injection, Staining, Electrophoresis, SDS Page, Western Blot, Isolation, Expressing

3) Product Images from "C/EBPα deficiency in podocytes aggravates podocyte senescence and kidney injury in aging mice"

Article Title: C/EBPα deficiency in podocytes aggravates podocyte senescence and kidney injury in aging mice

Journal: Cell Death & Disease

doi: 10.1038/s41419-019-1933-2

C/EBPα protects cultured podocytes from SASP in premature senescence. a Western blot analysis of synaptopodin and nephrin in immortalized mouse podocytes stimulated with or without 0.5 μM ADR for 24 h after transfection with the NGFR-C/EBPα or NGFR-vector plasmid for 24 h. b Semiquantitative average band intensity in ( a ). c Western blot analysis of TGF-β1, PAI-1, and NLRP3 in immortalized mouse podocytes that underwent the conditions indicated in ( a ). d Semiquantitative average band intensity in ( c ). e The mRNA expression of CTGF and VEGFA in the four indicated groups. f Western blot analysis of phospho-AMPK and phospho-mTOR in immortalized mouse podocytes exposed to the conditions indicated. g Semiquantitative average band intensity in ( f ). The results are presented as the means ± SEMs. GAPDH served as a loading control, and the value in the NGFR vector + control group was set to 1. * P
Figure Legend Snippet: C/EBPα protects cultured podocytes from SASP in premature senescence. a Western blot analysis of synaptopodin and nephrin in immortalized mouse podocytes stimulated with or without 0.5 μM ADR for 24 h after transfection with the NGFR-C/EBPα or NGFR-vector plasmid for 24 h. b Semiquantitative average band intensity in ( a ). c Western blot analysis of TGF-β1, PAI-1, and NLRP3 in immortalized mouse podocytes that underwent the conditions indicated in ( a ). d Semiquantitative average band intensity in ( c ). e The mRNA expression of CTGF and VEGFA in the four indicated groups. f Western blot analysis of phospho-AMPK and phospho-mTOR in immortalized mouse podocytes exposed to the conditions indicated. g Semiquantitative average band intensity in ( f ). The results are presented as the means ± SEMs. GAPDH served as a loading control, and the value in the NGFR vector + control group was set to 1. * P

Techniques Used: Cell Culture, Western Blot, Transfection, Plasmid Preparation, Expressing

4) Product Images from "Classical Estrogen Receptors and ER? Splice Variants in the Mouse"

Article Title: Classical Estrogen Receptors and ER? Splice Variants in the Mouse

Journal: PLoS ONE

doi: 10.1371/journal.pone.0070926

Representative immunofluorescence images localizing ERβ in mouse kidney. Green fluorescence represents ERβ in all images. Red fluorescence represents cell specific markers for co-localization (collecting duct marker, aquaporin 2; vascular smooth muscle mesangial cell marker, α-smooth muscle actin; podocyte cell marker, synaptopodin). Nuclei are stained blue with DAPI. Scale bar = 20 µm.
Figure Legend Snippet: Representative immunofluorescence images localizing ERβ in mouse kidney. Green fluorescence represents ERβ in all images. Red fluorescence represents cell specific markers for co-localization (collecting duct marker, aquaporin 2; vascular smooth muscle mesangial cell marker, α-smooth muscle actin; podocyte cell marker, synaptopodin). Nuclei are stained blue with DAPI. Scale bar = 20 µm.

Techniques Used: Immunofluorescence, Fluorescence, Marker, Staining

Representative immunofluorescence images localizing ERα36 in mouse kidney. Green fluorescence represents ERα36 in all images. Red fluorescence represents cell specific markers for co-localization (collecting duct marker, aquaporin 2; vascular smooth muscle mesangial cell marker, α-smooth muscle actin; podocyte cell marker, synaptopodin). Nuclei are stained blue with DAPI. Scale bar = 20 µm.
Figure Legend Snippet: Representative immunofluorescence images localizing ERα36 in mouse kidney. Green fluorescence represents ERα36 in all images. Red fluorescence represents cell specific markers for co-localization (collecting duct marker, aquaporin 2; vascular smooth muscle mesangial cell marker, α-smooth muscle actin; podocyte cell marker, synaptopodin). Nuclei are stained blue with DAPI. Scale bar = 20 µm.

Techniques Used: Immunofluorescence, Fluorescence, Marker, Staining

Representative immunofluorescence images localizing ERα66 in mouse kidney. Green fluorescence represents ERα66 in all images. Red fluorescence represents cell specific markers for co-localization (collecting duct marker, aquaporin 2; vascular smooth muscle mesangial cell marker, α-smooth muscle actin; podocyte cell marker, synaptopodin). Nuclei are stained blue with DAPI. Scale bar = 20 µm.
Figure Legend Snippet: Representative immunofluorescence images localizing ERα66 in mouse kidney. Green fluorescence represents ERα66 in all images. Red fluorescence represents cell specific markers for co-localization (collecting duct marker, aquaporin 2; vascular smooth muscle mesangial cell marker, α-smooth muscle actin; podocyte cell marker, synaptopodin). Nuclei are stained blue with DAPI. Scale bar = 20 µm.

Techniques Used: Immunofluorescence, Fluorescence, Marker, Staining

5) Product Images from "GLCCI1 is a novel component associated with the PI3K signaling pathway in podocyte foot processes"

Article Title: GLCCI1 is a novel component associated with the PI3K signaling pathway in podocyte foot processes

Journal: Experimental & Molecular Medicine

doi: 10.1038/emm.2016.28

The localization of glucocorticoid-induced transcript 1 (GLCCI1), podocyte-specific markers and proteins associated with the phosphoinositide 3-kinase (PI3K) signaling pathway in the rat glomerulus. ( a ) Staining of rat kidney sections with an antibody against GLCCI1 (red) is shown. The diabetic group showed low reactivity. However, GLCCI1 was regulated by wortmannin treatment. ( b – d ) The podocyte-specific proteins nephrin (green), podocin (red) and synaptopodin (green) showed a reactivity pattern similar to GLCCI1 following wortmannin treatment. ( e ) Serum/glucocorticoid-regulated kinase 1 (SGK1; red) was observed only in the diabetic group. We confirmed that SGK1 expression was decreased by wortmannin. ( f ) Forkhead box O3 (FOXO3A; red) was observed in the control group. The lower reactivity of FOXO3A was accompanied by the overexpression of SGK1 in the diabetic group. The localization of FOXO3A was regulated by wortmannin treatment. Original magnification: × 400. Scale bar, 50 μm.
Figure Legend Snippet: The localization of glucocorticoid-induced transcript 1 (GLCCI1), podocyte-specific markers and proteins associated with the phosphoinositide 3-kinase (PI3K) signaling pathway in the rat glomerulus. ( a ) Staining of rat kidney sections with an antibody against GLCCI1 (red) is shown. The diabetic group showed low reactivity. However, GLCCI1 was regulated by wortmannin treatment. ( b – d ) The podocyte-specific proteins nephrin (green), podocin (red) and synaptopodin (green) showed a reactivity pattern similar to GLCCI1 following wortmannin treatment. ( e ) Serum/glucocorticoid-regulated kinase 1 (SGK1; red) was observed only in the diabetic group. We confirmed that SGK1 expression was decreased by wortmannin. ( f ) Forkhead box O3 (FOXO3A; red) was observed in the control group. The lower reactivity of FOXO3A was accompanied by the overexpression of SGK1 in the diabetic group. The localization of FOXO3A was regulated by wortmannin treatment. Original magnification: × 400. Scale bar, 50 μm.

Techniques Used: Staining, Expressing, Over Expression

Colocalization of glucocorticoid-induced transcript 1 (GLCCI1) with the podocyte-specific proteins nephrin and synaptopodin. ( a ) Double labeling with the podocyte-specific marker nephrin (green) showed a complete overlap (merge, yellow) with the distribution of GLCCI1 (red) in the 5 m M and wortmannin-treated diabetic groups of cultured podocytes. ( b ) GLCCI1 (red) also colocalized with synaptopodin (green). We observed overlapping reactivity (yellow indicates red plus green) in the 5 m M and wortmannin-treated diabetic groups of cultured podocytes. ( c , d ) In rat glomeruli, we observed overlapping reactivity (yellow) via the colocalization of GLCCI1 (red) with nephrin and synaptopodin (green). Wortmannin ameliorated the lower reactivity in glomeruli from rats in the diabetic group. Original magnification: × 400. Scale bar, 50 μm.
Figure Legend Snippet: Colocalization of glucocorticoid-induced transcript 1 (GLCCI1) with the podocyte-specific proteins nephrin and synaptopodin. ( a ) Double labeling with the podocyte-specific marker nephrin (green) showed a complete overlap (merge, yellow) with the distribution of GLCCI1 (red) in the 5 m M and wortmannin-treated diabetic groups of cultured podocytes. ( b ) GLCCI1 (red) also colocalized with synaptopodin (green). We observed overlapping reactivity (yellow indicates red plus green) in the 5 m M and wortmannin-treated diabetic groups of cultured podocytes. ( c , d ) In rat glomeruli, we observed overlapping reactivity (yellow) via the colocalization of GLCCI1 (red) with nephrin and synaptopodin (green). Wortmannin ameliorated the lower reactivity in glomeruli from rats in the diabetic group. Original magnification: × 400. Scale bar, 50 μm.

Techniques Used: Labeling, Marker, Cell Culture

The localization of glucocorticoid-induced transcript 1 (GLCCI1), podocyte-specific markers and proteins involved in the phosphoinositide 3-kinase (PI3K) signaling pathway in podocytes. ( a ) In podocytes, GLCCI1 (red, Alexa 568 conjugated) was localized in the 4',6-diamidino-2-phenylindole (DAPI)-stained nuclei. No GLCCI1 localization was observed in the 25 m M D -glucose-treated podocytes. However, GLCCI1 was regulated by wortmannin treatment. ( b – d ) The podocyte-specific proteins nephrin (green, Alexa 488 conjugated), podocin (red) and synaptopodin (green) showed a reactivity pattern similar to GLCCI1 in podocytes treated with wortmannin. ( e ) Serum/glucocorticoid-regulated kinase 1 (SGK1; red) was observed only in the 25 m M group. We confirmed that treatment with wortmannin decreased SGK1 expression. ( f ) Forkhead box O3 (FOXO3A; red) was observed in the 5 m M group. A lower FOXO3A signal was accompanied by high reactivity of SGK1 in the 25 m M group. Localization of FOXO3A was regulated by treatment with wortmannin. Original magnification: × 400. Scale bar, 50 μm.
Figure Legend Snippet: The localization of glucocorticoid-induced transcript 1 (GLCCI1), podocyte-specific markers and proteins involved in the phosphoinositide 3-kinase (PI3K) signaling pathway in podocytes. ( a ) In podocytes, GLCCI1 (red, Alexa 568 conjugated) was localized in the 4',6-diamidino-2-phenylindole (DAPI)-stained nuclei. No GLCCI1 localization was observed in the 25 m M D -glucose-treated podocytes. However, GLCCI1 was regulated by wortmannin treatment. ( b – d ) The podocyte-specific proteins nephrin (green, Alexa 488 conjugated), podocin (red) and synaptopodin (green) showed a reactivity pattern similar to GLCCI1 in podocytes treated with wortmannin. ( e ) Serum/glucocorticoid-regulated kinase 1 (SGK1; red) was observed only in the 25 m M group. We confirmed that treatment with wortmannin decreased SGK1 expression. ( f ) Forkhead box O3 (FOXO3A; red) was observed in the 5 m M group. A lower FOXO3A signal was accompanied by high reactivity of SGK1 in the 25 m M group. Localization of FOXO3A was regulated by treatment with wortmannin. Original magnification: × 400. Scale bar, 50 μm.

Techniques Used: Staining, Expressing

Detection of glucocorticoid-induced transcript 1 (GLCCI1) and podocyte-specific marker gene expression by reverse transcription-PCR (RT-PCR). ( a ) Expression of the genes encoding GLCCI1, nephrin, podocin, synaptopodin and podocalyxin showed similar patterns in the podocytes. The expression levels differed significantly between the high glucose-induced and wortmannin-treated diabetic groups of podocytes. ( b ) The expression of the genes encoding GLCCI1, nephrin, podocin, synaptopodin and podocalyxin showed similar patterns in the kidneys of diabetic rats. In addition, wortmannin ameliorated the expression of all podocyte-specific proteins including GLCCI1. ( c , d ) The relative band intensity of proteins in podocytes and the kidneys of diabetic rats was observed by RT-PCR. The band intensity was measured using the Multi Gauge V3.0 software (Fuji Film). *** P
Figure Legend Snippet: Detection of glucocorticoid-induced transcript 1 (GLCCI1) and podocyte-specific marker gene expression by reverse transcription-PCR (RT-PCR). ( a ) Expression of the genes encoding GLCCI1, nephrin, podocin, synaptopodin and podocalyxin showed similar patterns in the podocytes. The expression levels differed significantly between the high glucose-induced and wortmannin-treated diabetic groups of podocytes. ( b ) The expression of the genes encoding GLCCI1, nephrin, podocin, synaptopodin and podocalyxin showed similar patterns in the kidneys of diabetic rats. In addition, wortmannin ameliorated the expression of all podocyte-specific proteins including GLCCI1. ( c , d ) The relative band intensity of proteins in podocytes and the kidneys of diabetic rats was observed by RT-PCR. The band intensity was measured using the Multi Gauge V3.0 software (Fuji Film). *** P

Techniques Used: Marker, Expressing, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Software

6) Product Images from "Age-related GSK3 β overexpression drives podocyte senescence and glomerular aging"

Article Title: Age-related GSK3 β overexpression drives podocyte senescence and glomerular aging

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI141848

Long-term lithium carbonate therapy in psychiatric patients inhibits GSK3β activity and attenuates cellular senescence in urinary exfoliated cells. ( A ) Schematic diagram depicts preparation of urinary exfoliated cells from psychiatric patients treated either with lithium carbonate [Li (+), n = 12] or without lithium carbonate [Li (–), n = 12]. Scale bars: 100 μm. ( B ) Immunofluorescent staining of urinary exfoliated cells for synaptopodin (red) with DAPI counterstaining for nuclei, as shown by fluorescence microscopy and differential interference contrast (DIC) microscopy. Arrowheads indicate synaptopodin-positive podocytes, while arrows indicate synaptopodin-negative urinary cells. Scale bars: 20 μm. ( C ) Multicolor immunofluorescent staining of urinary exfoliated cells for phosphorylated GSK3β at serine 9 (p-GSK3β S9 ), p16 INK4A , and WT-1. Arrows indicate WT-1–positive urinary podocytes with p-GSK3β S9-lo p16 hi staining pattern. Arrowheads indicate WT-1–positive urinary podocytes with p-GSK3β S9-hi p16 lo staining pattern. Scale bars: 100 μm. ( D ) Quantification of cells with high and low expression of p16 INK4A among all WT-1 + urinary cells. ** P
Figure Legend Snippet: Long-term lithium carbonate therapy in psychiatric patients inhibits GSK3β activity and attenuates cellular senescence in urinary exfoliated cells. ( A ) Schematic diagram depicts preparation of urinary exfoliated cells from psychiatric patients treated either with lithium carbonate [Li (+), n = 12] or without lithium carbonate [Li (–), n = 12]. Scale bars: 100 μm. ( B ) Immunofluorescent staining of urinary exfoliated cells for synaptopodin (red) with DAPI counterstaining for nuclei, as shown by fluorescence microscopy and differential interference contrast (DIC) microscopy. Arrowheads indicate synaptopodin-positive podocytes, while arrows indicate synaptopodin-negative urinary cells. Scale bars: 20 μm. ( C ) Multicolor immunofluorescent staining of urinary exfoliated cells for phosphorylated GSK3β at serine 9 (p-GSK3β S9 ), p16 INK4A , and WT-1. Arrows indicate WT-1–positive urinary podocytes with p-GSK3β S9-lo p16 hi staining pattern. Arrowheads indicate WT-1–positive urinary podocytes with p-GSK3β S9-hi p16 lo staining pattern. Scale bars: 100 μm. ( D ) Quantification of cells with high and low expression of p16 INK4A among all WT-1 + urinary cells. ** P

Techniques Used: Activity Assay, Staining, Fluorescence, Microscopy, Expressing

Cellular senescence and senescence-associated secretory phenotypes (SASPs) are mitigated in primary podocytes derived from KO mice and reinstated after GSK3β reconstitution. ( A ) Primary podocytes were cultured from glomeruli isolated from 12-month-old control mice (Con) and mice with podocyte-specific GSK3β knockout (KO). Representative micrographs show freshly isolated glomeruli and primary cultures of podocytes. Scale bars: 75 μm. ( B – E ) Primary podocytes were subjected to electroporation-based transfection with either an empty plasmid vector (EV) or a plasmid encoding the HA-conjugated WT GSK3β by using the Amaxa Nucleofection kit. ( B ) Cells were processed for SA-β-gal activity staining or immunofluorescent staining for synaptopodin (SYNPO; red) or γH2AX (green) followed by counterstaining with DAPI for nuclei or with rhodamine-phalloidin for F-actin (red). Scale bars: 20 μm (top 2 rows) and 30 μm (bottom row). ( C ) Cell lysates were processed for immunoblot analysis for indicated proteins, including SASP factors like fibronectin (FN) and PAI-1. β-Tubulin served as a loading control. ( D ) Absolute count of the number of γH2AX + cells expressed as percentages of the total number of cells per microscopic field. * P
Figure Legend Snippet: Cellular senescence and senescence-associated secretory phenotypes (SASPs) are mitigated in primary podocytes derived from KO mice and reinstated after GSK3β reconstitution. ( A ) Primary podocytes were cultured from glomeruli isolated from 12-month-old control mice (Con) and mice with podocyte-specific GSK3β knockout (KO). Representative micrographs show freshly isolated glomeruli and primary cultures of podocytes. Scale bars: 75 μm. ( B – E ) Primary podocytes were subjected to electroporation-based transfection with either an empty plasmid vector (EV) or a plasmid encoding the HA-conjugated WT GSK3β by using the Amaxa Nucleofection kit. ( B ) Cells were processed for SA-β-gal activity staining or immunofluorescent staining for synaptopodin (SYNPO; red) or γH2AX (green) followed by counterstaining with DAPI for nuclei or with rhodamine-phalloidin for F-actin (red). Scale bars: 20 μm (top 2 rows) and 30 μm (bottom row). ( C ) Cell lysates were processed for immunoblot analysis for indicated proteins, including SASP factors like fibronectin (FN) and PAI-1. β-Tubulin served as a loading control. ( D ) Absolute count of the number of γH2AX + cells expressed as percentages of the total number of cells per microscopic field. * P

Techniques Used: Derivative Assay, Mouse Assay, Cell Culture, Isolation, Knock-Out, Electroporation, Transfection, Plasmid Preparation, Activity Assay, Staining

GSK3β regulates the phosphorylation of p16 INK4A and p53, resulting in modulation of senescence signaling in podocytes. Conditionally immortalized murine podocytes were transiently lipotransfected with a control empty plasmid vector (EV), or plasmids encoding the HA-conjugated dominant-negative kinase dead (KD) mutant of GSK3β or constitutively active (S9A) mutant of GSK3β in the presence or absence of lithium chloride (LiCl, 10 mM) or an equal volume of vehicle. ( A ) After different treatments, cells were subjected to immunofluorescent staining for HA, which revealed a transfection efficiency of approximately 80%. Scale bar: 20 μm. ( B ) Whole cell lysates were processed for immunoprecipitation (IP) by using an anti-p16 INK4A or -p53 antibody, followed by immunoblot analysis (IB) of immunoprecipitates for phosphorylated serine (p-Ser), in parallel with input controls. ( C ) Representative immunoblot analysis of cell lysates for indicated molecules. β-Tubulin served as a loading control. ( D ) Cells were subjected to SA-β-gal activity staining, or to immunofluorescent staining for synaptopodin (SYNPO; red) or γH2AX (green) followed by counterstaining with DAPI for nuclei or with rhodamine-phalloidin for F-actin (red). Scale bars: 20 μm. ( E ) Absolute count of the number of γH2AX + cells as percentages of the total number of cells per microscopic field. * P
Figure Legend Snippet: GSK3β regulates the phosphorylation of p16 INK4A and p53, resulting in modulation of senescence signaling in podocytes. Conditionally immortalized murine podocytes were transiently lipotransfected with a control empty plasmid vector (EV), or plasmids encoding the HA-conjugated dominant-negative kinase dead (KD) mutant of GSK3β or constitutively active (S9A) mutant of GSK3β in the presence or absence of lithium chloride (LiCl, 10 mM) or an equal volume of vehicle. ( A ) After different treatments, cells were subjected to immunofluorescent staining for HA, which revealed a transfection efficiency of approximately 80%. Scale bar: 20 μm. ( B ) Whole cell lysates were processed for immunoprecipitation (IP) by using an anti-p16 INK4A or -p53 antibody, followed by immunoblot analysis (IB) of immunoprecipitates for phosphorylated serine (p-Ser), in parallel with input controls. ( C ) Representative immunoblot analysis of cell lysates for indicated molecules. β-Tubulin served as a loading control. ( D ) Cells were subjected to SA-β-gal activity staining, or to immunofluorescent staining for synaptopodin (SYNPO; red) or γH2AX (green) followed by counterstaining with DAPI for nuclei or with rhodamine-phalloidin for F-actin (red). Scale bars: 20 μm. ( E ) Absolute count of the number of γH2AX + cells as percentages of the total number of cells per microscopic field. * P

Techniques Used: Plasmid Preparation, Dominant Negative Mutation, Mutagenesis, Staining, Transfection, Immunoprecipitation, Activity Assay

7) Product Images from "Insulin-activated store-operated Ca2+ entry via Orai1 induces podocyte actin remodeling and causes proteinuria"

Article Title: Insulin-activated store-operated Ca2+ entry via Orai1 induces podocyte actin remodeling and causes proteinuria

Journal: Nature Communications

doi: 10.1038/s41467-021-26900-w

Orai1 is overexpressed in hyperinsulinemic db/db mice and CsA ameliorates slit-diaphragm disruption and albuminuria. a Time-dependent recovery of insulin-mediated actin rearrangement. Podocytes were incubated with insulin for 30 min (short-term) or 6 h (long-term), washed out (W/O), and further incubated in an insulin-free medium for 12 h before phalloidin staining. White arrows indicate actin rearrangement by insulin treatment. Scale bar = 50 μm. b Quantification of the actin fiber intensity in a . n = 3, 3, 4, 5, 4, and 3 dishes, twenty images in each dish. c , d Analysis of the serum levels of insulin (ng/ml) ( c ) and glucose (mg/dL) ( d ) in db/m and db/db mice at 9, 11, and 19 weeks of age ( n = 5 mice each). e Representative immunoblotting showing Orai1 and TRPC6 expression in the glomerulus of db/m and db/db mice at 11 and 19 weeks. f Densitometry of Orai1 (left) and TRPC6 (right) in e . Relative (Rel.) protein levels were normalized to db/m mice (11 weeks of age) n = 3 independent experiments. g Representative immunofluorescence images of synaptopodin (synap.; green) and Orai1 (red) in the glomeruli of db/m and db/db mice. Immunostaining was repeated five times independently from 5 mice each. Scale bar = 25 μm. h Immunoblotting analysis showing synaptopodin expression in the glomeruli of db/m and db/db mice at 11 and 19 weeks. i Quantification of the western blots in h . n = 3 independent experiments. j Effect of cyclosporin A (CsA, i.p . injection) on urine Alb/Cr ratio (left) and 24 h urinary albumin excretion (right) in db/m and db/db mice ( n = 5–7 images from mice each). k Immunofluorescence staining showing the effect of CsA on synaptopodin dissolution in db/db mice. Scale bar = 25 μm. l Summary of synaptopodin quantification in k . n = 30 images from 5 to 10 mice each. m Representative TEM images of foot processes from db/m and db/db mice with/without administration of CsA. Magnification is ×30,000. Scale bar = 1 μm. n , o GBM thickness ( n ) and an average number of foot processes per 1 μm length of GBM ( o ) in db/m and db/db mice with/without administration of CsA. Thirty images from 5–7 mice in each group. Bar graph data are expressed as mean ± SEM and were analyzed using two-way ANOVA followed by Tukey’s multiple comparisons test. ** p
Figure Legend Snippet: Orai1 is overexpressed in hyperinsulinemic db/db mice and CsA ameliorates slit-diaphragm disruption and albuminuria. a Time-dependent recovery of insulin-mediated actin rearrangement. Podocytes were incubated with insulin for 30 min (short-term) or 6 h (long-term), washed out (W/O), and further incubated in an insulin-free medium for 12 h before phalloidin staining. White arrows indicate actin rearrangement by insulin treatment. Scale bar = 50 μm. b Quantification of the actin fiber intensity in a . n = 3, 3, 4, 5, 4, and 3 dishes, twenty images in each dish. c , d Analysis of the serum levels of insulin (ng/ml) ( c ) and glucose (mg/dL) ( d ) in db/m and db/db mice at 9, 11, and 19 weeks of age ( n = 5 mice each). e Representative immunoblotting showing Orai1 and TRPC6 expression in the glomerulus of db/m and db/db mice at 11 and 19 weeks. f Densitometry of Orai1 (left) and TRPC6 (right) in e . Relative (Rel.) protein levels were normalized to db/m mice (11 weeks of age) n = 3 independent experiments. g Representative immunofluorescence images of synaptopodin (synap.; green) and Orai1 (red) in the glomeruli of db/m and db/db mice. Immunostaining was repeated five times independently from 5 mice each. Scale bar = 25 μm. h Immunoblotting analysis showing synaptopodin expression in the glomeruli of db/m and db/db mice at 11 and 19 weeks. i Quantification of the western blots in h . n = 3 independent experiments. j Effect of cyclosporin A (CsA, i.p . injection) on urine Alb/Cr ratio (left) and 24 h urinary albumin excretion (right) in db/m and db/db mice ( n = 5–7 images from mice each). k Immunofluorescence staining showing the effect of CsA on synaptopodin dissolution in db/db mice. Scale bar = 25 μm. l Summary of synaptopodin quantification in k . n = 30 images from 5 to 10 mice each. m Representative TEM images of foot processes from db/m and db/db mice with/without administration of CsA. Magnification is ×30,000. Scale bar = 1 μm. n , o GBM thickness ( n ) and an average number of foot processes per 1 μm length of GBM ( o ) in db/m and db/db mice with/without administration of CsA. Thirty images from 5–7 mice in each group. Bar graph data are expressed as mean ± SEM and were analyzed using two-way ANOVA followed by Tukey’s multiple comparisons test. ** p

Techniques Used: Mouse Assay, Incubation, Staining, Expressing, Immunofluorescence, Immunostaining, Western Blot, Injection, Transmission Electron Microscopy

Orai1 inhibition protects podocyte foot processes and proteinuria. a Representative traces of fura-2 ratio and the images of bright-field and fura-2 fluorescence microscopy (upper panel) of podocytes, carried out in situ on the acutely isolated mouse in db/m and db/db mice treated with GSK-7975A (5 mg/kg/day for 1 month). b Summary of the SOCE in a . n = 20 ( db/m + Vehicle), 22( db/db + Vehicle), and 17 ( db/db + GSK) glomeruli per each group, respectively. c Effect of SOCE inhibitors on albuminuria. Twenty-four-hour urinary Alb/Cr ratio (left) and albumin excretion (right) were measured in db/m and db/db mice. Vehicle (PBS), GSK-7975A (GSK, 5 mg/kg/every 2 days) or 2-APB (2 mg/kg/every 2 days) were inoculated i.p . from 8 to 12 weeks. n = 7 ( db/m + Veh), 4 ( db/m + GSK), 5 ( db/m + 2-APB), 5 ( db/db + Veh), 5 ( db/db + GSK), and 4 ( db/db + 2-APB). d Representative immunoblots representing the effects of SOCE inhibitors, GSK-7975A (upper) or 2-APB (lower), on synaptopodin expression from db/m and db/db mice. e Summary of quantification in d . n = 4 mice each (left). n = 4 ( db/m + Veh), 10 ( db/m + 2-APB), 5 ( db/db + Veh), and 7 ( db/db + 2-APB) (right). f Rescue of synaptopodin depletion by SOCE inhibitors (GSK or 2-APB). Synaptopodin staining (green) of mouse glomeruli from db/m and db/db mice with or without GSK or 2-APB i.p . injection. Scale bar = 25 μm. g Analysis of the relative fluorescence intensity in f . n = 4 ( db/db + Veh), 4 ( db/db + Veh), 5 ( db/db + GSK), and 6 ( db/db + 2-APB). Thirty images from mice in each group. h Representative TEM images of the foot processes of the podocytes of SOCE inhibitor-treated db/db mice. Magnification is ×30,000. Scale bar = 1 μm. i , j Summary of GBM thickness ( i ) and an average number of foot processes per 1 μm length of GBM ( j ) in h . n = 4 ( db/m + Veh), 10 ( db/db + Veh), 6 ( db/db + GSK), and 7 ( db/db + 2-APB) thirty images from mice in each group. Data are expressed as mean ± SEM and were analyzed using two-way ANOVA followed by Tukey’s multiple comparisons test. * p
Figure Legend Snippet: Orai1 inhibition protects podocyte foot processes and proteinuria. a Representative traces of fura-2 ratio and the images of bright-field and fura-2 fluorescence microscopy (upper panel) of podocytes, carried out in situ on the acutely isolated mouse in db/m and db/db mice treated with GSK-7975A (5 mg/kg/day for 1 month). b Summary of the SOCE in a . n = 20 ( db/m + Vehicle), 22( db/db + Vehicle), and 17 ( db/db + GSK) glomeruli per each group, respectively. c Effect of SOCE inhibitors on albuminuria. Twenty-four-hour urinary Alb/Cr ratio (left) and albumin excretion (right) were measured in db/m and db/db mice. Vehicle (PBS), GSK-7975A (GSK, 5 mg/kg/every 2 days) or 2-APB (2 mg/kg/every 2 days) were inoculated i.p . from 8 to 12 weeks. n = 7 ( db/m + Veh), 4 ( db/m + GSK), 5 ( db/m + 2-APB), 5 ( db/db + Veh), 5 ( db/db + GSK), and 4 ( db/db + 2-APB). d Representative immunoblots representing the effects of SOCE inhibitors, GSK-7975A (upper) or 2-APB (lower), on synaptopodin expression from db/m and db/db mice. e Summary of quantification in d . n = 4 mice each (left). n = 4 ( db/m + Veh), 10 ( db/m + 2-APB), 5 ( db/db + Veh), and 7 ( db/db + 2-APB) (right). f Rescue of synaptopodin depletion by SOCE inhibitors (GSK or 2-APB). Synaptopodin staining (green) of mouse glomeruli from db/m and db/db mice with or without GSK or 2-APB i.p . injection. Scale bar = 25 μm. g Analysis of the relative fluorescence intensity in f . n = 4 ( db/db + Veh), 4 ( db/db + Veh), 5 ( db/db + GSK), and 6 ( db/db + 2-APB). Thirty images from mice in each group. h Representative TEM images of the foot processes of the podocytes of SOCE inhibitor-treated db/db mice. Magnification is ×30,000. Scale bar = 1 μm. i , j Summary of GBM thickness ( i ) and an average number of foot processes per 1 μm length of GBM ( j ) in h . n = 4 ( db/m + Veh), 10 ( db/db + Veh), 6 ( db/db + GSK), and 7 ( db/db + 2-APB) thirty images from mice in each group. Data are expressed as mean ± SEM and were analyzed using two-way ANOVA followed by Tukey’s multiple comparisons test. * p

Techniques Used: Inhibition, Fluorescence, Microscopy, In Situ, Isolation, Mouse Assay, Western Blot, Expressing, Staining, Injection, Transmission Electron Microscopy

Working model of the effect of insulin signaling on podocyte homeostasis by the targeting of podocyte Orai1. Insulin stimulates Orai1 leading to an aberrant [Ca 2+ ] i signaling, which in turn activates calcineurin and causes synaptopodin degradation, actin cytoskeleton remodeling, increased contractility, and motility, and dysfunction of slit-diaphragm integrity. Acute (or short-term) stimulation of Orai1 by insulin receptor or receptor tyrosine kinases causes reversible physiological changes in the podocyte actin cytoskeleton. This may contribute to slit-diaphragm plasticity and/or survival against stress or injury. In contrast, chronic stimulation or hyperactivation of Orai1 activation leads to irreversible pathological changes such as podocyte foot process effacement and severe proteinuria.
Figure Legend Snippet: Working model of the effect of insulin signaling on podocyte homeostasis by the targeting of podocyte Orai1. Insulin stimulates Orai1 leading to an aberrant [Ca 2+ ] i signaling, which in turn activates calcineurin and causes synaptopodin degradation, actin cytoskeleton remodeling, increased contractility, and motility, and dysfunction of slit-diaphragm integrity. Acute (or short-term) stimulation of Orai1 by insulin receptor or receptor tyrosine kinases causes reversible physiological changes in the podocyte actin cytoskeleton. This may contribute to slit-diaphragm plasticity and/or survival against stress or injury. In contrast, chronic stimulation or hyperactivation of Orai1 activation leads to irreversible pathological changes such as podocyte foot process effacement and severe proteinuria.

Techniques Used: Activation Assay

Insulin stimulates Orai1-mediated store-operated Ca 2+ entry (SOCE), aggravating actin rearrangement and transepithelial albumin leakage in mouse podocytes. a Vehicle ( n = 29 cells) or serum growth factors including serum (10%) ( n = 26 cells), insulin (100 nM for 1 h, n = 27 cells), and IGF-1 (10 nM for 1 h, n = 29 cells) increased SOCE in podocytes. To examine insulin effects, cells or isolated glomeruli were incubated in a serum-free medium for ~16 h before insulin treatment for the indicated duration. b Representative SOCE traces with the images of bright-field and fura-2 fluorescence (upper panel) microscopy of insulin-treated podocytes (100 nM) carried out in situ on acutely isolated mouse glomeruli. c , d Effect of SOCE inhibitors [GSK (GSK-7975A; 3 μM) or 2-APB (100 μM)] on insulin-stimulated SOCE in isolated (iso.) glomeruli ( c ) and mouse (m.) podocytes ( d ). n = 33 (Vehicle), 77 (Insulin), 74 (Insulin+GSK), and 33 (Insulin+2-APB) glomeruli ( c ) and 19 (Vehicle), 17 (Insulin), 15 (Insulin+GSK), and 21 (Insulin+2-APB) cells ( d ) per group. e Representative SOCE traces showing the effect of Orai1 knockdown by siRNA on insulin-stimulated SOCE in cultured mouse podocytes. f Summary of the SOCE in e . n = 14 (siCtrl+Veh), 14 (siCtrl+Insulin), 20 (siOrai1+Veh), and 18 (siOrai1+Insulin) cells per each group. g Dose–response of insulin on SOCE in cultured mouse podocytes. n = 21, 21, 32, 10, 33, 15, 19 cells for each point, respectively. EC 50 of insulin is approximately 0.95 nM. h , i Effect of Orai1-mediated SOCE inhibition by siRNA Orai1 (siOrai1) or GSK or 2-APB ( h ) and effect of cyclosporine A (CsA; 10 μM) ( i ) on insulin-mediated actin rearrangement (white arrow). Scale bar = 50 μm. The experiments were repeated twice and several cells were analyzed for each experiment. j Effect of SOCE inhibitors and CsA on insulin-mediated depletion of synaptopodin (Synap.) in cultured mouse podocytes. k Effect of siRNA Orai1 on the insulin-induced dissolution of synaptopodin. l , m . Summary of relative synaptopodin expression in the j and k , respectively. n = 3 independent experiments. n Collagen contraction assay showing insulin-stimulated podocyte retraction, which is rescued by the inhibition of Orai1-calcineurin pathway. This assay was repeated two times independently. o Effect of Orai1 knockdown and GSK and CsA treatment on insulin-mediated transepithelial albumin leakage in mouse podocytes. n = 4 independent experiments. Bar graphs are expressed as mean ± SEM and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. * p
Figure Legend Snippet: Insulin stimulates Orai1-mediated store-operated Ca 2+ entry (SOCE), aggravating actin rearrangement and transepithelial albumin leakage in mouse podocytes. a Vehicle ( n = 29 cells) or serum growth factors including serum (10%) ( n = 26 cells), insulin (100 nM for 1 h, n = 27 cells), and IGF-1 (10 nM for 1 h, n = 29 cells) increased SOCE in podocytes. To examine insulin effects, cells or isolated glomeruli were incubated in a serum-free medium for ~16 h before insulin treatment for the indicated duration. b Representative SOCE traces with the images of bright-field and fura-2 fluorescence (upper panel) microscopy of insulin-treated podocytes (100 nM) carried out in situ on acutely isolated mouse glomeruli. c , d Effect of SOCE inhibitors [GSK (GSK-7975A; 3 μM) or 2-APB (100 μM)] on insulin-stimulated SOCE in isolated (iso.) glomeruli ( c ) and mouse (m.) podocytes ( d ). n = 33 (Vehicle), 77 (Insulin), 74 (Insulin+GSK), and 33 (Insulin+2-APB) glomeruli ( c ) and 19 (Vehicle), 17 (Insulin), 15 (Insulin+GSK), and 21 (Insulin+2-APB) cells ( d ) per group. e Representative SOCE traces showing the effect of Orai1 knockdown by siRNA on insulin-stimulated SOCE in cultured mouse podocytes. f Summary of the SOCE in e . n = 14 (siCtrl+Veh), 14 (siCtrl+Insulin), 20 (siOrai1+Veh), and 18 (siOrai1+Insulin) cells per each group. g Dose–response of insulin on SOCE in cultured mouse podocytes. n = 21, 21, 32, 10, 33, 15, 19 cells for each point, respectively. EC 50 of insulin is approximately 0.95 nM. h , i Effect of Orai1-mediated SOCE inhibition by siRNA Orai1 (siOrai1) or GSK or 2-APB ( h ) and effect of cyclosporine A (CsA; 10 μM) ( i ) on insulin-mediated actin rearrangement (white arrow). Scale bar = 50 μm. The experiments were repeated twice and several cells were analyzed for each experiment. j Effect of SOCE inhibitors and CsA on insulin-mediated depletion of synaptopodin (Synap.) in cultured mouse podocytes. k Effect of siRNA Orai1 on the insulin-induced dissolution of synaptopodin. l , m . Summary of relative synaptopodin expression in the j and k , respectively. n = 3 independent experiments. n Collagen contraction assay showing insulin-stimulated podocyte retraction, which is rescued by the inhibition of Orai1-calcineurin pathway. This assay was repeated two times independently. o Effect of Orai1 knockdown and GSK and CsA treatment on insulin-mediated transepithelial albumin leakage in mouse podocytes. n = 4 independent experiments. Bar graphs are expressed as mean ± SEM and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. * p

Techniques Used: Isolation, Incubation, Fluorescence, Microscopy, In Situ, Cell Culture, Inhibition, Expressing, Contraction Assay

Insulin activation targeting the Orai1 channel induces albuminuria in mice. a , b mRNA ( a ) and protein ( b ) expression levels of Orai1 in the isolated glomeruli and medulla of wild-type (WT), Cre+ ;Orai1 fl/+ , and Cre+ ;Orai1 fl/fl mice. n = 3 independent experiments. c Immunofluorescence image displaying Orai1 (green) and synaptopodin (red; Synap.) expression in the isolated glomeruli of Cre+ ;Orai1 fl/+ and Cre+ ;Orai1 fl/fl mice. Scale bar = 25 μm. All images were selected among 3 mice in each group by independent experiments. d H E staining of the kidney sections of the WT, Cre+ ;Orai1 fl/+ , and Cre+ ;Orai1 fl/fl mice. e Representative traces of SOCE with the images of bright-field and fura-2 fluorescence (upper panel) microscopy carried out in situ on acutely isolated mouse glomeruli from WT, Cre + ;Orai1 fl/+ , and Cre + ;Orai1 fl/fl mice. f Quantification of SOCE in e . n = 30 (WT), 25 ( Cre+ ;Orai1 fl/+ ), and 29 ( Cre + ;Orai1 fl/fl ) glomeruli per group. g Representative traces of fura-2 ratio showing insulin effects on SOCE in acutely isolated mouse glomeruli from Cre + ;Orai1 fl/+ and Cre+ ;Orai1 fl/fl mice. h Quantification of SOCE in g . n = 60, 26, 24, and 48 glomeruli per each group, respectively. i Effects of insulin administration (i.p.; 5 U/kg) on 24 h urinary albumin/creatinine ratio (Alb/Cr Ratio) (left) and albumin excretion (right) in Cre+ ;Orai1 fl/+ and Cre+ ;Orai1 fl/fl mice. n = 8, 7, 8, and 7 mice per group. j Immunoblotting analysis showing insulin effects on synaptopodin in isolated glomeruli from Cre + ;Orai1 fl/+ and Cre+ ;Orai1 fl/fl mice. k Densitometry of relative (Rel.) synaptopodin expression in j . n = 8, 7, 8, and 7 mice per each group. l Immunoblotting analysis showing the expression of exogenous Orai1 protein (detected by anti-Flag antibody) 48 h after in vivo gene delivery of Flag-tagged-Orai1. m Immunostaining of mCherry-tagged Orai1 (red; mCh-Orai1) and synaptopodin (green; Synap.) in mice injected with Orai1 transgene. Scale bar = 25 μm. Immunostaining was repeated two times from 3 mice each. n Effect of transgenic Orai1 overexpression and insulin administration ( i.p .; 5 U/kg) on urinary Alb/Cr ratio (left) and albumin excretion. n = 5 mice each. o Immunoblotting analysis showing the effect of transgenic Orai1 overexpression and insulin inoculation (i.p.; 5 U/kg) on synaptopodin expression. p Densitometry of relative (Rel.) synaptopodin levels in o . n = 5 mice each. q Representative TEM images of foot processes from in vivo gene delivery of Flag-tagged-Orai1 with/without administration of insulin. EM repeated in 3 mice each. Magnification is ×30,000. Scale bar = 1 μm. Bar graphs are expressed as mean ± SEM and analyzed by two-way ANOVA followed by Tukey’s multiple comparisons test. * p
Figure Legend Snippet: Insulin activation targeting the Orai1 channel induces albuminuria in mice. a , b mRNA ( a ) and protein ( b ) expression levels of Orai1 in the isolated glomeruli and medulla of wild-type (WT), Cre+ ;Orai1 fl/+ , and Cre+ ;Orai1 fl/fl mice. n = 3 independent experiments. c Immunofluorescence image displaying Orai1 (green) and synaptopodin (red; Synap.) expression in the isolated glomeruli of Cre+ ;Orai1 fl/+ and Cre+ ;Orai1 fl/fl mice. Scale bar = 25 μm. All images were selected among 3 mice in each group by independent experiments. d H E staining of the kidney sections of the WT, Cre+ ;Orai1 fl/+ , and Cre+ ;Orai1 fl/fl mice. e Representative traces of SOCE with the images of bright-field and fura-2 fluorescence (upper panel) microscopy carried out in situ on acutely isolated mouse glomeruli from WT, Cre + ;Orai1 fl/+ , and Cre + ;Orai1 fl/fl mice. f Quantification of SOCE in e . n = 30 (WT), 25 ( Cre+ ;Orai1 fl/+ ), and 29 ( Cre + ;Orai1 fl/fl ) glomeruli per group. g Representative traces of fura-2 ratio showing insulin effects on SOCE in acutely isolated mouse glomeruli from Cre + ;Orai1 fl/+ and Cre+ ;Orai1 fl/fl mice. h Quantification of SOCE in g . n = 60, 26, 24, and 48 glomeruli per each group, respectively. i Effects of insulin administration (i.p.; 5 U/kg) on 24 h urinary albumin/creatinine ratio (Alb/Cr Ratio) (left) and albumin excretion (right) in Cre+ ;Orai1 fl/+ and Cre+ ;Orai1 fl/fl mice. n = 8, 7, 8, and 7 mice per group. j Immunoblotting analysis showing insulin effects on synaptopodin in isolated glomeruli from Cre + ;Orai1 fl/+ and Cre+ ;Orai1 fl/fl mice. k Densitometry of relative (Rel.) synaptopodin expression in j . n = 8, 7, 8, and 7 mice per each group. l Immunoblotting analysis showing the expression of exogenous Orai1 protein (detected by anti-Flag antibody) 48 h after in vivo gene delivery of Flag-tagged-Orai1. m Immunostaining of mCherry-tagged Orai1 (red; mCh-Orai1) and synaptopodin (green; Synap.) in mice injected with Orai1 transgene. Scale bar = 25 μm. Immunostaining was repeated two times from 3 mice each. n Effect of transgenic Orai1 overexpression and insulin administration ( i.p .; 5 U/kg) on urinary Alb/Cr ratio (left) and albumin excretion. n = 5 mice each. o Immunoblotting analysis showing the effect of transgenic Orai1 overexpression and insulin inoculation (i.p.; 5 U/kg) on synaptopodin expression. p Densitometry of relative (Rel.) synaptopodin levels in o . n = 5 mice each. q Representative TEM images of foot processes from in vivo gene delivery of Flag-tagged-Orai1 with/without administration of insulin. EM repeated in 3 mice each. Magnification is ×30,000. Scale bar = 1 μm. Bar graphs are expressed as mean ± SEM and analyzed by two-way ANOVA followed by Tukey’s multiple comparisons test. * p

Techniques Used: Activation Assay, Mouse Assay, Expressing, Isolation, Immunofluorescence, Staining, Fluorescence, Microscopy, In Situ, In Vivo, Immunostaining, Injection, Transgenic Assay, Over Expression, Transmission Electron Microscopy

8) Product Images from "Cdc42 deficiency induces podocyte apoptosis by inhibiting the Nwasp/stress fibers/YAP pathway"

Article Title: Cdc42 deficiency induces podocyte apoptosis by inhibiting the Nwasp/stress fibers/YAP pathway

Journal: Cell Death & Disease

doi: 10.1038/cddis.2016.51

Cdc42 and Nwasp were decreased in injured podocytes. ( a ) The mRNA expression of Cdc42 and Nwasp were decreased in HG, LPS or ADR-injured podocytes. ( b, c ) The protein expression of Cdc42 and Nwasp were decreased in HG, LPS or ADR-injured podocytes. ( d ) Cdc42 activity was decreased in HG, LPS or ADR-injured podocytes. ( e ) Representative micrographs of dual-color fluorescence staining of kidney glomeruli for Cdc42 (red) and synaptopodin (green) from control, LPS and ADR mice. Magnification × 400, scale bar=25 μ m. ( f ) Representative micrographs of dual-color fluorescence staining of kidney glomeruli for Cdc42 (red) and synaptopodin (green) from control, DN and FSGS patients. Magnification × 400, scale bar=25 μ m. All in vitro studies result in MA (as an osmotic control) and the Con group show hardly any difference ( a–d ). All data were from at least three independent experiments. * P
Figure Legend Snippet: Cdc42 and Nwasp were decreased in injured podocytes. ( a ) The mRNA expression of Cdc42 and Nwasp were decreased in HG, LPS or ADR-injured podocytes. ( b, c ) The protein expression of Cdc42 and Nwasp were decreased in HG, LPS or ADR-injured podocytes. ( d ) Cdc42 activity was decreased in HG, LPS or ADR-injured podocytes. ( e ) Representative micrographs of dual-color fluorescence staining of kidney glomeruli for Cdc42 (red) and synaptopodin (green) from control, LPS and ADR mice. Magnification × 400, scale bar=25 μ m. ( f ) Representative micrographs of dual-color fluorescence staining of kidney glomeruli for Cdc42 (red) and synaptopodin (green) from control, DN and FSGS patients. Magnification × 400, scale bar=25 μ m. All in vitro studies result in MA (as an osmotic control) and the Con group show hardly any difference ( a–d ). All data were from at least three independent experiments. * P

Techniques Used: Expressing, Activity Assay, Fluorescence, Staining, Mouse Assay, In Vitro

Loss of Cdc42/Nwasp decreased stress fiber formation and YAP expression. ( a ) Typical confocal images of culture podocytes showing expression of YAP (green), Phalloidin-stained stress fiber (red) and DAPI-stained nuclei (blue). Compared to siCon, nuclear and cytoplasmic YAP and stress fiber were obviously reduced in Cdc42 siRNA or Nwasp siRNA-treated podocytes. ( b ) The mRNA expression of YAP was decreased in Cdc42 siRNA and Nwasp siRNA-treated podocytes. ( c–f ) The total and nuclear protein expression of YAP was decreased in Cdc42 siRNA or Nwasp siRNA-treated podocytes. ( g ) The mRNA expression of Nwasp was decreased in Cdc42 siRNA-treated podocytes. ( h–i ) Cdc42 and Nwasp protein expression were reduced to about 21.8%, 15.4% in Cdc42 siRNA and Nwasp siRNA-treated podocytes, respectively. Nwasp protein expression was obviously decreased in Cdc42 knockdown podocytes comparing to siCon, as Cdc42 protein expression was not changed in Nwasp knockdown podocytes. ( j ) On top: typical images of dual-color fluorescence staining of kidney glomeruli for synaptopodin (green) and Cdc42 (red) from control, Dmso and ML-141 (Cdc42-specific inhibitor) mice. Cdc42 was hardly seen in podocytes from ML-141 mice. Below: representative micrographs of dual-color fluorescence staining of kidney glomeruli for WT-1 (green) and YAP (red) from control, Dmso and ML-141 mice. Nuclear YAP was decreased in podocytes from ML-141 mice comparing to Con. Magnification × 400, scale bar=25 μ m. All above data were from at least three independent experiments. * P
Figure Legend Snippet: Loss of Cdc42/Nwasp decreased stress fiber formation and YAP expression. ( a ) Typical confocal images of culture podocytes showing expression of YAP (green), Phalloidin-stained stress fiber (red) and DAPI-stained nuclei (blue). Compared to siCon, nuclear and cytoplasmic YAP and stress fiber were obviously reduced in Cdc42 siRNA or Nwasp siRNA-treated podocytes. ( b ) The mRNA expression of YAP was decreased in Cdc42 siRNA and Nwasp siRNA-treated podocytes. ( c–f ) The total and nuclear protein expression of YAP was decreased in Cdc42 siRNA or Nwasp siRNA-treated podocytes. ( g ) The mRNA expression of Nwasp was decreased in Cdc42 siRNA-treated podocytes. ( h–i ) Cdc42 and Nwasp protein expression were reduced to about 21.8%, 15.4% in Cdc42 siRNA and Nwasp siRNA-treated podocytes, respectively. Nwasp protein expression was obviously decreased in Cdc42 knockdown podocytes comparing to siCon, as Cdc42 protein expression was not changed in Nwasp knockdown podocytes. ( j ) On top: typical images of dual-color fluorescence staining of kidney glomeruli for synaptopodin (green) and Cdc42 (red) from control, Dmso and ML-141 (Cdc42-specific inhibitor) mice. Cdc42 was hardly seen in podocytes from ML-141 mice. Below: representative micrographs of dual-color fluorescence staining of kidney glomeruli for WT-1 (green) and YAP (red) from control, Dmso and ML-141 mice. Nuclear YAP was decreased in podocytes from ML-141 mice comparing to Con. Magnification × 400, scale bar=25 μ m. All above data were from at least three independent experiments. * P

Techniques Used: Expressing, Staining, Fluorescence, Mouse Assay

9) Product Images from "Genetic Ablation of Calcium-independent Phospholipase A2γ Exacerbates Glomerular Injury in Adriamycin Nephrosis in Mice"

Article Title: Genetic Ablation of Calcium-independent Phospholipase A2γ Exacerbates Glomerular Injury in Adriamycin Nephrosis in Mice

Journal: Scientific Reports

doi: 10.1038/s41598-019-52834-x

Effect of iPLA 2 γ on podocyte number and differentiation in adriamycin nephrosis. Kidneys were harvested from control (Ctrl) and iPLA 2 γ KO mice 4 weeks after adriamycin administration. ( a , b ) Kidney sections were stained with antibodies to WT1, synaptopodin, podocalyxin and nephrin. ( a ) Representative IF staining. ( b ) WT1 counts and quantification of IF intensity. The number of WT1 positive nuclei (reflecting number of podocytes) was lower in KO mice. *P
Figure Legend Snippet: Effect of iPLA 2 γ on podocyte number and differentiation in adriamycin nephrosis. Kidneys were harvested from control (Ctrl) and iPLA 2 γ KO mice 4 weeks after adriamycin administration. ( a , b ) Kidney sections were stained with antibodies to WT1, synaptopodin, podocalyxin and nephrin. ( a ) Representative IF staining. ( b ) WT1 counts and quantification of IF intensity. The number of WT1 positive nuclei (reflecting number of podocytes) was lower in KO mice. *P

Techniques Used: Mouse Assay, Staining

10) Product Images from "A Homozygous Missense Mutation in the Ciliary Gene TTC21B Causes Familial FSGS"

Article Title: A Homozygous Missense Mutation in the Ciliary Gene TTC21B Causes Familial FSGS

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2013101126

IFT139, expressed at the base of primary cilium in tubules, fetal and undifferentiated podocytes, relocalizes along the microtubule network in mature and differentiated podocytes. (A) Immunolocalization of IFT139 in adult control human kidney using lotus tetragonolobus (LTA) and peanut agglutinin lectin (PNA) to identify proximal and distal tubules, respectively, revealed that IFT139 is expressed predominantly in distal tubules and glomeruli. Scale bar: 50 µ m. (B) Western blot showing expression of IFT139 in total kidney and isolated glomeruli or tubules from adult wild-type mice of mixed background. (C) Immunolabeling of IFT139; acetylated α -tubulin, a ciliary component; and nephrin, a podocyte marker, in fetal (14 weeks) and mature kidneys. IFT139 is expressed in developing glomeruli, notably at the base of primary cilia (arrow). In contrast, we observed a rearrangement of the acetylated α -tubulin network in mature glomeruli, and IFT139 colocalizes with this network. Scale bar: 50 µ m. (D) Immunolocalization of IFT139 in podocytes after 3 and 14 days of differentiation (D-3 and D-14). IFT139 localizes at the base of the cilium in undifferentiated podocytes and is redistributed along the acetylated α -tubulin network in differentiated podocytes, labeled by synaptopodin. Scale bar: 20 µ m. (E) Percentage of ciliated cells in cultured podocytes demonstrated that differentiated podocytes lose their primary cilium. For statistical analysis, four independent experimentations were performed. A t test was conducted; the mean±SEM is shown (* P
Figure Legend Snippet: IFT139, expressed at the base of primary cilium in tubules, fetal and undifferentiated podocytes, relocalizes along the microtubule network in mature and differentiated podocytes. (A) Immunolocalization of IFT139 in adult control human kidney using lotus tetragonolobus (LTA) and peanut agglutinin lectin (PNA) to identify proximal and distal tubules, respectively, revealed that IFT139 is expressed predominantly in distal tubules and glomeruli. Scale bar: 50 µ m. (B) Western blot showing expression of IFT139 in total kidney and isolated glomeruli or tubules from adult wild-type mice of mixed background. (C) Immunolabeling of IFT139; acetylated α -tubulin, a ciliary component; and nephrin, a podocyte marker, in fetal (14 weeks) and mature kidneys. IFT139 is expressed in developing glomeruli, notably at the base of primary cilia (arrow). In contrast, we observed a rearrangement of the acetylated α -tubulin network in mature glomeruli, and IFT139 colocalizes with this network. Scale bar: 50 µ m. (D) Immunolocalization of IFT139 in podocytes after 3 and 14 days of differentiation (D-3 and D-14). IFT139 localizes at the base of the cilium in undifferentiated podocytes and is redistributed along the acetylated α -tubulin network in differentiated podocytes, labeled by synaptopodin. Scale bar: 20 µ m. (E) Percentage of ciliated cells in cultured podocytes demonstrated that differentiated podocytes lose their primary cilium. For statistical analysis, four independent experimentations were performed. A t test was conducted; the mean±SEM is shown (* P

Techniques Used: Western Blot, Expressing, Isolation, Mouse Assay, Immunolabeling, Marker, Labeling, Cell Culture

11) Product Images from "Klotho May Ameliorate Proteinuria by Targeting TRPC6 Channels in Podocytes"

Article Title: Klotho May Ameliorate Proteinuria by Targeting TRPC6 Channels in Podocytes

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2015080888

Klotho ameliorates albuminuria in mice induced by podocyte-specific overexpression of Trpc6 . (A) Western blot analysis (detected by anti-Flag antibody [left panel] and by anti-TRPC6 antibody [right panel]) of cell lysates from podocytes and HEK cells transfected with podocyte-specific, Flag-tagged TRPC6 (Np-Flag-TRPC6) or empty pCDNA3 vector (Vec). (B) Expression of Flag-TRPC6 protein by immunofluorescence in podocytes (marked by synaptopodin) in mice injected with Trpc6 transgene. (C) Immunoblot analysis of urine samples from mice which received i.p. injection of His-tagged recombinant klotho or vehicle. His-tagged recombinant klotho or vehicle was inoculated in mice i.p. at 24 hours after gene delivery. Twenty-four-hour urine samples were collected from mice by using metabolic cage before and 24 hours after gene delivery. Right panel: His-tagged klotho protein in urine was immunoprecipitated using anti-His antibody followed by immunoblotting using anti-klotho antibody. Left panel: His-tagged klotho or IgG was diluted in PBS and subjected to immunoprecipitation by anti-His antibody as for urine samples. (D and E) Twenty-four-hour urinary albumin excretion (D) and urine albumin-to-creatinine ratio (E) from mice before (basal) and 24–48 hours after in vivo hydrodynamic gene delivery of vector (Vec) or Flag-TRPC6 (Neph-C6) and with i.p. injection of klotho or vehicle. # denotes P
Figure Legend Snippet: Klotho ameliorates albuminuria in mice induced by podocyte-specific overexpression of Trpc6 . (A) Western blot analysis (detected by anti-Flag antibody [left panel] and by anti-TRPC6 antibody [right panel]) of cell lysates from podocytes and HEK cells transfected with podocyte-specific, Flag-tagged TRPC6 (Np-Flag-TRPC6) or empty pCDNA3 vector (Vec). (B) Expression of Flag-TRPC6 protein by immunofluorescence in podocytes (marked by synaptopodin) in mice injected with Trpc6 transgene. (C) Immunoblot analysis of urine samples from mice which received i.p. injection of His-tagged recombinant klotho or vehicle. His-tagged recombinant klotho or vehicle was inoculated in mice i.p. at 24 hours after gene delivery. Twenty-four-hour urine samples were collected from mice by using metabolic cage before and 24 hours after gene delivery. Right panel: His-tagged klotho protein in urine was immunoprecipitated using anti-His antibody followed by immunoblotting using anti-klotho antibody. Left panel: His-tagged klotho or IgG was diluted in PBS and subjected to immunoprecipitation by anti-His antibody as for urine samples. (D and E) Twenty-four-hour urinary albumin excretion (D) and urine albumin-to-creatinine ratio (E) from mice before (basal) and 24–48 hours after in vivo hydrodynamic gene delivery of vector (Vec) or Flag-TRPC6 (Neph-C6) and with i.p. injection of klotho or vehicle. # denotes P

Techniques Used: Mouse Assay, Over Expression, Western Blot, Transfection, Plasmid Preparation, Expressing, Immunofluorescence, Injection, Recombinant, Immunoprecipitation, In Vivo

12) Product Images from "Klotho May Ameliorate Proteinuria by Targeting TRPC6 Channels in Podocytes"

Article Title: Klotho May Ameliorate Proteinuria by Targeting TRPC6 Channels in Podocytes

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2015080888

Klotho ameliorates albuminuria in mice induced by podocyte-specific overexpression of Trpc6 . (A) Western blot analysis (detected by anti-Flag antibody [left panel] and by anti-TRPC6 antibody [right panel]) of cell lysates from podocytes and HEK cells transfected with podocyte-specific, Flag-tagged TRPC6 (Np-Flag-TRPC6) or empty pCDNA3 vector (Vec). (B) Expression of Flag-TRPC6 protein by immunofluorescence in podocytes (marked by synaptopodin) in mice injected with Trpc6 transgene. (C) Immunoblot analysis of urine samples from mice which received i.p. injection of His-tagged recombinant klotho or vehicle. His-tagged recombinant klotho or vehicle was inoculated in mice i.p. at 24 hours after gene delivery. Twenty-four-hour urine samples were collected from mice by using metabolic cage before and 24 hours after gene delivery. Right panel: His-tagged klotho protein in urine was immunoprecipitated using anti-His antibody followed by immunoblotting using anti-klotho antibody. Left panel: His-tagged klotho or IgG was diluted in PBS and subjected to immunoprecipitation by anti-His antibody as for urine samples. (D and E) Twenty-four-hour urinary albumin excretion (D) and urine albumin-to-creatinine ratio (E) from mice before (basal) and 24–48 hours after in vivo hydrodynamic gene delivery of vector (Vec) or Flag-TRPC6 (Neph-C6) and with i.p. injection of klotho or vehicle. # denotes P
Figure Legend Snippet: Klotho ameliorates albuminuria in mice induced by podocyte-specific overexpression of Trpc6 . (A) Western blot analysis (detected by anti-Flag antibody [left panel] and by anti-TRPC6 antibody [right panel]) of cell lysates from podocytes and HEK cells transfected with podocyte-specific, Flag-tagged TRPC6 (Np-Flag-TRPC6) or empty pCDNA3 vector (Vec). (B) Expression of Flag-TRPC6 protein by immunofluorescence in podocytes (marked by synaptopodin) in mice injected with Trpc6 transgene. (C) Immunoblot analysis of urine samples from mice which received i.p. injection of His-tagged recombinant klotho or vehicle. His-tagged recombinant klotho or vehicle was inoculated in mice i.p. at 24 hours after gene delivery. Twenty-four-hour urine samples were collected from mice by using metabolic cage before and 24 hours after gene delivery. Right panel: His-tagged klotho protein in urine was immunoprecipitated using anti-His antibody followed by immunoblotting using anti-klotho antibody. Left panel: His-tagged klotho or IgG was diluted in PBS and subjected to immunoprecipitation by anti-His antibody as for urine samples. (D and E) Twenty-four-hour urinary albumin excretion (D) and urine albumin-to-creatinine ratio (E) from mice before (basal) and 24–48 hours after in vivo hydrodynamic gene delivery of vector (Vec) or Flag-TRPC6 (Neph-C6) and with i.p. injection of klotho or vehicle. # denotes P

Techniques Used: Mouse Assay, Over Expression, Western Blot, Transfection, Plasmid Preparation, Expressing, Immunofluorescence, Injection, Recombinant, Immunoprecipitation, In Vivo

13) Product Images from "Krüppel-like factor 4 is a negative regulator of STAT3-induced glomerular epithelial cell proliferation"

Article Title: Krüppel-like factor 4 is a negative regulator of STAT3-induced glomerular epithelial cell proliferation

Journal: JCI Insight

doi: 10.1172/jci.insight.98214

Podocin-Cre Klf4 fl/fl mice exhibit significant podocyte injury after NTS treatment. Podocin-Cre Klf4 fl/fl and Podocin-Cre Klf4 +/+ mice were treated with nephrotoxic serum (NTS) or VEH for 7 days. ( A ) Representative images of immunostaining for Nephrin (upper panel), Synaptopodin (Synpo; middle panel) and WT1 (lower panel) (×20). The glomerular region was selected and intensity of staining (OD) is shown as a relative fold change to untreated Podocin-Cre Klf4 +/+ mice for ( B ) Nephrin ( n = 5 in each group) and ( C ) Synpo ( n = 3–4 in each group). ( D ) Number of WT1 + cells per glomerular cross-sectional area ( n = 5 in each group). * P
Figure Legend Snippet: Podocin-Cre Klf4 fl/fl mice exhibit significant podocyte injury after NTS treatment. Podocin-Cre Klf4 fl/fl and Podocin-Cre Klf4 +/+ mice were treated with nephrotoxic serum (NTS) or VEH for 7 days. ( A ) Representative images of immunostaining for Nephrin (upper panel), Synaptopodin (Synpo; middle panel) and WT1 (lower panel) (×20). The glomerular region was selected and intensity of staining (OD) is shown as a relative fold change to untreated Podocin-Cre Klf4 +/+ mice for ( B ) Nephrin ( n = 5 in each group) and ( C ) Synpo ( n = 3–4 in each group). ( D ) Number of WT1 + cells per glomerular cross-sectional area ( n = 5 in each group). * P

Techniques Used: Mouse Assay, Immunostaining, Staining

Podocin-Cre Klf4 fl/fl (FVB/N) mice exhibit significant podocyte injury. ( A ) Representative images of immunostaining for Nephrin (upper panel), Synaptopodin (Synpo) (middle panel), and WT1 (lower panel) in Podocin-Cre Klf4 fl/fl (FVB/N) and Podocin-Cre Klf4 +/+ (FVB/N) mice (×20). ( B ) The glomerular region was selected, and intensity (OD) was measured and quantified as a relative fold change to untreated Podocin-Cre Klf4 +/+ (FVB/N) mice for Nephrin (upper panel) and Synpo (middle panel) ( n = 5 in each group, 30 glomeruli per mouse). The number of WT1 + cells per glomerular cross-sectional area (lower panel) ( n = 4–5 in each group, 30 glomeruli per mouse). * P
Figure Legend Snippet: Podocin-Cre Klf4 fl/fl (FVB/N) mice exhibit significant podocyte injury. ( A ) Representative images of immunostaining for Nephrin (upper panel), Synaptopodin (Synpo) (middle panel), and WT1 (lower panel) in Podocin-Cre Klf4 fl/fl (FVB/N) and Podocin-Cre Klf4 +/+ (FVB/N) mice (×20). ( B ) The glomerular region was selected, and intensity (OD) was measured and quantified as a relative fold change to untreated Podocin-Cre Klf4 +/+ (FVB/N) mice for Nephrin (upper panel) and Synpo (middle panel) ( n = 5 in each group, 30 glomeruli per mouse). The number of WT1 + cells per glomerular cross-sectional area (lower panel) ( n = 4–5 in each group, 30 glomeruli per mouse). * P

Techniques Used: Mouse Assay, Immunostaining

14) Product Images from "An inhibitor of spleen tyrosine kinase suppresses experimental crescentic glomerulonephritis"

Article Title: An inhibitor of spleen tyrosine kinase suppresses experimental crescentic glomerulonephritis

Journal: International Journal of Immunopathology and Pharmacology

doi: 10.1177/2058738418783404

Analysis of SYK expression on day 14 of vehicle-treated NTN by confocal microscopy. (a) Double staining for SYK and OX-42+ macrophages shows considerable overlapping indicating that SYK expression on day 14 NTN is largely restricted to macrophages. (b) Double staining for SYK and R73+ T cells indicates no overlap. (c) Double staining for SYK and OX-7+ mesangial cells indicates no overlap. (d) Double staining for SYK and synaptopodin+ podocytes indicates no overlap. Original magnification ×600.
Figure Legend Snippet: Analysis of SYK expression on day 14 of vehicle-treated NTN by confocal microscopy. (a) Double staining for SYK and OX-42+ macrophages shows considerable overlapping indicating that SYK expression on day 14 NTN is largely restricted to macrophages. (b) Double staining for SYK and R73+ T cells indicates no overlap. (c) Double staining for SYK and OX-7+ mesangial cells indicates no overlap. (d) Double staining for SYK and synaptopodin+ podocytes indicates no overlap. Original magnification ×600.

Techniques Used: Expressing, Confocal Microscopy, Double Staining

15) Product Images from "The HIV protease inhibitor darunavir prevents kidney injury via HIV-independent mechanisms"

Article Title: The HIV protease inhibitor darunavir prevents kidney injury via HIV-independent mechanisms

Journal: Scientific Reports

doi: 10.1038/s41598-019-52278-3

DRV reduced molecular markers of injury in Tg26 mice. Immunofluorescence microscopy demonstrated strong Synpo, Nephrin, and WT1 expression in podocytes in wild-type mice and markedly reduced expression in vehicle control- and AZT-treated Tg26 mice ( A ). Synpo, Nephrin, and WT1 expression in DRV- or DRV + AZT-treated Tg26 mice was similar to wild-type mice ( A ). Quantitative assessment of Synpo ( B ) and WT1 ( C ) demonstrated significantly higher expression in wild-type, DRV and DRV + AZT-treated Tg26 mice compared to vehicle control and AZT-treated Tg26 mice. Immunofluorescence microscopy revealed rare Ki-67-positive cells in wild-type kidneys but increased expression in Tg26 mice, particularly in proximal tubules, treated with vehicle control or AZT ( A , D ). The percentage of Ki-67-positive cells was reduced in DRV and DRV + AZT treated Tg26 mice ( A , D ) to levels similar to wild-type mice ( D ). Scale bar, 50 µm. *p
Figure Legend Snippet: DRV reduced molecular markers of injury in Tg26 mice. Immunofluorescence microscopy demonstrated strong Synpo, Nephrin, and WT1 expression in podocytes in wild-type mice and markedly reduced expression in vehicle control- and AZT-treated Tg26 mice ( A ). Synpo, Nephrin, and WT1 expression in DRV- or DRV + AZT-treated Tg26 mice was similar to wild-type mice ( A ). Quantitative assessment of Synpo ( B ) and WT1 ( C ) demonstrated significantly higher expression in wild-type, DRV and DRV + AZT-treated Tg26 mice compared to vehicle control and AZT-treated Tg26 mice. Immunofluorescence microscopy revealed rare Ki-67-positive cells in wild-type kidneys but increased expression in Tg26 mice, particularly in proximal tubules, treated with vehicle control or AZT ( A , D ). The percentage of Ki-67-positive cells was reduced in DRV and DRV + AZT treated Tg26 mice ( A , D ) to levels similar to wild-type mice ( D ). Scale bar, 50 µm. *p

Techniques Used: Mouse Assay, Immunofluorescence, Microscopy, Expressing

16) Product Images from "Genetic Ablation of Calcium-independent Phospholipase A2γ Induces Glomerular Injury in Mice *"

Article Title: Genetic Ablation of Calcium-independent Phospholipase A2γ Induces Glomerular Injury in Mice *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.696781

Effect of iPLA 2 γ deletion on expression of nephrin (fully glycosylated and ER forms), podocalyxin, GRP94, synaptopodin ( synpo ), phospho-AMPK ( pAMPK ), AMPK, and LC3-I and -II. Glomeruli were isolated from 10–11-month-old WT and iPLA 2 γ-KO
Figure Legend Snippet: Effect of iPLA 2 γ deletion on expression of nephrin (fully glycosylated and ER forms), podocalyxin, GRP94, synaptopodin ( synpo ), phospho-AMPK ( pAMPK ), AMPK, and LC3-I and -II. Glomeruli were isolated from 10–11-month-old WT and iPLA 2 γ-KO

Techniques Used: Expressing, Isolation

Expression of synaptopodin, nephrin, podocalyxin, and WT1 in 10–11-month-old WT and iPLA 2 γ-KO mice. A , kidney sections were stained with specific antibodies and were examined by immunofluorescence microscopy. Bar , 20 μm. B–D
Figure Legend Snippet: Expression of synaptopodin, nephrin, podocalyxin, and WT1 in 10–11-month-old WT and iPLA 2 γ-KO mice. A , kidney sections were stained with specific antibodies and were examined by immunofluorescence microscopy. Bar , 20 μm. B–D

Techniques Used: Expressing, Mouse Assay, Staining, Immunofluorescence, Microscopy

17) Product Images from "Characterization and Culture of Fetal Rhesus Monkey Renal Cortical Cells"

Article Title: Characterization and Culture of Fetal Rhesus Monkey Renal Cortical Cells

Journal: Pediatric research

doi: 10.1203/PDR.0b013e3181b45565

Immunocytochemistry - Cultured cortical cells on fibronectin and renal ECM Cells from passage 5 cultured on fibronectin contained more synaptopodin positive cells (red) (arrow) than those cultured on other substrates. Nuclei stained with DAPI (blue) (A-D). Synaptopodin positive cells were positive for nestin (green) and showed morphology consistent with mature podocytes (A-C). (A-B, 20x, bar = 100 μm; C, 60x, bar = 50 μm; D, 100x, bar = 20 μm).
Figure Legend Snippet: Immunocytochemistry - Cultured cortical cells on fibronectin and renal ECM Cells from passage 5 cultured on fibronectin contained more synaptopodin positive cells (red) (arrow) than those cultured on other substrates. Nuclei stained with DAPI (blue) (A-D). Synaptopodin positive cells were positive for nestin (green) and showed morphology consistent with mature podocytes (A-C). (A-B, 20x, bar = 100 μm; C, 60x, bar = 50 μm; D, 100x, bar = 20 μm).

Techniques Used: Immunocytochemistry, Cell Culture, Staining

Quantitative PCR - Synaptopodin and nestin While synaptopodin expression increased (A), nestin expression decreased (B) in cells grown on all substrates studied (black column - P1, white column - P5). Furthermore, podocyte specific expression was present in cells at P5 (C) (black - plastic, white - renal ECM) (D) (black - collagen I, white - collagen IV, gray - laminin, hatched - fibronectin). Passage 1 (P1) compared to passage 5 (P5) (*p
Figure Legend Snippet: Quantitative PCR - Synaptopodin and nestin While synaptopodin expression increased (A), nestin expression decreased (B) in cells grown on all substrates studied (black column - P1, white column - P5). Furthermore, podocyte specific expression was present in cells at P5 (C) (black - plastic, white - renal ECM) (D) (black - collagen I, white - collagen IV, gray - laminin, hatched - fibronectin). Passage 1 (P1) compared to passage 5 (P5) (*p

Techniques Used: Real-time Polymerase Chain Reaction, Expressing

Renal morphology and immunohistochemistry - Early third trimester fetal monkey kidney and nephrogenic zone (A) The fetal monkey kidney in the early third trimester shows a clear nephrogenic zone in the cortical region with the ureteric bud (arrowhead) and the metenephric mesenchyme (arrow) evident. The four stages of developing glomeruli are also present and represent the vesicle (a), S-shape (b), capillary (c), and mature (d) glomerulus. Hematoxylin and eosin (H E). (B-C) The nephrogenic zone is shown with the nuclei stained with DAPI (blue), metanephric mesenchyme (arrowheads), developing podocytes, and mature podocytes (arrows) positive for synaptopodin (green) and nestin (red). (D-I) Mature podocytes in terminally differentiated glomeruli are positive for nestin (red) and synaptopodin (green). (J-O) The endothelial marker CD31 (green) is not evident with the nestin positive cells (red) in these specimens (A-C, 20x, bar = 100 μm; D-F, 40x, bar = 50 μm; G-I, 100x, bar = 20 μm; J-L, 40x, bar = 50 μm; M-O, 100x, bar = 20 μm).
Figure Legend Snippet: Renal morphology and immunohistochemistry - Early third trimester fetal monkey kidney and nephrogenic zone (A) The fetal monkey kidney in the early third trimester shows a clear nephrogenic zone in the cortical region with the ureteric bud (arrowhead) and the metenephric mesenchyme (arrow) evident. The four stages of developing glomeruli are also present and represent the vesicle (a), S-shape (b), capillary (c), and mature (d) glomerulus. Hematoxylin and eosin (H E). (B-C) The nephrogenic zone is shown with the nuclei stained with DAPI (blue), metanephric mesenchyme (arrowheads), developing podocytes, and mature podocytes (arrows) positive for synaptopodin (green) and nestin (red). (D-I) Mature podocytes in terminally differentiated glomeruli are positive for nestin (red) and synaptopodin (green). (J-O) The endothelial marker CD31 (green) is not evident with the nestin positive cells (red) in these specimens (A-C, 20x, bar = 100 μm; D-F, 40x, bar = 50 μm; G-I, 100x, bar = 20 μm; J-L, 40x, bar = 50 μm; M-O, 100x, bar = 20 μm).

Techniques Used: Immunohistochemistry, Staining, Marker

18) Product Images from "Inducible ATF3–NFAT axis aggravates podocyte injury"

Article Title: Inducible ATF3–NFAT axis aggravates podocyte injury

Journal: Journal of Molecular Medicine (Berlin, Germany)

doi: 10.1007/s00109-017-1601-x

The translocation of ATF3 to the nucleus was increased in injured podocytes. a Nuclear ATF3 protein (red) was increased after 2 h-treatment of HG, Ionomycin or LPS in cultured podocytes, as shown by immunofluorescence with the podocyte marker synaptopodin (synpo, green) and DAPI-stained nuclei (blue) ( n = 3). b After nuclear fractionation, nuclear protein is isolated and prepared. Western blotting showed an increased ATF3 protein in nuclei of podocytes treated by HG, Ionomycin, or LPS treated for 2 h ( n = 4). Data were from at least three independent experiments. * P
Figure Legend Snippet: The translocation of ATF3 to the nucleus was increased in injured podocytes. a Nuclear ATF3 protein (red) was increased after 2 h-treatment of HG, Ionomycin or LPS in cultured podocytes, as shown by immunofluorescence with the podocyte marker synaptopodin (synpo, green) and DAPI-stained nuclei (blue) ( n = 3). b After nuclear fractionation, nuclear protein is isolated and prepared. Western blotting showed an increased ATF3 protein in nuclei of podocytes treated by HG, Ionomycin, or LPS treated for 2 h ( n = 4). Data were from at least three independent experiments. * P

Techniques Used: Translocation Assay, Cell Culture, Immunofluorescence, Marker, Staining, Fractionation, Isolation, Western Blot

ATF3 is upregulated in podocytes in proteinuric patients and experimental proteinuric models. a , b ATF3 protein (red) is found in podocytes, as shown by double immunofluorescence with the podocyte marker synaptopodin (synpo, green) resulting in a partial yellow overlap. ATF3 expression is low in normal glomeruli from patients without glomerular disease. In contrast, ATF3 protein is increased in podocytes of proteinuric patients with minimal change disease (MCD, n = 4), focal segmental glomerulosclerosis (FSGS, n = 4), or diabetic nephropathy (DN, n = 6); similarly, western blotting showed an elevated level of ATF3 protein in patients with MCD, FSGS, or DN. c , d ATF3 is induced in podocytes in murine models of proteinuria. Compared with the control (C57 or BKS), ATF3 expression is elevated in the murine model of transient proteinuria (LPS-treated C57 mice) and the murine model of diabetic nephropathy (db/db mice) ( n = 3) (color figure online)
Figure Legend Snippet: ATF3 is upregulated in podocytes in proteinuric patients and experimental proteinuric models. a , b ATF3 protein (red) is found in podocytes, as shown by double immunofluorescence with the podocyte marker synaptopodin (synpo, green) resulting in a partial yellow overlap. ATF3 expression is low in normal glomeruli from patients without glomerular disease. In contrast, ATF3 protein is increased in podocytes of proteinuric patients with minimal change disease (MCD, n = 4), focal segmental glomerulosclerosis (FSGS, n = 4), or diabetic nephropathy (DN, n = 6); similarly, western blotting showed an elevated level of ATF3 protein in patients with MCD, FSGS, or DN. c , d ATF3 is induced in podocytes in murine models of proteinuria. Compared with the control (C57 or BKS), ATF3 expression is elevated in the murine model of transient proteinuria (LPS-treated C57 mice) and the murine model of diabetic nephropathy (db/db mice) ( n = 3) (color figure online)

Techniques Used: Immunofluorescence, Marker, Expressing, Western Blot, Mouse Assay

19) Product Images from "Morphine Induces Albuminuria by Compromising Podocyte Integrity"

Article Title: Morphine Induces Albuminuria by Compromising Podocyte Integrity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0055748

H 2 O 2 decreases podocyte expression of SD molecules. A. Human podocytes were treated with 1, 10, or 50 µM H 2 O 2 for 24 h, and the cell lysates were collected for Western blots. B–E. Quantification of the expression of nephrin ( B ), synaptopodin ( C ), CD2AP ( D ), and podocin ( E ) in A , and the results (mean ± SD) represent three independent samples. * p
Figure Legend Snippet: H 2 O 2 decreases podocyte expression of SD molecules. A. Human podocytes were treated with 1, 10, or 50 µM H 2 O 2 for 24 h, and the cell lysates were collected for Western blots. B–E. Quantification of the expression of nephrin ( B ), synaptopodin ( C ), CD2AP ( D ), and podocin ( E ) in A , and the results (mean ± SD) represent three independent samples. * p

Techniques Used: Expressing, Western Blot

Morphine affects podocytes through K and M receptors. A. Human podocytes were treated with agonists specifically for DOR (labeled as D), KOR (K), or MOR (M) for 24 h, and the cell lysates were collected for Western blots. B–E. Quantification of the expression of nephrin ( B ), synaptopodin ( C ), CD2AP ( D ), and podocin ( E ) in A , and the results (mean ± SD) represent three independent samples. * p
Figure Legend Snippet: Morphine affects podocytes through K and M receptors. A. Human podocytes were treated with agonists specifically for DOR (labeled as D), KOR (K), or MOR (M) for 24 h, and the cell lysates were collected for Western blots. B–E. Quantification of the expression of nephrin ( B ), synaptopodin ( C ), CD2AP ( D ), and podocin ( E ) in A , and the results (mean ± SD) represent three independent samples. * p

Techniques Used: Labeling, Western Blot, Expressing

SOD and catalase attenuate morphine-induced decrease of podcoyte expression of SD molecules. A. Human podocytes were pretreated with SOD or catalase for 1 h before 10 −6 M morphine was added. After another 24 h incubation at 37°C, the cell lysates were collected for Western blot. B–E. Quantification of the expression of nephrin ( B ), synaptopodin ( C ), CD2AP ( D ), and podocin ( E ) in A , and the results (mean ± SD) represent three independent samples. * p
Figure Legend Snippet: SOD and catalase attenuate morphine-induced decrease of podcoyte expression of SD molecules. A. Human podocytes were pretreated with SOD or catalase for 1 h before 10 −6 M morphine was added. After another 24 h incubation at 37°C, the cell lysates were collected for Western blot. B–E. Quantification of the expression of nephrin ( B ), synaptopodin ( C ), CD2AP ( D ), and podocin ( E ) in A , and the results (mean ± SD) represent three independent samples. * p

Techniques Used: Expressing, Incubation, Western Blot

Morphine decreases podocyte expression of SD molecules. A. Differentiated human podocytes were treated with 10 −8 or 10 −6 M morphine for 24 h, and the cell lysates were collected for Western blots. B–E. Quantification of the expression of nephrin ( B ), synaptopodin ( C ), CD2AP ( D ), and podocin ( E ) in A , and the results (mean ± SD) represent three independent samples. * p
Figure Legend Snippet: Morphine decreases podocyte expression of SD molecules. A. Differentiated human podocytes were treated with 10 −8 or 10 −6 M morphine for 24 h, and the cell lysates were collected for Western blots. B–E. Quantification of the expression of nephrin ( B ), synaptopodin ( C ), CD2AP ( D ), and podocin ( E ) in A , and the results (mean ± SD) represent three independent samples. * p

Techniques Used: Expressing, Western Blot

Morphine-receiving mice display alterations in SD molecules and effeacement of foot processes of podocytes. A. Two-month-old mice were administrated either normal saline or morphine for 3 days. Frozen sections were prepared for immuno-fluorescence staining. B. kidneys were homogenized, and the tissue lysates were subjected to Western blot to detect the changes of nephrin and synaptopodin. C–D. Quantification of the expression of nephrin ( C ) and synaptopodin ( D ), and the results (mean ± SD) represent three independent samples. E. A representative microphotograph from transmission electron microscopy studies. Foot processes are indicated by arrows. Foot processes are well delineated and are not fused in the control, while they are fused (effaced) in morphine-receiving mice. The scale bar is 500 nm. Mag. 270,000X.
Figure Legend Snippet: Morphine-receiving mice display alterations in SD molecules and effeacement of foot processes of podocytes. A. Two-month-old mice were administrated either normal saline or morphine for 3 days. Frozen sections were prepared for immuno-fluorescence staining. B. kidneys were homogenized, and the tissue lysates were subjected to Western blot to detect the changes of nephrin and synaptopodin. C–D. Quantification of the expression of nephrin ( C ) and synaptopodin ( D ), and the results (mean ± SD) represent three independent samples. E. A representative microphotograph from transmission electron microscopy studies. Foot processes are indicated by arrows. Foot processes are well delineated and are not fused in the control, while they are fused (effaced) in morphine-receiving mice. The scale bar is 500 nm. Mag. 270,000X.

Techniques Used: Mouse Assay, Fluorescence, Staining, Western Blot, Expressing, Transmission Assay, Electron Microscopy

Inhibition of AKT, p38, and JNK attenuates morphine-induced decrease of SD molecules expression. A. Human podocytes were pretreated with of LY294002 (LY), SB203580 (SB), or SP600125 (SP) for 1 h before 10 −6 M morphine was added. After another 24 h incubation at 37°C, the cell lysate were collected for Western blot. B–E. Quantification of the expression of nephrin ( B ), synaptopodin ( C ), CD2AP ( D ), and podocin ( E ) in A , and the results (mean ± SD) represent three independent samples. * p
Figure Legend Snippet: Inhibition of AKT, p38, and JNK attenuates morphine-induced decrease of SD molecules expression. A. Human podocytes were pretreated with of LY294002 (LY), SB203580 (SB), or SP600125 (SP) for 1 h before 10 −6 M morphine was added. After another 24 h incubation at 37°C, the cell lysate were collected for Western blot. B–E. Quantification of the expression of nephrin ( B ), synaptopodin ( C ), CD2AP ( D ), and podocin ( E ) in A , and the results (mean ± SD) represent three independent samples. * p

Techniques Used: Inhibition, Expressing, Incubation, Western Blot

20) Product Images from "An inhibitor of spleen tyrosine kinase suppresses experimental crescentic glomerulonephritis"

Article Title: An inhibitor of spleen tyrosine kinase suppresses experimental crescentic glomerulonephritis

Journal: International Journal of Immunopathology and Pharmacology

doi: 10.1177/2058738418783404

Analysis of SYK expression on day 14 of vehicle-treated NTN by confocal microscopy. (a) Double staining for SYK and OX-42+ macrophages shows considerable overlapping indicating that SYK expression on day 14 NTN is largely restricted to macrophages. (b) Double staining for SYK and R73+ T cells indicates no overlap. (c) Double staining for SYK and OX-7+ mesangial cells indicates no overlap. (d) Double staining for SYK and synaptopodin+ podocytes indicates no overlap. Original magnification ×600.
Figure Legend Snippet: Analysis of SYK expression on day 14 of vehicle-treated NTN by confocal microscopy. (a) Double staining for SYK and OX-42+ macrophages shows considerable overlapping indicating that SYK expression on day 14 NTN is largely restricted to macrophages. (b) Double staining for SYK and R73+ T cells indicates no overlap. (c) Double staining for SYK and OX-7+ mesangial cells indicates no overlap. (d) Double staining for SYK and synaptopodin+ podocytes indicates no overlap. Original magnification ×600.

Techniques Used: Expressing, Confocal Microscopy, Double Staining

21) Product Images from "Origin of Parietal Podocytes in Atubular Glomeruli Mapped by Lineage Tracing"

Article Title: Origin of Parietal Podocytes in Atubular Glomeruli Mapped by Lineage Tracing

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2013040376

Podocyte and PEC marker expression in humans. (A–A’’’) Immunofluorescence double stainings of an apparently normal kidney of an 18-year-old old healthy man show no colocalization of the podocyte marker synaptopodin and
Figure Legend Snippet: Podocyte and PEC marker expression in humans. (A–A’’’) Immunofluorescence double stainings of an apparently normal kidney of an 18-year-old old healthy man show no colocalization of the podocyte marker synaptopodin and

Techniques Used: Marker, Expressing, Immunofluorescence

Statistical analysis of the proportion of Bowman’s capsules covered by synaptopodin-positive cells (presumptive parietal podocytes). More than half of the glomeruli do not show parietal podocytes. In a significant portion of glomeruli (approximately
Figure Legend Snippet: Statistical analysis of the proportion of Bowman’s capsules covered by synaptopodin-positive cells (presumptive parietal podocytes). More than half of the glomeruli do not show parietal podocytes. In a significant portion of glomeruli (approximately

Techniques Used:

Cysts induced by electrocoagulation are glomerular cysts. (A) In normal glomeruli, synaptopodin (A, arrow) and SSeCKS (A’, arrow) are specifically detected in podocytes and PECs, respectively. The boundary from synaptopodin-positive podocytes
Figure Legend Snippet: Cysts induced by electrocoagulation are glomerular cysts. (A) In normal glomeruli, synaptopodin (A, arrow) and SSeCKS (A’, arrow) are specifically detected in podocytes and PECs, respectively. The boundary from synaptopodin-positive podocytes

Techniques Used:

Parietal podocytes in atubular glomeruli. (A and B) Bowman's capsules of atubular glomeruli are lined by synaptopodin-positive parietal podocytes regardless of the cyst diameter. (C–F) Influence of genetic background (38 Sv129 mice versus 36 C57BL/6
Figure Legend Snippet: Parietal podocytes in atubular glomeruli. (A and B) Bowman's capsules of atubular glomeruli are lined by synaptopodin-positive parietal podocytes regardless of the cyst diameter. (C–F) Influence of genetic background (38 Sv129 mice versus 36 C57BL/6

Techniques Used: Mouse Assay

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    Santa Cruz Biotechnology goat anti synaptopodin
    Aggravation of podocyte injury upon autophagy inhibition. (A–B).The Western blot results confirmed that the expression of <t>synaptopodin</t> and LC3 II was lower in ATG7 (a key protein of autophagy) RNAi-mediated knock down differentiated MPCs (siRNA+R+P) than in the siRNA negative control group (siCon+R+P). Both groups were pretreated with rapamycin and treated with PAN,* P
    Goat Anti Synaptopodin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology goat anti synpo
    MAPT phosphorylated at Ser262 increased in neuronal processes when <t>BAG3</t> or <t>SYNPO</t> was knocked down in mature neurons. ( A ) Representative blots of MAPT and phosphorylated MAPT (p-Thr231, p-Ser262 and p-Ser396/Ser404) in neurons transduced with scramble (scr), shBag3 or shSynpo lentivirus. ( B ) Quantitation of the levels of MAPT or phosphorylated MAPT in BAG3 or SYNPO knockdown neurons from 3 independent experiments. Data were normalized to the loading control ACTB and then compared to scramble controls. Data were shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test. *, p
    Goat Anti Synpo, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti synpo/product/Santa Cruz Biotechnology
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    Image Search Results


    Aggravation of podocyte injury upon autophagy inhibition. (A–B).The Western blot results confirmed that the expression of synaptopodin and LC3 II was lower in ATG7 (a key protein of autophagy) RNAi-mediated knock down differentiated MPCs (siRNA+R+P) than in the siRNA negative control group (siCon+R+P). Both groups were pretreated with rapamycin and treated with PAN,* P

    Journal: PLoS ONE

    Article Title: Rapamycin Upregulates Autophagy by Inhibiting the mTOR-ULK1 Pathway, Resulting in Reduced Podocyte Injury

    doi: 10.1371/journal.pone.0063799

    Figure Lengend Snippet: Aggravation of podocyte injury upon autophagy inhibition. (A–B).The Western blot results confirmed that the expression of synaptopodin and LC3 II was lower in ATG7 (a key protein of autophagy) RNAi-mediated knock down differentiated MPCs (siRNA+R+P) than in the siRNA negative control group (siCon+R+P). Both groups were pretreated with rapamycin and treated with PAN,* P

    Article Snippet: The following primary antibodies were used: rabbit anti-synaptopodin and goat anti-synaptopodin (Santa Cruz Biotechnology, Santa Cruz, CA); anti-p-mTOR (Ser2448) (Cell Signaling, Danvers, MA); anti-p-70S6K (Thr389) (Cell Signaling); anti-p-4EBP1 (Ser65) (Cell Signaling); anti-p-ULK1 (Ser757) (Cell Signaling); rabbit anti-LC3 (Sigma-Aldrich) and moduse anti-LC3 (MBL Co. NaKa-ku Nagoya, Japan); and mouse anti-β-actin (Sigma-Aldrich).

    Techniques: Inhibition, Western Blot, Expressing, Negative Control

    Rapamycin reduced podocyte injury by inhibiting the mTOR-ULK1 signaling pathway. (A–B).Western blot analysis of cellular proteins showed recovery of synaptopodin and LC3 II expression, a decrease in mTOR activity, and a decrease in ULK1 phosphorylation in rapamycin-pretreated cells (P+R) compared to PAN-treated cells in the absence of rapamycin (PAN), * P

    Journal: PLoS ONE

    Article Title: Rapamycin Upregulates Autophagy by Inhibiting the mTOR-ULK1 Pathway, Resulting in Reduced Podocyte Injury

    doi: 10.1371/journal.pone.0063799

    Figure Lengend Snippet: Rapamycin reduced podocyte injury by inhibiting the mTOR-ULK1 signaling pathway. (A–B).Western blot analysis of cellular proteins showed recovery of synaptopodin and LC3 II expression, a decrease in mTOR activity, and a decrease in ULK1 phosphorylation in rapamycin-pretreated cells (P+R) compared to PAN-treated cells in the absence of rapamycin (PAN), * P

    Article Snippet: The following primary antibodies were used: rabbit anti-synaptopodin and goat anti-synaptopodin (Santa Cruz Biotechnology, Santa Cruz, CA); anti-p-mTOR (Ser2448) (Cell Signaling, Danvers, MA); anti-p-70S6K (Thr389) (Cell Signaling); anti-p-4EBP1 (Ser65) (Cell Signaling); anti-p-ULK1 (Ser757) (Cell Signaling); rabbit anti-LC3 (Sigma-Aldrich) and moduse anti-LC3 (MBL Co. NaKa-ku Nagoya, Japan); and mouse anti-β-actin (Sigma-Aldrich).

    Techniques: Western Blot, Expressing, Activity Assay

    Preparation of passive Heymann nephritis rat model. (A).At day 1, 7, 14, 21, and 28 after antiserum injection, rats were sacrificed and the renal cortex was removed. Cryosections of the renal cortex were stained with goat anti-rat IgG-fluorescein isothiocyanate (FITC). Microscopic examination revealed that there were IgG depositions in the glomeruli of PHN rats compared to those of control rats. Depositions gradually increased over time, peaking on day 14. Magnification = × 400 (B). 24-hr urine was collected on days 0 (control), 1, 7, 14, 21, and 28, and analyzed for urinary albumin content using Coomassie Brilliant Blue G-250. (C). Electrophoresis followed by Coomassie blue staining. Each group urine sample was subjected to SDS-PAGE. Obvious albuminuria was apparent from day 7. (D-E).Western blot analysis of isolated glomerular protein on day 14 showed significantly reduced expression of synaptopodin in PHN rats compared to that in controls, * P

    Journal: PLoS ONE

    Article Title: Rapamycin Upregulates Autophagy by Inhibiting the mTOR-ULK1 Pathway, Resulting in Reduced Podocyte Injury

    doi: 10.1371/journal.pone.0063799

    Figure Lengend Snippet: Preparation of passive Heymann nephritis rat model. (A).At day 1, 7, 14, 21, and 28 after antiserum injection, rats were sacrificed and the renal cortex was removed. Cryosections of the renal cortex were stained with goat anti-rat IgG-fluorescein isothiocyanate (FITC). Microscopic examination revealed that there were IgG depositions in the glomeruli of PHN rats compared to those of control rats. Depositions gradually increased over time, peaking on day 14. Magnification = × 400 (B). 24-hr urine was collected on days 0 (control), 1, 7, 14, 21, and 28, and analyzed for urinary albumin content using Coomassie Brilliant Blue G-250. (C). Electrophoresis followed by Coomassie blue staining. Each group urine sample was subjected to SDS-PAGE. Obvious albuminuria was apparent from day 7. (D-E).Western blot analysis of isolated glomerular protein on day 14 showed significantly reduced expression of synaptopodin in PHN rats compared to that in controls, * P

    Article Snippet: The following primary antibodies were used: rabbit anti-synaptopodin and goat anti-synaptopodin (Santa Cruz Biotechnology, Santa Cruz, CA); anti-p-mTOR (Ser2448) (Cell Signaling, Danvers, MA); anti-p-70S6K (Thr389) (Cell Signaling); anti-p-4EBP1 (Ser65) (Cell Signaling); anti-p-ULK1 (Ser757) (Cell Signaling); rabbit anti-LC3 (Sigma-Aldrich) and moduse anti-LC3 (MBL Co. NaKa-ku Nagoya, Japan); and mouse anti-β-actin (Sigma-Aldrich).

    Techniques: Injection, Staining, Electrophoresis, SDS Page, Western Blot, Isolation, Expressing

    MAPT phosphorylated at Ser262 increased in neuronal processes when BAG3 or SYNPO was knocked down in mature neurons. ( A ) Representative blots of MAPT and phosphorylated MAPT (p-Thr231, p-Ser262 and p-Ser396/Ser404) in neurons transduced with scramble (scr), shBag3 or shSynpo lentivirus. ( B ) Quantitation of the levels of MAPT or phosphorylated MAPT in BAG3 or SYNPO knockdown neurons from 3 independent experiments. Data were normalized to the loading control ACTB and then compared to scramble controls. Data were shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test. *, p

    Journal: bioRxiv

    Article Title: BAG3 and SYNPO (synaptopodin) facilitate phospho-MAPT/Tau degradation via autophagy in neuronal processes

    doi: 10.1101/518597

    Figure Lengend Snippet: MAPT phosphorylated at Ser262 increased in neuronal processes when BAG3 or SYNPO was knocked down in mature neurons. ( A ) Representative blots of MAPT and phosphorylated MAPT (p-Thr231, p-Ser262 and p-Ser396/Ser404) in neurons transduced with scramble (scr), shBag3 or shSynpo lentivirus. ( B ) Quantitation of the levels of MAPT or phosphorylated MAPT in BAG3 or SYNPO knockdown neurons from 3 independent experiments. Data were normalized to the loading control ACTB and then compared to scramble controls. Data were shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test. *, p

    Article Snippet: Primary antibodies were diluted in blocking solutions as follows: rabbit anti-BAG3, 1:3000; mouse anti-FLAG, 1:1000; goat anti-SYNPO, 1:200; mouse anti-SYNPO, 1:1000; rabbit anti-SQSTM1, 1:1000; mouse anti-GAPDH, 1:10000; rabbit anti-LC3B, 1:5000; mouse anti-ACTB, 1:10000; rabbit anti-VPS18, 1:1000; rabbit anti-MAPT, 1:10,000; mouse anti-p-Thr231 (AT180), 1:1000; mouse anti-p-Ser262 (12E8), 1:1000; mouse anti-p-Ser396/Ser404 (PHF1), 1:5000, followed by incubation at 4°C overnight.

    Techniques: Transduction, Quantitation Assay

    Phosphorylated MAPT Ser262 accumulates in autophagosomes at post-synaptic densities when either BAG3 or SYNPO expression is decreased. ( A ) Representative images of LC3B and DLG4/PSD95 colocalization in dendrites. Neurons were treated with either DMSO or bafilomycin A1 (BafA1) for 4 h before fixing and immunostaining. ( B ) Representative images of p-Ser262 and DLG4 co-staining in dendrites of neurons transduced with scramble (scr), shBag3 or shSynpo lentivirus. Arrow heads denote the overlapping of fluorescence. ( C ) Quantification of colocalization using Mander’s colocalization coefficient and object based analysis. In each condition, 18-20 neurons from 2 independent experiments were counted. 1-3 processes from each neuron were chosen for analysis. Data were shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test. **, p

    Journal: bioRxiv

    Article Title: BAG3 and SYNPO (synaptopodin) facilitate phospho-MAPT/Tau degradation via autophagy in neuronal processes

    doi: 10.1101/518597

    Figure Lengend Snippet: Phosphorylated MAPT Ser262 accumulates in autophagosomes at post-synaptic densities when either BAG3 or SYNPO expression is decreased. ( A ) Representative images of LC3B and DLG4/PSD95 colocalization in dendrites. Neurons were treated with either DMSO or bafilomycin A1 (BafA1) for 4 h before fixing and immunostaining. ( B ) Representative images of p-Ser262 and DLG4 co-staining in dendrites of neurons transduced with scramble (scr), shBag3 or shSynpo lentivirus. Arrow heads denote the overlapping of fluorescence. ( C ) Quantification of colocalization using Mander’s colocalization coefficient and object based analysis. In each condition, 18-20 neurons from 2 independent experiments were counted. 1-3 processes from each neuron were chosen for analysis. Data were shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test. **, p

    Article Snippet: Primary antibodies were diluted in blocking solutions as follows: rabbit anti-BAG3, 1:3000; mouse anti-FLAG, 1:1000; goat anti-SYNPO, 1:200; mouse anti-SYNPO, 1:1000; rabbit anti-SQSTM1, 1:1000; mouse anti-GAPDH, 1:10000; rabbit anti-LC3B, 1:5000; mouse anti-ACTB, 1:10000; rabbit anti-VPS18, 1:1000; rabbit anti-MAPT, 1:10,000; mouse anti-p-Thr231 (AT180), 1:1000; mouse anti-p-Ser262 (12E8), 1:1000; mouse anti-p-Ser396/Ser404 (PHF1), 1:5000, followed by incubation at 4°C overnight.

    Techniques: Expressing, Immunostaining, Staining, Transduction, Fluorescence

    BAG3 interacts with SYNPO in mature neurons. ( A ) Immunoprecipitation of endogenous BAG3 from mature rat cortical neuronal lysates. SYNPO was detected in the isolated bound fractions. ( B ) Immunoprecipitation of endogenous SYNPO from mature rat cortical neurons. Both BAG3 and SQSTM1 were detected in the precipitated fraction. ( C ) Co-immunoprecipitation of SQSTM1 and SYNPO is independent of BAG3 in mature rat neurons.

    Journal: bioRxiv

    Article Title: BAG3 and SYNPO (synaptopodin) facilitate phospho-MAPT/Tau degradation via autophagy in neuronal processes

    doi: 10.1101/518597

    Figure Lengend Snippet: BAG3 interacts with SYNPO in mature neurons. ( A ) Immunoprecipitation of endogenous BAG3 from mature rat cortical neuronal lysates. SYNPO was detected in the isolated bound fractions. ( B ) Immunoprecipitation of endogenous SYNPO from mature rat cortical neurons. Both BAG3 and SQSTM1 were detected in the precipitated fraction. ( C ) Co-immunoprecipitation of SQSTM1 and SYNPO is independent of BAG3 in mature rat neurons.

    Article Snippet: Primary antibodies were diluted in blocking solutions as follows: rabbit anti-BAG3, 1:3000; mouse anti-FLAG, 1:1000; goat anti-SYNPO, 1:200; mouse anti-SYNPO, 1:1000; rabbit anti-SQSTM1, 1:1000; mouse anti-GAPDH, 1:10000; rabbit anti-LC3B, 1:5000; mouse anti-ACTB, 1:10000; rabbit anti-VPS18, 1:1000; rabbit anti-MAPT, 1:10,000; mouse anti-p-Thr231 (AT180), 1:1000; mouse anti-p-Ser262 (12E8), 1:1000; mouse anti-p-Ser396/Ser404 (PHF1), 1:5000, followed by incubation at 4°C overnight.

    Techniques: Immunoprecipitation, Isolation

    Loss of BAG3 or SYNPO does not affect the initiation or maturation of autophagosomes. ( A ) Schematic representation of proteinase K (PK) protection assay. This panel was adapted and reproduced from [ 46 ]. ( B ) Autophagic cargo receptor SQSTM1 was protected from PK digestion unless the detergent Triton X-100 (TX-100) was present. Lysates from scramble (scr), BAG3 or SYNPO knockdown neurons treated with or without 10 μM chloroquine (CQ) were subjected to PK protection assays. VPS18 was used as a cytosolic control. ( C ) Quantification of the amount of PK protected SQSTM1 in each condition. Percentage of PK protected SQSTM1 was the ratio of SQSTM1 in the presence PK but in the absence of TX-100 relative to its untreated control in a given condition. Data are shown as mean ± SEM. Statistical analysis was performed using two-way ANOVA with Tukey’s post hoc test. **, p

    Journal: bioRxiv

    Article Title: BAG3 and SYNPO (synaptopodin) facilitate phospho-MAPT/Tau degradation via autophagy in neuronal processes

    doi: 10.1101/518597

    Figure Lengend Snippet: Loss of BAG3 or SYNPO does not affect the initiation or maturation of autophagosomes. ( A ) Schematic representation of proteinase K (PK) protection assay. This panel was adapted and reproduced from [ 46 ]. ( B ) Autophagic cargo receptor SQSTM1 was protected from PK digestion unless the detergent Triton X-100 (TX-100) was present. Lysates from scramble (scr), BAG3 or SYNPO knockdown neurons treated with or without 10 μM chloroquine (CQ) were subjected to PK protection assays. VPS18 was used as a cytosolic control. ( C ) Quantification of the amount of PK protected SQSTM1 in each condition. Percentage of PK protected SQSTM1 was the ratio of SQSTM1 in the presence PK but in the absence of TX-100 relative to its untreated control in a given condition. Data are shown as mean ± SEM. Statistical analysis was performed using two-way ANOVA with Tukey’s post hoc test. **, p

    Article Snippet: Primary antibodies were diluted in blocking solutions as follows: rabbit anti-BAG3, 1:3000; mouse anti-FLAG, 1:1000; goat anti-SYNPO, 1:200; mouse anti-SYNPO, 1:1000; rabbit anti-SQSTM1, 1:1000; mouse anti-GAPDH, 1:10000; rabbit anti-LC3B, 1:5000; mouse anti-ACTB, 1:10000; rabbit anti-VPS18, 1:1000; rabbit anti-MAPT, 1:10,000; mouse anti-p-Thr231 (AT180), 1:1000; mouse anti-p-Ser262 (12E8), 1:1000; mouse anti-p-Ser396/Ser404 (PHF1), 1:5000, followed by incubation at 4°C overnight.

    Techniques:

    Colocalization of SYNPO with BAG3, SQSTM1 and HSPA/HSP70. Cortical neurons were immunostained for SYNPO and BAG3, SQSTM1, or HSPA/HSP70. Immunofluorescence of SYNPO overlaps with BAG3, SQSTM1 or HSPA/HSP70 in neuronal processes ( A ) and soma ( B ). Corresponding line scans are shown on the right; arrowheads indicate the areas of overlapping of intensity. Quantification of colocalization using Pearson’s correlation coefficient ( C ) and object-based analysis ( D ). In each condition, 10-30 neurons from 3 independent experiments were used for quantification. Data were plotted as mean ± SEM. As MAP2 appears in a continuous localization within neuronal dendrites and barely overlaps with SYNPO (see also Figure S1 ), the colocalization between SYNPO and BAG3, SQSTM1 and HSPA/HSP70, respectively, was compared to SYNPO and MAP2 using one-way ANOVA followed by Dunnett’s multiple comparisons test. ****, p

    Journal: bioRxiv

    Article Title: BAG3 and SYNPO (synaptopodin) facilitate phospho-MAPT/Tau degradation via autophagy in neuronal processes

    doi: 10.1101/518597

    Figure Lengend Snippet: Colocalization of SYNPO with BAG3, SQSTM1 and HSPA/HSP70. Cortical neurons were immunostained for SYNPO and BAG3, SQSTM1, or HSPA/HSP70. Immunofluorescence of SYNPO overlaps with BAG3, SQSTM1 or HSPA/HSP70 in neuronal processes ( A ) and soma ( B ). Corresponding line scans are shown on the right; arrowheads indicate the areas of overlapping of intensity. Quantification of colocalization using Pearson’s correlation coefficient ( C ) and object-based analysis ( D ). In each condition, 10-30 neurons from 3 independent experiments were used for quantification. Data were plotted as mean ± SEM. As MAP2 appears in a continuous localization within neuronal dendrites and barely overlaps with SYNPO (see also Figure S1 ), the colocalization between SYNPO and BAG3, SQSTM1 and HSPA/HSP70, respectively, was compared to SYNPO and MAP2 using one-way ANOVA followed by Dunnett’s multiple comparisons test. ****, p

    Article Snippet: Primary antibodies were diluted in blocking solutions as follows: rabbit anti-BAG3, 1:3000; mouse anti-FLAG, 1:1000; goat anti-SYNPO, 1:200; mouse anti-SYNPO, 1:1000; rabbit anti-SQSTM1, 1:1000; mouse anti-GAPDH, 1:10000; rabbit anti-LC3B, 1:5000; mouse anti-ACTB, 1:10000; rabbit anti-VPS18, 1:1000; rabbit anti-MAPT, 1:10,000; mouse anti-p-Thr231 (AT180), 1:1000; mouse anti-p-Ser262 (12E8), 1:1000; mouse anti-p-Ser396/Ser404 (PHF1), 1:5000, followed by incubation at 4°C overnight.

    Techniques: Immunofluorescence

    Loss of BAG3 or SYNPO reduces LC3B-II and SQSTM1 turnover. ( A ) LC3B-I/II levels in primary cortical neurons transduced with lentivirus expressing shBag3 or a scrambled (scr) version. Neurons were treated with or without 10 μM chloroquine (CQ) for 16h. ( B ) LC3B blots of neurons transduced with lentivirus expressing shRNA for rat Synpo or a scrambled ( scr ) version. Neurons were treated as ( A ). ( C ) Quantifications of LC3B-II in BAG3 and SYNPO knockdown neurons in the absence or presence of CQ treatment. LC3B-II was normalized to the loading control ACTB then compared to the scrambled condition. Graph show mean ± SEM of 4-6 samples from 3 independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. *, p

    Journal: bioRxiv

    Article Title: BAG3 and SYNPO (synaptopodin) facilitate phospho-MAPT/Tau degradation via autophagy in neuronal processes

    doi: 10.1101/518597

    Figure Lengend Snippet: Loss of BAG3 or SYNPO reduces LC3B-II and SQSTM1 turnover. ( A ) LC3B-I/II levels in primary cortical neurons transduced with lentivirus expressing shBag3 or a scrambled (scr) version. Neurons were treated with or without 10 μM chloroquine (CQ) for 16h. ( B ) LC3B blots of neurons transduced with lentivirus expressing shRNA for rat Synpo or a scrambled ( scr ) version. Neurons were treated as ( A ). ( C ) Quantifications of LC3B-II in BAG3 and SYNPO knockdown neurons in the absence or presence of CQ treatment. LC3B-II was normalized to the loading control ACTB then compared to the scrambled condition. Graph show mean ± SEM of 4-6 samples from 3 independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. *, p

    Article Snippet: Primary antibodies were diluted in blocking solutions as follows: rabbit anti-BAG3, 1:3000; mouse anti-FLAG, 1:1000; goat anti-SYNPO, 1:200; mouse anti-SYNPO, 1:1000; rabbit anti-SQSTM1, 1:1000; mouse anti-GAPDH, 1:10000; rabbit anti-LC3B, 1:5000; mouse anti-ACTB, 1:10000; rabbit anti-VPS18, 1:1000; rabbit anti-MAPT, 1:10,000; mouse anti-p-Thr231 (AT180), 1:1000; mouse anti-p-Ser262 (12E8), 1:1000; mouse anti-p-Ser396/Ser404 (PHF1), 1:5000, followed by incubation at 4°C overnight.

    Techniques: Transduction, Expressing, shRNA

    Colocalization of BAG3, SYNPO or SQSTM1 with endogenous MAP1LC3B/LC3B in neuronal processes. ( A ) Neurons were co-immunostained for LC3B and BAG3, SYNPO or SQSTM1, respectively. Overlap of BAG3, SYNPO or SQSTM1 with LC3B puncta was observed in neuronal processes. SYN1 was used as a negative control. The corresponding line scans are shown at right. Arrowheads denote areas of overlap. Scale bar: 10 μm; scale bar in the high magnification inserts: 2 μm. ( B ) Quantification of colocalization using Pearson’s correlation coefficient. ( C ) Quantification of colocalization using object based analysis. In each condition, 12-20 neurons from 3 independent experiments were used for quantification. Graphs were plotted as mean ± SEM. Colocalization between LC3B and SYNPO, BAG3 and SQSTM1, respectively, was compared to LC3B and SYN1 using one-way ANOVA followed by Dunnett’s multiple comparisons test. ****, p

    Journal: bioRxiv

    Article Title: BAG3 and SYNPO (synaptopodin) facilitate phospho-MAPT/Tau degradation via autophagy in neuronal processes

    doi: 10.1101/518597

    Figure Lengend Snippet: Colocalization of BAG3, SYNPO or SQSTM1 with endogenous MAP1LC3B/LC3B in neuronal processes. ( A ) Neurons were co-immunostained for LC3B and BAG3, SYNPO or SQSTM1, respectively. Overlap of BAG3, SYNPO or SQSTM1 with LC3B puncta was observed in neuronal processes. SYN1 was used as a negative control. The corresponding line scans are shown at right. Arrowheads denote areas of overlap. Scale bar: 10 μm; scale bar in the high magnification inserts: 2 μm. ( B ) Quantification of colocalization using Pearson’s correlation coefficient. ( C ) Quantification of colocalization using object based analysis. In each condition, 12-20 neurons from 3 independent experiments were used for quantification. Graphs were plotted as mean ± SEM. Colocalization between LC3B and SYNPO, BAG3 and SQSTM1, respectively, was compared to LC3B and SYN1 using one-way ANOVA followed by Dunnett’s multiple comparisons test. ****, p

    Article Snippet: Primary antibodies were diluted in blocking solutions as follows: rabbit anti-BAG3, 1:3000; mouse anti-FLAG, 1:1000; goat anti-SYNPO, 1:200; mouse anti-SYNPO, 1:1000; rabbit anti-SQSTM1, 1:1000; mouse anti-GAPDH, 1:10000; rabbit anti-LC3B, 1:5000; mouse anti-ACTB, 1:10000; rabbit anti-VPS18, 1:1000; rabbit anti-MAPT, 1:10,000; mouse anti-p-Thr231 (AT180), 1:1000; mouse anti-p-Ser262 (12E8), 1:1000; mouse anti-p-Ser396/Ser404 (PHF1), 1:5000, followed by incubation at 4°C overnight.

    Techniques: Negative Control

    The BAG3 WW domain and SYNPO PPxY motifs are required for their interaction. ( A ) BAG3 is a multi-domain protein, which contains a WW domain at its amino-terminus for binding PPxY motifs in partner proteins. In a previously performed peptide array screen for BAG3 WW domain interacting proteins of the human proteome [ 18 ], 12-mer peptides of SYNPO2 (aa 615-626) and SYNPO (aa 333-344), respectively, were strongly recognized by the WW domain of the co-chaperone BAG3. ( B ) HeLa cells were transiently transfected with empty plasmid or plasmid constructs for the expression of FLAG-tagged SYNPO or mutant forms with inactivating mutations in the PPxY motifs, as indicated followed by immunoprecipitation with an anti-FLAG antibody (IP). Isolated immune complexes were probed for the presence of endogenous BAG3. Input samples correspond to 32 μg of protein. ( C ) Similar to the experimental approach described under ( B ), BAG3 complexes were isolated from HeLa cells expressing a wild-type form of the BAG3 co-chaperone or a form with an inactivated WW domain (BAG3-WAWA). Isolated complexes were analyzed for the presence of endogenous SYNPO.

    Journal: bioRxiv

    Article Title: BAG3 and SYNPO (synaptopodin) facilitate phospho-MAPT/Tau degradation via autophagy in neuronal processes

    doi: 10.1101/518597

    Figure Lengend Snippet: The BAG3 WW domain and SYNPO PPxY motifs are required for their interaction. ( A ) BAG3 is a multi-domain protein, which contains a WW domain at its amino-terminus for binding PPxY motifs in partner proteins. In a previously performed peptide array screen for BAG3 WW domain interacting proteins of the human proteome [ 18 ], 12-mer peptides of SYNPO2 (aa 615-626) and SYNPO (aa 333-344), respectively, were strongly recognized by the WW domain of the co-chaperone BAG3. ( B ) HeLa cells were transiently transfected with empty plasmid or plasmid constructs for the expression of FLAG-tagged SYNPO or mutant forms with inactivating mutations in the PPxY motifs, as indicated followed by immunoprecipitation with an anti-FLAG antibody (IP). Isolated immune complexes were probed for the presence of endogenous BAG3. Input samples correspond to 32 μg of protein. ( C ) Similar to the experimental approach described under ( B ), BAG3 complexes were isolated from HeLa cells expressing a wild-type form of the BAG3 co-chaperone or a form with an inactivated WW domain (BAG3-WAWA). Isolated complexes were analyzed for the presence of endogenous SYNPO.

    Article Snippet: Primary antibodies were diluted in blocking solutions as follows: rabbit anti-BAG3, 1:3000; mouse anti-FLAG, 1:1000; goat anti-SYNPO, 1:200; mouse anti-SYNPO, 1:1000; rabbit anti-SQSTM1, 1:1000; mouse anti-GAPDH, 1:10000; rabbit anti-LC3B, 1:5000; mouse anti-ACTB, 1:10000; rabbit anti-VPS18, 1:1000; rabbit anti-MAPT, 1:10,000; mouse anti-p-Thr231 (AT180), 1:1000; mouse anti-p-Ser262 (12E8), 1:1000; mouse anti-p-Ser396/Ser404 (PHF1), 1:5000, followed by incubation at 4°C overnight.

    Techniques: Binding Assay, Peptide Microarray, Transfection, Plasmid Preparation, Construct, Expressing, Mutagenesis, Immunoprecipitation, Isolation

    SYNPO or BAG3 knockdown does not affect lysosomal function. ( A ) Maturation of CTSL (cathepsin L) in either BAG3 or SYNPO knockdown neurons by immunoblotting. pro., precursor CTSL; im, immature CTSL; m, mature CTSL. Neurons treated with 10 μM chloroquine (CQ) were used as a positive control. ( B ) Quantification of precursor CTSL:mature CTSL ratio. Graph shows mean ± SEM. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test. **, p

    Journal: bioRxiv

    Article Title: BAG3 and SYNPO (synaptopodin) facilitate phospho-MAPT/Tau degradation via autophagy in neuronal processes

    doi: 10.1101/518597

    Figure Lengend Snippet: SYNPO or BAG3 knockdown does not affect lysosomal function. ( A ) Maturation of CTSL (cathepsin L) in either BAG3 or SYNPO knockdown neurons by immunoblotting. pro., precursor CTSL; im, immature CTSL; m, mature CTSL. Neurons treated with 10 μM chloroquine (CQ) were used as a positive control. ( B ) Quantification of precursor CTSL:mature CTSL ratio. Graph shows mean ± SEM. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test. **, p

    Article Snippet: Primary antibodies were diluted in blocking solutions as follows: rabbit anti-BAG3, 1:3000; mouse anti-FLAG, 1:1000; goat anti-SYNPO, 1:200; mouse anti-SYNPO, 1:1000; rabbit anti-SQSTM1, 1:1000; mouse anti-GAPDH, 1:10000; rabbit anti-LC3B, 1:5000; mouse anti-ACTB, 1:10000; rabbit anti-VPS18, 1:1000; rabbit anti-MAPT, 1:10,000; mouse anti-p-Thr231 (AT180), 1:1000; mouse anti-p-Ser262 (12E8), 1:1000; mouse anti-p-Ser396/Ser404 (PHF1), 1:5000, followed by incubation at 4°C overnight.

    Techniques: Positive Control

    BAG3 or SYNPO knockdown blocks the autophagic flux of autophagy in neuronal processes. Representative maximal-projections of confocal z-stack images of neuronal soma ( A ) and processes ( B ). Neurons treated with 100 nM bafilomycin A1 (BafA1) for 4 h were used as positive controls. Scale bar: 10 μm. ( C ) Quantification of autophagosomes (green) and autolysosomes (red only) under the conditions of ( A ) and ( B ). The total number of green particles (autophagosomes) and red particles (autophagosomes plus autolysosomes) were counted as described in Materials and Methods. Red only particles (autolysosomes) were determined by subtracting the number of green particles from the respective number of red particles. Data were obtained from 20-30 neurons of 3 independent experiments. One to three processes from each neuron were chosen for analysis. Data were shown as mean ± SEM. Statistical analysis was performed using two-way ANOVA with Dunnett’s post hoc test. *, p

    Journal: bioRxiv

    Article Title: BAG3 and SYNPO (synaptopodin) facilitate phospho-MAPT/Tau degradation via autophagy in neuronal processes

    doi: 10.1101/518597

    Figure Lengend Snippet: BAG3 or SYNPO knockdown blocks the autophagic flux of autophagy in neuronal processes. Representative maximal-projections of confocal z-stack images of neuronal soma ( A ) and processes ( B ). Neurons treated with 100 nM bafilomycin A1 (BafA1) for 4 h were used as positive controls. Scale bar: 10 μm. ( C ) Quantification of autophagosomes (green) and autolysosomes (red only) under the conditions of ( A ) and ( B ). The total number of green particles (autophagosomes) and red particles (autophagosomes plus autolysosomes) were counted as described in Materials and Methods. Red only particles (autolysosomes) were determined by subtracting the number of green particles from the respective number of red particles. Data were obtained from 20-30 neurons of 3 independent experiments. One to three processes from each neuron were chosen for analysis. Data were shown as mean ± SEM. Statistical analysis was performed using two-way ANOVA with Dunnett’s post hoc test. *, p

    Article Snippet: Primary antibodies were diluted in blocking solutions as follows: rabbit anti-BAG3, 1:3000; mouse anti-FLAG, 1:1000; goat anti-SYNPO, 1:200; mouse anti-SYNPO, 1:1000; rabbit anti-SQSTM1, 1:1000; mouse anti-GAPDH, 1:10000; rabbit anti-LC3B, 1:5000; mouse anti-ACTB, 1:10000; rabbit anti-VPS18, 1:1000; rabbit anti-MAPT, 1:10,000; mouse anti-p-Thr231 (AT180), 1:1000; mouse anti-p-Ser262 (12E8), 1:1000; mouse anti-p-Ser396/Ser404 (PHF1), 1:5000, followed by incubation at 4°C overnight.

    Techniques: