anti synpo  (Santa Cruz Biotechnology)

Journal: Frontiers in Pharmacology

Article Title: Sanqi Oral Solution Mitigates Proteinuria in Rat Passive Heymann Nephritis and Blocks Podocyte Apoptosis via Nrf2/HO-1 Pathway

doi: 10.3389/fphar.2021.727874

Figure Lengend Snippet: SQ alleviated glomerular podocyte injury in PHN rats. Effects of SQ and CP on foot process width (magnification × 12,000, red arrows) and synaptopodin expression (magnification × 400) were measured by TEM and immunofluorescence staining (A) . With the treatment of SQ and CP, restored glomerular podocytic foot processes (B) and synaptopodin expression (C) were seen in PHN rats ( n = 6). Data are represented as mean ± SD from independent groups. ** p

Article Snippet: Primary antibodies against C5b-9 (sc-66190) and synaptopodin (sc-515842) were purchased from Santa Cruz Biotechnology (Dallas, Texas, United States).

Techniques: Expressing, Transmission Electron Microscopy, Immunofluorescence, Staining

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

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

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

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

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

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

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:

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

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

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

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

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

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: