Journal: Scientific reports

Article Title: Inhibition of SHIP2 in CD2AP-deficient podocytes ameliorates reactive oxygen species generation but aggravates apoptosis.

doi: 10.1038/s41598-017-10512-w

Figure Lengend Snippet: Figure 1. ROS generation and apoptosis are increased in CD2AP−/− podocytes. (A) ROS production is increased in the absence of CD2AP (CD2AP−/−) compared to wild type (WT) podocytes as observed by DCFH-DA fluorescent probe assay. Hoechst 33342 was used for normalization. (B) Flow cytometry with annexin V and 7-AAD double labelling indicates that absence of CD2AP induces apoptosis. Reintroduction of CD2AP into CD2AP−/− podocytes rescues CD2AP−/− podocytes from apoptosis. (C) Representative immunoblot of CD2AP expression in WT podocytes, and in CD2AP−/− podocytes infected with lentiviruses containing an empty vector (EV) or human CD2AP cDNA. (D,E) Immunoblotting and quantification reveals that phosphorylation of AKT on T308 (p-T308) is reduced in CD2AP−/− podocytes compared to WT podocytes. Total AKT (panAKT) is used for normalization. Full width blot of (D) is shown in Supplemental Fig. S1A. (F) In-cell Western and quantification shows no difference in the phosphorylation of AKT on S473 (p- S473) in relation to total AKT (panAKT) between WT and CD2AP−/− podocytes. Both p-S473 and panAKT were normalized to nuclear marker DRAQ5TM. (G) In-cell Western and quantification reveals that the ratio of phosphorylated ERK (p-ERK) to total ERK is significantly lower in CD2AP−/− podocytes compared to WT podocytes. Both p-ERK and total ERK were normalized to nuclear marker DRAQ5TM. The experiments were performed three times with three replicates in each experiment. The bars show the mean expression in arbitrary units (error bars STDEV). *p < 0.05, ***p < 0.001, Student’s t-test.

Article Snippet: Untagged human CD2AP cDNA (OriGene Technologies, Rockville, MD, USA) was cloned into pSIN18.cppt.hEF1_p.WPRE vector44.

Techniques: Flow Cytometry, Western Blot, Expressing, Infection, Plasmid Preparation, Phospho-proteomics, In-Cell ELISA, Marker

Journal: Scientific reports

Article Title: Inhibition of SHIP2 in CD2AP-deficient podocytes ameliorates reactive oxygen species generation but aggravates apoptosis.

doi: 10.1038/s41598-017-10512-w

Figure Lengend Snippet: Figure 2. Inhibition of SHIP2 activity reduces ROS production in CD2AP knockout podocytes but does not protect from apoptosis. (A) Representative immunoblot for SHIP2 in wild type (WT) and CD2AP−/− podocytes. Tubulin is included as a loading control. (B) Quantification of SHIP2 expression level in WT and CD2AP−/− podocytes in three replicate blots as in (A) shows an increase in SHIP2 expression. Tubulin was used for normalisation. (C) SHIP2 activity is increased in the absence of CD2AP. Treatment of CD2AP−/− podocytes with SHIP2 inhibitor AS1949490 reduces SHIP2 activity. (D) Representative immunoblot for SHIP2 of immunoprecipitations carried out with SHIP2 IgG (IP SHIP2) or goat IgG (IP IgG) as a control from 500 µg of protein lysates prepared from WT podocytes and CD2AP−/− podocytes treated or not with AS1949490. Similar immunoprecipitations were performed to enrich SHIP2 for activity assays. Full width blot is shown in Supplemental Fig. S1B. (E) Inhibition of SHIP2 prevents the increase in ROS generation induced by the absence of CD2AP, as observed by DCFH-DA fluorescent probe assay. Hoechst 33342 was used for normalization. (F) Flow cytometry with annexin V labelling indicates that AS1949490 treatment of CD2AP−/− podocytes induces apoptosis. (G) Representative immunoblot for PDK1 and CDK2 in WT and CD2AP−/− podocytes. Tubulin is included as a loading control. (H) Quantification of PDK1 and CDK2 in three replicate blots as in (G) in WT and CD2AP−/− podocytes shows a decrease in PDK1 and CDK2 expression in the absence of CD2AP. (I) SHIP2 inhibitor AS1949490 treatment reduces the expression of PDK1 in CD2AP−/− podocytes but not in WT podocytes. The experiments were performed three times with four (A–D,F–I) or 32 (E) replicates in each experiment. The bars show the mean expression in arbitrary units (error bars STDEV). *p < 0.05, ***p < 0.001, Student’s t-test.

Article Snippet: Untagged human CD2AP cDNA (OriGene Technologies, Rockville, MD, USA) was cloned into pSIN18.cppt.hEF1_p.WPRE vector44.

Techniques: Inhibition, Activity Assay, Knock-Out, Western Blot, Control, Expressing, Flow Cytometry

Journal: Scientific reports

Article Title: Inhibition of SHIP2 in CD2AP-deficient podocytes ameliorates reactive oxygen species generation but aggravates apoptosis.

doi: 10.1038/s41598-017-10512-w

Figure Lengend Snippet: Figure 3. SHIP2 inhibition diminishes activation of AKT and ERK signalling pathways in CD2AP-deficient podocytes. (A) Representative immunoblot used for quantifying T308 phosphorylation of AKT in WT and CD2AP−/− podocytes treated or not with AS1949490. (B) Quantification of three replicate immunoblots as in (A) reveals that AS1949490 treatment increases T308 phosphorylation of AKT (p-T308) in both WT and CD2AP−/− podocytes. (C) In-cell Western and quantification shows that AS1949490 treatment increases S473 phosphorylation of AKT (p-S473) in WT podocytes but not in CD2AP−/− podocytes. Phosphorylated AKT (S473) and total AKT (panAKT) were both normalized to nuclear marker DRAQ5TM. (D) Representative immunoblot used for quantifying p-PDK1 in WT and CD2AP−/− podocytes treated or not with AS1949490. (E) Quantification of three replicate immunoblots as in (D) reveals that AS1949490 treatment increases relative phosphorylation of PDK1 in both WT and CD2AP−/− podocytes. p-PDK1 and total PDK1 were normalized to tubulin before calculation of the ratio. (F) In-cell Western and quantification reveals that AS1949490 treatment increases the phosphorylation of ERK (p-ERK) in both WT and CD2AP−/− podocytes, but the response remains significantly lower in the absence of CD2AP. The experiments were performed three times with three replicates in each experiment. The bars show the mean expression in arbitrary units (error bars STDEV). *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t-test.

Article Snippet: Untagged human CD2AP cDNA (OriGene Technologies, Rockville, MD, USA) was cloned into pSIN18.cppt.hEF1_p.WPRE vector44.

Techniques: Inhibition, Activation Assay, Western Blot, Phospho-proteomics, In-Cell ELISA, Marker, Expressing

Journal: Scientific reports

Article Title: Inhibition of SHIP2 in CD2AP-deficient podocytes ameliorates reactive oxygen species generation but aggravates apoptosis.

doi: 10.1038/s41598-017-10512-w

Figure Lengend Snippet: Figure 4. Absence of CD2AP in mouse kidneys leads to an increase in ROS. (A) Representative image of a 3 weeks old wild type mouse kidney section stained with anti-8-OHdG IgG. (B) Representative image of a 3 weeks old CD2AP−/− mouse kidney section stained with anti-8-OHdG IgG. (C) Quantification reveals 3.5- fold increase in 8-OHdG staining in glomeruli of CD2AP−/− mice reflecting oxidative damage to DNA and RNA. Scale bar (A,B): 40 µm. In (C), the bars show the difference in arbitrary units between relative mask areas quantified with the HistoQuant module (error bars SEM). ***p < 0.001, Student’s t-test.

Article Snippet: Untagged human CD2AP cDNA (OriGene Technologies, Rockville, MD, USA) was cloned into pSIN18.cppt.hEF1_p.WPRE vector44.

Techniques: Staining

Journal: Scientific reports

Article Title: Inhibition of SHIP2 in CD2AP-deficient podocytes ameliorates reactive oxygen species generation but aggravates apoptosis.

doi: 10.1038/s41598-017-10512-w

Figure Lengend Snippet: Figure 5. PA-treatment decreases CD2AP expression and increases SHIP2 activity, ROS production and apoptosis in cultured human podocytes. (A) Representative immunoblot for CD2AP after PA-treatment. Tubulin is included as a loading control. (B) Quantification of CD2AP after PA-treatment in immunoblots like in (A) shows a decrease in CD2AP expression. (C) Quantification of SHIP2 expression in podocytes treated with PA by In-Cell Western shows that the expression of SHIP2 is increased by PA-treatment. DRAQ5TM was used for normalization. (D) PA-treatment increases SHIP2 activity in human podocytes. (E) PA increases ROS generation as visualized by an increase in the intensity of DCFH-DA fluorescent probe. Hoechst 33342 was used for normalization. (F) Flow cytometry of cultured human podocytes stained with annexin V and 7-AAD double labelling shows that PA-treatment increases podocyte apoptosis. The experiments were performed three times with four (B,D,F), 24 (C) or 48 (E) replicates in each experiment. The bars (B–F) show the mean expression in arbitrary units (error bars STDEV). ***p < 0.001, Student’s t-test.

Article Snippet: Untagged human CD2AP cDNA (OriGene Technologies, Rockville, MD, USA) was cloned into pSIN18.cppt.hEF1_p.WPRE vector44.

Techniques: Expressing, Activity Assay, Cell Culture, Western Blot, Control, In-Cell ELISA, Flow Cytometry, Staining

Journal: Scientific reports

Article Title: Inhibition of SHIP2 in CD2AP-deficient podocytes ameliorates reactive oxygen species generation but aggravates apoptosis.

doi: 10.1038/s41598-017-10512-w

Figure Lengend Snippet: Figure 7. CD2AP overexpression restores PDK1 and CDK2 expression and reduces ROS generation and podocyte apoptosis induced by PA in cultured human podocytes. (A) Representative immunoblot for CD2AP, PDK1 and CDK2 in cultured human podocytes infected with lentiviruses containing an empty vector (EV) or human CD2AP cDNA (CD2AP OE), and with or without PA-treatment. Tubulin is included as a loading control. (B) Quantification of CD2AP, PDK1, and CDK2 expression level in Western blots as in (A) shows that PA-treatment reduces the expression of CD2AP, PDK1, and CDK2 and that CD2AP overexpression prevents PA-induced downregulation of CD2AP. Overexpression of CD2AP alone does not affect the expression of PDK1 or CDK2. (C) Overexpression of CD2AP prevents PA-induced increase in ROS generation as visualized by DCFH-DA fluorescent probe assay. Overexpression of CD2AP alone does not affect ROS generation. Hoechst 33342 was used for normalization. (D) Flow cytometry of cultured human podocytes stained with annexin V and 7-AAD confirms that overexpression of CD2AP protects podocytes from PA-induced apoptosis. The experiments were performed three times with three (A–B,D) or 24 (C) replicates in each experiment. The bars (b–d) show the mean expression in arbitrary units (error bars STDEV). *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t-test.

Article Snippet: Untagged human CD2AP cDNA (OriGene Technologies, Rockville, MD, USA) was cloned into pSIN18.cppt.hEF1_p.WPRE vector44.

Techniques: Over Expression, Expressing, Cell Culture, Western Blot, Infection, Plasmid Preparation, Control, Flow Cytometry, Staining

Journal: Scientific reports

Article Title: Inhibition of SHIP2 in CD2AP-deficient podocytes ameliorates reactive oxygen species generation but aggravates apoptosis.

doi: 10.1038/s41598-017-10512-w

Figure Lengend Snippet: Figure 8. CD2AP expression appears to be reduced and SHIP2 expression increased in kidneys of PA-induced nephrotic rats. (A–C) In normal rat glomerulus CD2AP is expressed in podocytes and partially co-localizes with nephrin. (D–F) In PA-induced nephrotic rats 10 days after PA administration, both CD2AP and nephrin appear weak and show patch-like accumulation of the signal. (G–I) In normal rat glomerulus weak signal for SHIP2 is observed in podocytes as visualized by double labelling for nephrin. (J–L) In PA-induced nephrotic rats 10 days after PA administration, SHIP2 staining appears in diffuse, patch-like accumulations similarly as nephrin. Scale bar: 20 µm.

Article Snippet: Untagged human CD2AP cDNA (OriGene Technologies, Rockville, MD, USA) was cloned into pSIN18.cppt.hEF1_p.WPRE vector44.

Techniques: Expressing, Staining

Journal: Scientific reports

Article Title: Inhibition of SHIP2 in CD2AP-deficient podocytes ameliorates reactive oxygen species generation but aggravates apoptosis.

doi: 10.1038/s41598-017-10512-w

Figure Lengend Snippet: Figure 9. Schematic model illustrating the effect of SHIP2 inhibition in CD2AP-deficient podocytes. (A) Wild type podocytes in basal state. CD2AP associates with SHIP2. CD2AP inhibits the production of ROS, and SHIP2 negatively regulates the pathways leading to AKT and ERK phosphorylation. The balance between the prosurvival and proapoptotic signals is maintained. (B) CD2AP−/− podocytes in basal state. The expression and activity of SHIP2 and generation of ROS are increased in the absence of CD2AP. Increased SHIP2 activity leads to low phosphorylation level of AKT and ERK. The expression of PDK1 is downregulated contributing to degreased phosphorylation of T308 of AKT. Decrease in prosurvival signalling manifests as increased apoptosis of the cells. (C) Inhibition of SHIP2 activity with AS1949490 in CD2AP−/− podocytes. Inhibition of the activity of SHIP2 leads to reduced generation of ROS. AS1949490 attenuates the potential of SHIP2 to negatively regulate AKT and ERK activity, yet the phosphorylation of S473 of AKT does not increase. Despite of low expression level of PDK1, the phosphorylation of T308 of AKT increases. As phosphorylation of both sites of AKT is required for its full activity, remains the balance between prosurvival and proapoptotic signalling disrupted and podocytes undergo apoptosis. It is also possible that increased ERK activation may contribute to an increase in apoptosis (see Discussion for details).

Article Snippet: Untagged human CD2AP cDNA (OriGene Technologies, Rockville, MD, USA) was cloned into pSIN18.cppt.hEF1_p.WPRE vector44.

Techniques: Inhibition, Phospho-proteomics, Expressing, Activity Assay, Activation Assay

Journal: Nature Communications

Article Title: Noncanonical function of folate through folate receptor 1 during neural tube formation

doi: 10.1038/s41467-024-45775-1

Figure Lengend Snippet: a Neural plate stage Xenopus laevis embryos were processed for co-immunoprecipitation (IP) assays. Example of Western blot assay from immunoprecipitates with FOLR1 or GFP (control) antibodies and probed for CD2AP or FOLR1. Similar results were observed in N = 3 independent experiments. b Neural plate stage Xenopus laevis embryos were fixed and processed for immunostaining. Images are transverse single z-sections of immunostained neural plate showing apical colocalization of phospho-CD2AP (p-CD2AP) and p-c-Cbl with C-cadherin. Scale bar, 10 μm. Similar results were observed in N = 3 independent experiments. c Two-cell stage Xenopus laevis embryos were bilaterally microinjected with 9.9 pmol of Control-morpholino (Control, Control-MO), 2.6 pmol CD2AP-MO1 (CD2AP KD1) or 7.4–9.9 pmol CD2AP-MO2 (CD2AP KD2/KD) per blastomere until neural tube closed in control embryos, when they were fixed and photomicrographed. Examples of whole embryos in each group. Arrowheads indicate open neural tube (neural tube defect, NTD). Numbers are embryos presenting closed (green) or open (purple, NTD) neural tube. Bar graph represents proportion of phenotypes in each group. d Two-cell stage Xenopus laevis embryos were unilaterally microinjected with 9.9 pmol Control-MO (Control) and 7.4–9.9 pmol CD2AP-MO2 (CD2AP KD) along with GFP and mCherry mRNA, respectively, and allowed to develop until they reached mid-neural plate stages, when they were fixed and processed for immunostaining. Image is a transverse section of the neural plate, immunostained for GFP (Control), mCherry (CD2AP KD) and C-cadherin. Double arrows indicate apical surface length of medial superficial Control (white) and CD2AP KD (magenta) neural plate cells. Scale bar, 20 μm. Graph shows individual and mean ± SD apical length of superficial neural plate cells per embryo, n of cells = 75 in each half of the neural plate, N of embryos = 4. **** p < 0.0001, two-tailed paired t -test. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were: anti-phospho-tyr-4,8,10 CD2AP, 1:500 (Rockland, cat. # 600-401-J96), anti-phospho-tyr-674 c-Cbl, 1:500 (MyBioSource, cat. # MBS820886), anti-C-cadherin, 1:100 (Developmental Studies Hybridoma Bank, cat. # 6B6), anti-EEA1, 1:1000 (Origene, cat. # AB0006-200), anti-ubiquitin, 1:500 (Stress Marq, cat. # SPC-119B), anti-Rab7, 1:500 (Cell Signaling, cat. # 9367), anti-LAMP1, 1:500 (Abcam, cat. # ab24170), anti-GFP, 1:750 (Abcam, cat. # ab13970), anti-mCherry, 1:750 (Biorbyt, cat. # orb11618), anti-SOX2, 1:300 (Cat # AF2018, R&D Systems), anti-α-tubulin, 1:500 (Abcam, cat. # ab15246).

Techniques: Immunoprecipitation, Western Blot, Control, Immunostaining, Two Tailed Test

Journal: Nature Communications

Article Title: Noncanonical function of folate through folate receptor 1 during neural tube formation

doi: 10.1038/s41467-024-45775-1

Figure Lengend Snippet: a Two-cell stage Xenopus laevis embryos were bilaterally microinjected with hEEA1-GFP and membrane mCherry mRNAs and unilaterally microinjected with 9.9 pmol CD2AP-MO (CD2AP KD) per blastomere along with fluorescent tracer and allowed to develop until they reached early neural plate stages (stage 13-14), when they were time-lapse imaged with an acquisition rate of 1 frame/6 min. Image is maximum intensity projection of single time frame. Dashed line indicates border between morpholino-injected and wild-type (WT) neural plate. Inset shows neural plate injected side showing tracer in blue. Graphs show distribution between both halves of the neural plate (in %) of the number of EEA1-GFP vesicles and area fraction of labeled endosomes per neural plate cell surface. Two-tailed paired t -test, n = 28 cells analyzed in each group from N = 5 embryos per group. Scale bar, 20 μm. b Two-cell stage Xenopus laevis embryos were bilaterally microinjected with 7.4 pmol Control-MO (Control) or CD2AP-MO (CD2AP KD) per blastomere and allowed to grow until they reached mid-neural plate stages (stage 15–17) when neural plate was dissected and processed for Western blot assays. Image is an example of Western blot assay. Graph shows individual and mean ± SD percent of optical density (OD) for C-cadherin immunoblot band normalized with GAPDH protein band OD and compared to controls. Two-tailed ratio t -test, n = 28 and 24 neural plates for Control and CD2AP KD groups, respectively, N = 5 independent experiments. In ( a , b ), * p < 0.05, ** p < 0.01, *** p < 0.001. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were: anti-phospho-tyr-4,8,10 CD2AP, 1:500 (Rockland, cat. # 600-401-J96), anti-phospho-tyr-674 c-Cbl, 1:500 (MyBioSource, cat. # MBS820886), anti-C-cadherin, 1:100 (Developmental Studies Hybridoma Bank, cat. # 6B6), anti-EEA1, 1:1000 (Origene, cat. # AB0006-200), anti-ubiquitin, 1:500 (Stress Marq, cat. # SPC-119B), anti-Rab7, 1:500 (Cell Signaling, cat. # 9367), anti-LAMP1, 1:500 (Abcam, cat. # ab24170), anti-GFP, 1:750 (Abcam, cat. # ab13970), anti-mCherry, 1:750 (Biorbyt, cat. # orb11618), anti-SOX2, 1:300 (Cat # AF2018, R&D Systems), anti-α-tubulin, 1:500 (Abcam, cat. # ab15246).

Techniques: Membrane, Injection, Labeling, Two Tailed Test, Control, Western Blot

Journal: Nature Communications

Article Title: Noncanonical function of folate through folate receptor 1 during neural tube formation

doi: 10.1038/s41467-024-45775-1

Figure Lengend Snippet: Two-cell stage Xenopus laevis embryos were microinjected with 14.8 pmol Control-morpholino (MO, Control, a , b ), 3.2 pmol FOLR1-MO (FOLR1 KD, a ) or 14.8 pmol CD2AP-MO2 (CD2AP KD, b ) per embryo and incubated with saline or proteasome and lysosome inhibitors at the end of gastrulation (stage 12) until neural plate stages (stage 17) when they were processed for Western blot assays. Images are examples of Western blot assays. Graphs show individual and mean ± SD percent of optical density (OD) for CD2AP ( a ) or FOLR1 ( b ) immunoblot band normalized with GAPDH protein band OD and compared to controls. In ( a ), n = 34 and 42 neural plates for Control and FOLR1 KD, respectively, N = 7 independent experiments; n = 20 and 26 neural plates for Control+inhibitors and FOLR1 KD+inhibitors groups, respectively, N = 3 independent experiments. In ( b ) n = 16 and 20 for Control and CD2AP KD groups, respectively and n = 16 and 18 neural plates for Control+inhibitors and CD2AP KD+inhibitors, respectively, N = 3 independent experiments. In ( a , b ) * p < 0.05, *** p < 0.001, ns: not significant, two-tailed ratio t -test. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were: anti-phospho-tyr-4,8,10 CD2AP, 1:500 (Rockland, cat. # 600-401-J96), anti-phospho-tyr-674 c-Cbl, 1:500 (MyBioSource, cat. # MBS820886), anti-C-cadherin, 1:100 (Developmental Studies Hybridoma Bank, cat. # 6B6), anti-EEA1, 1:1000 (Origene, cat. # AB0006-200), anti-ubiquitin, 1:500 (Stress Marq, cat. # SPC-119B), anti-Rab7, 1:500 (Cell Signaling, cat. # 9367), anti-LAMP1, 1:500 (Abcam, cat. # ab24170), anti-GFP, 1:750 (Abcam, cat. # ab13970), anti-mCherry, 1:750 (Biorbyt, cat. # orb11618), anti-SOX2, 1:300 (Cat # AF2018, R&D Systems), anti-α-tubulin, 1:500 (Abcam, cat. # ab15246).

Techniques: Control, Incubation, Saline, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Noncanonical function of folate through folate receptor 1 during neural tube formation

doi: 10.1038/s41467-024-45775-1

Figure Lengend Snippet: a – c Neural plate from mid neural plate stage Xenopus laevis embryos was dissected and dissociated cells plated in vitro. After 2 h, cells were loaded with the Ca 2+ sensor Fluo4-AM and time-lapse imaged. a Example of 1-h recording of neural plate cell Ca 2+ activity. b , c Folinic acid ( b , c ), folic acid ( c ) or vehicle was added to neural plate cells in culture during time-lapse imaging and the Ca 2+ response was recorded in the first minute post addition. b Example of acute transient elicited by 100 μM folinic acid. c Graph shows mean ± SEM folinic- or folic acid-responsive neural plate cells compared to total number of cells with spontaneous Ca 2+ transients in 30 min recording, N = 3 independent experiments. Two-tailed one sample t and Wilcoxon test. d Two-cell stage embryos were bilaterally microinjected with GCaMP6s mRNA and grown until early and mid-neural plate stages when they were time-lapse imaged before and after addition of vehicle or 300 μM folinic acid. Image shows example of embryo with cells exhibiting Ca 2+ transients indicated with circles. Graph shows individual and mean ± SD percent change in Ca 2+ transient frequency before and after addition of vehicle or folinic acid, n = 4, 5, 6 and 7 embryos for Early-Vehicle, Early-Folinic acid, Mid-Vehicle and Mid-Folinic acid groups, respectively. One-sample two-tailed t -test, compared to hypothetical value of 100%. e – h Two-cell stage embryos were bilaterally microinjected with GCaMP6s mRNA ( e – h ) and unilaterally microinjected with 9.9 pmol FOLR1-MO1 (FOLR1 KD1/KD) or 1.6 pmol FOLR1-MO2 (FOLR1 KD2) per blastomere ( e ) and grown until mid-neural plate stages when they were time-lapse imaged. Image in ( e ) shows example of embryo with cells exhibiting Ca 2+ transients indicated with circles in WT and FOLR1 KD1 halves. Graphs show individual Ca 2+ transient frequency (transients/5 min) in WT and FOLR1 KD1 or KD2 halves ( e , n = 6 embryos) and in WT embryos before and after addition of vehicle ( f , n = 7 embryos), 50 μM folic acid ( g , n = 6 embryos), Na + and voltage-gated Ca 2+ channel blockers (VGC block : 0.02% tricaine+10 μM nitrendipine+25 μM TTA-2, h , n = 5 embryos), or a mix of folic acid and VGC block ( h , n = 5 embryos). Two-tailed paired t -test ( e – g ) and 1-way ANOVA-Tukey multiple comparisons test ( h ). In ( c – h ), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant. i Model of FOLR1 and CD2AP regulation of neural tube formation. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were: anti-phospho-tyr-4,8,10 CD2AP, 1:500 (Rockland, cat. # 600-401-J96), anti-phospho-tyr-674 c-Cbl, 1:500 (MyBioSource, cat. # MBS820886), anti-C-cadherin, 1:100 (Developmental Studies Hybridoma Bank, cat. # 6B6), anti-EEA1, 1:1000 (Origene, cat. # AB0006-200), anti-ubiquitin, 1:500 (Stress Marq, cat. # SPC-119B), anti-Rab7, 1:500 (Cell Signaling, cat. # 9367), anti-LAMP1, 1:500 (Abcam, cat. # ab24170), anti-GFP, 1:750 (Abcam, cat. # ab13970), anti-mCherry, 1:750 (Biorbyt, cat. # orb11618), anti-SOX2, 1:300 (Cat # AF2018, R&D Systems), anti-α-tubulin, 1:500 (Abcam, cat. # ab15246).

Techniques: In Vitro, Activity Assay, Imaging, Two Tailed Test, Blocking Assay

Journal: Human Molecular Genetics

Article Title: A Drosophila model system to assess the function of human monogenic podocyte mutations that cause nephrotic syndrome

doi: 10.1093/hmg/ddw428

Figure Lengend Snippet: Human monogenic Nephrotic Syndrome (NS) genes and their Drosophila orthologs

Article Snippet: DNA cloning and generation of transgenic fly strains A normal human CD2AP cDNA was obtained from OriGene, which encodes the common 639 a.a. isoform with NCBI reference sequence NP_036252 .

Techniques: Filtration, Binding Assay, Clinical Proteomics, Membrane, Glycoproteomics

Journal: Human Molecular Genetics

Article Title: A Drosophila model system to assess the function of human monogenic podocyte mutations that cause nephrotic syndrome

doi: 10.1093/hmg/ddw428

Figure Lengend Snippet: Cindr is required for nephrocyte function, fly survival, and nephrocyte cell ultrastructure; Cindr gene silencing can be rescued by a normal but not a patient-derived mutant allele of human CD2AP; Cindr interacts with Mec2 in nephrocytes. (A). ANF-RFP uptake visualized by fluorescence microscopy in nephrocytes of Control flies (genotype Hand-GFP; Dot-Gal4), Cindr (CD2AP)-KD flies in which Cindr expression was silenced in nephrocytes with a Cindr targeting RNAi construct (genotype Hand-GFP; Dot-Gal4; UAS-Cindr-RNAi), Cindr-KD + CD2AP-WT flies in which a transgenic wild type allele of human CD2AP was expressed simultaneously with Cindr -targeting RNAi (genotype Hand-GFP; Dot-Gal4; UAS-Cindr-RNAi; UAS-CD2AP), and Cindr-KD + CD2AP-K301M flies in which a transgenic mutant allele of human CD2AP was expressed simultaneously with Cindr -targeting RNAi (genotype Hand-GFP; Dot-Gal4; UAS-Cindr-RNAi; UAS-CD2AP-K301M). The panels show merged ANF-RFP (54) and GFP (green, mostly nuclear) fluorescence. (B) Quantification of nephrocyte RFP levels relative to Control flies. For quantification, ≥20 nephrocytes were analysed from each of three female flies per genotype. The results are presented as mean ± s.e.m. Statistical significance (*) was defined as P < 0.05. (C) Adult fly survival curves illustrating effects on longevity of Cindr silencing and extent of rescue from expression of WT and mutant alleles of human CD2AP. 50 flies of each genotype were maintained at 29°C (to increase Gal4 driven RNAi transgene expression) and mortality was recorded every 48 hours until all flies were dead. Triplicate samples were analysed. (D) Average adult lifespan showing effects of Cindr silencing and rescue by WT and mutant alleles of human CD2AP. 50 flies of each genotype were maintained at 29°C (to increase Gal4 driven RNAi transgene expression) and mortality was recorded every 48 hours until all flies were dead. Experiments were performed in triplicate. The results are presented as mean ± s.e.m. Statistical significance (*) was defined as P < 0.05. (E) Transmission electron microscopy showing effects on nephrocyte NSD (arrowheads) and lacunar channel (*) ultrastructure of Cindr gene silencing and the extent of rescue from expression of WT and mutant alleles of human CD2AP. In Control nephrocytes NSDs were regularly and precisely spaced along the entire circumference of the cell and spanned the "“mouth”" of each lacunar channel. Silencing Cindr gene expression led to fusion of NSDs and the loss of lacunar channels. Expression of WT human CD2AP significantly rescued the ultrastructural defects, while the mutant CD2AP allele had very little effect. (F). Functional interaction between Cindr and Mec2 in nephrocytes demonstrated by Cindr+/-; Mec2+/- double heterozygote synergistic enhancement of deleterious effect on uptake of fluorescent Dextran particles in comparison to Cindr+/- or Mec2+/- single heterozygotes. (G). Quantitative analysis of fluorescent Dextran levels in nephrocytes of the indicated genotype, expressed relative to Control. For quantification, ≥20 nephrocytes were analysed from each of three female flies per genotype. The results are presented as mean ± s.e.m. Statistical significance (*) was defined as P < 0.05.

Article Snippet: DNA cloning and generation of transgenic fly strains A normal human CD2AP cDNA was obtained from OriGene, which encodes the common 639 a.a. isoform with NCBI reference sequence NP_036252 .

Techniques: Derivative Assay, Mutagenesis, Fluorescence, Microscopy, Control, Expressing, Construct, Transgenic Assay, Transmission Assay, Electron Microscopy, Gene Expression, Functional Assay, Comparison

Journal: Frontiers in Pharmacology

Article Title: Shensu IV maintains the integrity of the glomerular filtration barrier and exerts renal protective effects by regulating endogenous hydrogen sulfide levels

doi: 10.3389/fphar.2024.1447249

Figure Lengend Snippet: Primer sequence for RT-qPCR.

Article Snippet: Membranes were blocked with 5% skim milk (Solarbio) to prevent nonspecific binding and then incubated with primary antibodies against CD2AP (1:2000; A01756-2, BOSTER, Wuhan, China), nephrin (1:2000; A01756-2, BOSTER), CBS (1:10,000; 14787-1-AP, Proteintech, Wuhan, China), CSE (1: 4,000; 12217-1-AP, Proteintech), PI3K (1:2000; 60225-1-Ig, Proteintech), p-PI3K (1:2000; bs-3332R, BOSTER), AKT (1:10,000; 60203-2-Ig, Proteintech), p-AKT (1: 10,000; 66,444-1-lg, Proteintech), NOX4 (1:8,000; 14347-1-AP, Proteintech), and GAPDH (1:40,000; 60004-1-Ig, Proteintech).

Techniques: Sequencing

Journal: Frontiers in Pharmacology

Article Title: Shensu IV maintains the integrity of the glomerular filtration barrier and exerts renal protective effects by regulating endogenous hydrogen sulfide levels

doi: 10.3389/fphar.2024.1447249

Figure Lengend Snippet: Shensu IV regulates the PI3K/AKT signaling pathway through H2S. (A) The effects of Shensu IV and NaHS on the mRNA expression of CD2AP, nephrin, CBS, CSE, NOX4, PI3K, and AKT in renal tissue of PAN rats were analyzed by RT-qPCR. (B) Western blot analysis of the effects of Shensu IV and NaHS on the protein levels of CD2AP, nephrin, CBS, CSE, NOX4, PI3K, p-PI3K,AKT,p-AKT in renal tissue of PAN rats. * P < 0.05, ** P < 0.01, *** P < 0.001. Abbreviations: CD2AP, CD2-associated protein; CBS, Cystathionine β-synthase; CSE, Cystathionine γ-lyase; PI3K, Phosphoinositide 3-Kinase; AKT, Protein Kinase B.

Article Snippet: Membranes were blocked with 5% skim milk (Solarbio) to prevent nonspecific binding and then incubated with primary antibodies against CD2AP (1:2000; A01756-2, BOSTER, Wuhan, China), nephrin (1:2000; A01756-2, BOSTER), CBS (1:10,000; 14787-1-AP, Proteintech, Wuhan, China), CSE (1: 4,000; 12217-1-AP, Proteintech), PI3K (1:2000; 60225-1-Ig, Proteintech), p-PI3K (1:2000; bs-3332R, BOSTER), AKT (1:10,000; 60203-2-Ig, Proteintech), p-AKT (1: 10,000; 66,444-1-lg, Proteintech), NOX4 (1:8,000; 14347-1-AP, Proteintech), and GAPDH (1:40,000; 60004-1-Ig, Proteintech).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Shensu IV maintains the integrity of the glomerular filtration barrier and exerts renal protective effects by regulating endogenous hydrogen sulfide levels

doi: 10.3389/fphar.2024.1447249

Figure Lengend Snippet: Shensu IV regulates the PI3K/AKT signaling pathway through H2S in podocytes. (A) The effects of Shensu IV and NaHS on the mRNA expression of CD2AP, nephrin, CBS, CSE, NOX4, PI3K, and AKT in podocytes were analyzed by RT-qPCR. (B) Western blot analysis of the effects of Shensu IV and NaHS on the protein levels of CD2AP, nephrin, CBS, CSE, NOX4, PI3K, p-PI3K,AKT,p-AKT in PAN-induced podocyocytes. * P < 0.05, ** P < 0.01, *** P < 0.001. Abbreviations: CD2AP, CD2-associated protein; CBS, Cystathionine β-synthase; CSE, Cystathionine γ-lyase; PI3K, Phosphoinositide 3-Kinase; AKT, Protein Kinase B.

Article Snippet: Membranes were blocked with 5% skim milk (Solarbio) to prevent nonspecific binding and then incubated with primary antibodies against CD2AP (1:2000; A01756-2, BOSTER, Wuhan, China), nephrin (1:2000; A01756-2, BOSTER), CBS (1:10,000; 14787-1-AP, Proteintech, Wuhan, China), CSE (1: 4,000; 12217-1-AP, Proteintech), PI3K (1:2000; 60225-1-Ig, Proteintech), p-PI3K (1:2000; bs-3332R, BOSTER), AKT (1:10,000; 60203-2-Ig, Proteintech), p-AKT (1: 10,000; 66,444-1-lg, Proteintech), NOX4 (1:8,000; 14347-1-AP, Proteintech), and GAPDH (1:40,000; 60004-1-Ig, Proteintech).

Techniques: Expressing, Quantitative RT-PCR, Western Blot