Journal: International Journal of Molecular Medicine

Article Title: Exogenous spermine attenuates diabetic kidney injury in rats by inhibiting AMPK/mTOR signaling pathway

doi: 10.3892/ijmm.2021.4860

Figure Lengend Snippet: Spermine attenuates podocyte injury and promotes autophagy in T1D rats. (A) Representative immunoblotting of nephrin, CD2-AP and podocin protein levels in kidney sections. (B) Representative immunohistochemical staining for podocin in kidney sections (scale bar, 100 µ m) and its quantification. (C) Representative immunoblotting for Atg5, P62, Beclin1 and LC3II/LCI in kidney sections. (D) Representative immunohistochemical staining for LC3 in kidney sections (scale bar, 100 µ m) and its quantification. Data are expressed as the mean ± standard error of the mean (n=6). * P<0.05 vs. the control group; # P<0.05 vs. the T1D group. T1D, type 1 diabetic; CD-2AP, CD2-associated protein; Atg5, autophagy protein 5; LC3, microtube-associated proteins 1A/1B light chain 3; Sp, spermine-treated group.

Article Snippet: After blocking with TBS with 0.1% Tween-20 (TBST) containing 5% non-fat dry milk for 1 h at 4°C, the membrane was incubated overnight at 4°C with antibodies against ODC (1:1,000; cat. no. ab97395; Abcam), SSAT (1:1,000; cat. no. ab105220; Abcam), nephrin (1:400; cat. no. BA1669; Boster Biological Technology), podocin (1:400; cat. no. BA3416; Boster Biological Technology), CD2-associated protein (CD-2AP; 1:1,000; cat. no. bs-0512R; BIOSS), microtube-associated proteins 1A/1B light chain 3 (LC3; 1:1,000; cat. no. 3868T; Cell Signaling Technology, Inc.), Beclin 1 (1:1,000; cat. no. 3495T; Cell Signaling Technology, Inc.), P62 (1:1,000; cat. no. 8025T; Cell Signaling Technology, Inc.), autophagy protein 5 (Atg5; 1:1,000; cat. no. 12994T; Cell Signaling Technology, Inc.), cleaved caspase-3 (1:1,000; cat. no. 9661T; Cell Signaling Technology, Inc.), total (t)-mTOR (1:1,000; cat. no. A00003-2; Boster Biological Technology), phosphorylated (p)-mTOR (1:600; cat. no. BM4840; Boster Biological Technology), t-AMPK (1:1,000; cat. no. A00994-6; Boster Biological Technology), and p-AMPK (1:1,000; cat. no. P00994; Boster Biological Technology).

Techniques: Western Blot, Immunohistochemical staining, Staining

Journal: International Journal of Molecular Medicine

Article Title: Exogenous spermine attenuates diabetic kidney injury in rats by inhibiting AMPK/mTOR signaling pathway

doi: 10.3892/ijmm.2021.4860

Figure Lengend Snippet: Spermine reduces HG-induced apoptosis via activating autophagy in podocytes. (A) Cell Counting Kit-8 assays were performed using podocytes incubated under HG conditions in the presence of spermine or rapamycin. (B) Hoechst 33342 staining (scale bar, 500 µ m). (C) Representative immunoblotting analyses of the proteins levels of nephrin, CD-2AP and podocin protein levels. (D) Representative images of immunofluorescence staining for nephrin under different culture conditions (scale bar, 200 µ m). (E) Representative immunoblotting analyses for P62, Beclin1, LC3II/LC3I and cleaved caspase-3. Data are expressed as the mean ± standard error of the mean (n=6-8). * P<0.05 vs. the NG group; # P<0.05 vs. the HG group. HG, high glucose; CD-2AP, CD2-associated protein; LC3, microtube-associated proteins 1A/1B light chain 3; Sp, spermine-treated group; NG, normal glucose.

Article Snippet: After blocking with TBS with 0.1% Tween-20 (TBST) containing 5% non-fat dry milk for 1 h at 4°C, the membrane was incubated overnight at 4°C with antibodies against ODC (1:1,000; cat. no. ab97395; Abcam), SSAT (1:1,000; cat. no. ab105220; Abcam), nephrin (1:400; cat. no. BA1669; Boster Biological Technology), podocin (1:400; cat. no. BA3416; Boster Biological Technology), CD2-associated protein (CD-2AP; 1:1,000; cat. no. bs-0512R; BIOSS), microtube-associated proteins 1A/1B light chain 3 (LC3; 1:1,000; cat. no. 3868T; Cell Signaling Technology, Inc.), Beclin 1 (1:1,000; cat. no. 3495T; Cell Signaling Technology, Inc.), P62 (1:1,000; cat. no. 8025T; Cell Signaling Technology, Inc.), autophagy protein 5 (Atg5; 1:1,000; cat. no. 12994T; Cell Signaling Technology, Inc.), cleaved caspase-3 (1:1,000; cat. no. 9661T; Cell Signaling Technology, Inc.), total (t)-mTOR (1:1,000; cat. no. A00003-2; Boster Biological Technology), phosphorylated (p)-mTOR (1:600; cat. no. BM4840; Boster Biological Technology), t-AMPK (1:1,000; cat. no. A00994-6; Boster Biological Technology), and p-AMPK (1:1,000; cat. no. P00994; Boster Biological Technology).

Techniques: Cell Counting, Incubation, Staining, Western Blot, Immunofluorescence

Journal: International immunopharmacology

Article Title: Minnelide combined with Angptl3 knockout completely protects mice with adriamycin nephropathy via suppression of TGF-β1-Smad2 and p53 pathways.

doi: 10.1016/j.intimp.2022.109656

Figure Lengend Snippet: Fig. 2. Minnelide alleviates podocyte injury in Angptl3 knockout mice with adriamycin nephropathy. (A) Immunofluorescence of nephrin, podocin, and cd2ap in kidney tissues of three groups of mice. (B and C) The protein expression levels of nephrin, podocin, and cd2ap in kidney tissues of three groups of mice were measured by western blot. n = 3, ****P < 0.0001 vs Angptl3-/- group; #P < 0.05, ##P < 0.01 and ####P < 0.0001 vs ADR + Angptl3-/- group.

Article Snippet: The membrane was incubated overnight at 4 ◦C with the following primary antibodies: nephrin (1:500, Invitrogen, PA5-106921), podocin (1:500, Proteintech, 20384–1-AP), cd2ap (1:500, Invitrogen, PA5-51894), α-SMA (1:1000, Servicebio, GB111364), TGF-β1 (1:500, Servicebio, GB111876), pSmad2 (1:500, Servicebio, GB11343), pp53 (1:500, Servicebio, B. Ji et al. International Immunopharmacology 115 (2023) 109656 GB114061), p53 (1:500, Proteintech, 10442–1-AP), Bax (1:500, Abcam, ab216494), Bcl-2 (1:500, Abcam, ab196495), and Cleaved-caspase3 (1:500, Servicebio, GB11532).

Techniques: Knock-Out, Immunofluorescence, Expressing, Western Blot

Journal: International immunopharmacology

Article Title: Minnelide combined with Angptl3 knockout completely protects mice with adriamycin nephropathy via suppression of TGF-β1-Smad2 and p53 pathways.

doi: 10.1016/j.intimp.2022.109656

Figure Lengend Snippet: Fig. 8. Triptolide attenuates podocyte injury in Angptl3 knockout primary podocytes. (A) Immunofluorescence of nephrin, podocin, and cd2ap in Angptl3 knockout primary podocytes. (B) The expression of podocin and cd2ap proteins in Angptl3 knockout primary podocytes was measured by western blot. n = 3. ****P < 0.0001 vs Angptl3-/- group; ##P < 0.01 and ####P < 0.0001 vs ADR + Angptl3-/- group.

Article Snippet: The membrane was incubated overnight at 4 ◦C with the following primary antibodies: nephrin (1:500, Invitrogen, PA5-106921), podocin (1:500, Proteintech, 20384–1-AP), cd2ap (1:500, Invitrogen, PA5-51894), α-SMA (1:1000, Servicebio, GB111364), TGF-β1 (1:500, Servicebio, GB111876), pSmad2 (1:500, Servicebio, GB11343), pp53 (1:500, Servicebio, B. Ji et al. International Immunopharmacology 115 (2023) 109656 GB114061), p53 (1:500, Proteintech, 10442–1-AP), Bax (1:500, Abcam, ab216494), Bcl-2 (1:500, Abcam, ab196495), and Cleaved-caspase3 (1:500, Servicebio, GB11532).

Techniques: Knock-Out, Immunofluorescence, Expressing, Western Blot

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: 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. . (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 DRAQ5 TM . (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 DRAQ5 TM . 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 vector .

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: 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. . ( 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 vector .

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: 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 DRAQ5 TM . (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 vector .

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: 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 vector .

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: 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. DRAQ5 TM 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 vector .

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: 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 vector .

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: 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 vector .

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: 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 vector .

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

Journal: Journal of the American Society of Nephrology

Article Title: TRPC6 Binds to and Activates Calpain, Independent of Its Channel Activity, and Regulates Podocyte Cytoskeleton, Cell Adhesion, and Motility

doi: 10.1681/asn.2018070729

Figure Lengend Snippet: Figure 1. A GFP tagged TRPC6 construct is functional when expressed in a TRPC6 KO podocyte cell line. (A) Podocyte markers CD2- associated protein (CD2AP), synaptopodin, Wills tumor protein 1 (WT1), podocin, and nephrin are expressed in TRPC6 knockout (T6K) cells. TRPC6 is not expressed. (B) A TRPC6 construct with an extracellular green fluorescent protein (GFP) tag at amino acid 561 was generated and reintroduced to the T6K cells. This is demonstrated by western blotting and immunofluorescence (magenta, actin; GFP, green; DAPI, blue). Disease-causing and DN forms of the GFP-tagged TRPC6 construct were also generated and introduced to T6K cells. (C and D) Biotinylation and TIRF microscopy demonstrating that WT TRPC6-GFP was able to traffic to the plasma membrane. CD99 is a membrane protein and was used as a control in both experiments. (E) Patch clamp analysis of channel function. Pooled data of change in Ihold caused by 6-minute 1 mM AngII perfusion. AngII perfusion causes a rapid change in Ihold in WT (236.2610.5 pA at 8-minute timepoint, open sym- bols, n=5) but not the null mutant (8.763.4 pA at 8-minute timepoint, closed symbols, n=4) or T6K (23.564.3 pA at 8-minute timepoint, triangle symbols, n=4) cells. Gray vertical bar represents perfusion of AngII. All symbols represent the mean6SEM. (F) Summary box plot (boxes, 25th–75th percentile; lines, median) showing changes in Ihold caused by 1 mM AngII perfusion at the 8-minute timepoint in (E). *P,0.05, unpaired t test. Con, control.

Article Snippet: Briefly, cells were Antibody Supplier and Catalog Number TRPC6 Cell Signaling Technology #16716 Caldesmon-1 Cell Signaling Technology #2980 Calpain 1 Cell Signaling Technology #2556 Calpain 2 Cell Signaling Technology #2539S Calpain 1 Abcam #ab28258 Talin-1 Cell Signaling Technology #4021 Phospho-p44/p42 (ERK1/2) Cell Signaling Technology #4370S FAK Cell Signaling Technology #3285S Phospho-FAK (Tyr397) Cell Signaling Technology #8556S Synaptopodin Santa Cruz #sc-515842 WT1 Cell Signaling technology #13580 Podocin Abcam #ab50339 CD2AP Cell Signaling #5478 Nephrin Acris #BP5030 GFP Sigma #11814460001 CD99 Kind gift from Professor George Banting University of Bristol.

Techniques: Construct, Functional Assay, Knock-Out, Generated, Western Blot, Microscopy, Clinical Proteomics, Membrane, Control, Patch Clamp, Mutagenesis

Journal: Nature communications

Article Title: 3D organoid-derived human glomeruli for personalised podocyte disease modelling and drug screening.

doi: 10.1038/s41467-018-07594-z

Figure Lengend Snippet: Fig. 6 Organoid Glomeruli model of congenital nephrotic syndrome in vitro. a Description of the NPHS1 variants identified in the patient modelled, diagnosed with congenital nephrotic syndrome (CNS). b–d Immunostaining of OrgGloms isolated from control organoids and CNS patient organoids show reduced NEPHRIN and PODOCIN protein levels in the organoids derived from patient-iPSC, representative images shown of >3 biological replicates. Scale bars 10 µm. e Higher power immunofluorescent images show the polarised co-localisation of NEPHRIN with NEPH1 (solid white arrowheads) and PODOCIN in control OrgGloms. This polarisation is lost in CNS OrgGloms due to the absence of NEPHRIN (white arrows). Scale bars 10 µm. f Quantitative analysis of fluorescence intensities from independent OrgGlom biological replicates performed using one control and two distinct patient-derived CNS iPSC clones. Organoid glomeruli generated from both patient-derived iPSC clones show significant reduction in NEPHRIN and PODOCIN protein levels. Two-way ANOVA p < 0.0001; error bars = SEM. Biological replicates. NEPHRIN (controls, n = 20; CNS, n = 56); PODOCIN (controls, n = 14; CNS, n = 22); CD2AP (controls, n = 8; CNS, n = 15); NEPH1 (controls, n = 10; CNS, n = 17). Significant difference assessed by Sidak’s multiple comparisons test between cell lines; F-value = 112; DF = 1. NEPHRIN: control vs CNS#1, p < 0.0001; control vs CNS#2, p < 0.0001; CNS#1 vs CNS#2, p > 0.9999. PODOCIN: control vs CNS#1, p < 0.0001; control vs CNS#2, p < 0.0001; CNS#1 vs CNS#2, p = 0.9995. CD2AP: control vs CNS#1, p = 0.0007; control vs CNS#2, p = 0.0016; CNS#1 vs CNS#2, p = 0.9980. NEPH1: control vs CNS#1, p = 0.5320; control vs CNS#2, p = 0.9994; CNS#1 vs CNS#2, p = 0.9992. g Quantitative western blot analysis of NEPHRIN and PODOCIN protein levels within independent biological replicates confirms the significant depletion of these proteins in OrgGloms derived from CNS iPSCs

Article Snippet: The following primary antibodies were used for immunofluorescence at a concentration of 1:200: CD2AP (sc-9137; Santa Cruz), NEPHRIN (AF4269; R&D Systems), PODOCIN (P0372; Sigma Aldrich), SYNAPTOPODIN (sc-21537; Santa Cruz), PODOCALYXIN (39-3800; Thermo Fisher), WT1 (m3561; Dako), PCADHERIN (sc-7893; Santa Cruz), GLUT4 (MAB1262; R&D systems), FcRN (sc46328; Santa Cruz), NEPH1 (HPA030458; Atlas Antibodies), Caspase-3 (9661; Cell Signalling), E-CADHERIN (610181; BD Biosciences), LTL (B-1325; Vector Laboratories), LAMA5 (ab77175; Abcam).

Techniques: In Vitro, Immunostaining, Isolation, Control, Derivative Assay, Clone Assay, Generated, Western Blot

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Tangzhiqing Granules Alleviate Podocyte Epithelial-Mesenchymal Transition in Kidney of Diabetic Rats

doi: 10.1155/2017/1479136

Figure Lengend Snippet: Immunohistochemical staining for nephrin, CD2AP, desmin, and α -SMA accumulation in renal cortical tissues of the five groups. (a) Immunohistochemical staining for nephrin, CD2AP, desmin, and α -SMA; (b) mean density for nephrin; (c) mean density for CD2AP; (d) mean density for desmin; (e) mean density for α -SMA. The data were expressed as mean ± SD; ∗ P < 0.05 versus control group; # P < 0.05 compared to DM group.

Article Snippet: CD2 associated protein antibody (CD2AP) was purchased from Signalway Antibody LLC (MD, USA).

Techniques: Immunohistochemical staining, Staining, Control

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Tangzhiqing Granules Alleviate Podocyte Epithelial-Mesenchymal Transition in Kidney of Diabetic Rats

doi: 10.1155/2017/1479136

Figure Lengend Snippet: Effect of Tangzhiqing granules on expression of nephrin, CD2AP, desmin, and α -SMA protein and mRNA in renal cortex tissues in five groups. Nephrin, CD2AP, desmin, and α -SMA protein expression levels were analyzed by western blotting (a). Relative protein expression levels of nephrin, CD2AP, desmin, and α -SMA were shown in (b–e). Data were presented as mean ± SD and normalized to β -actin protein expression. The mRNA expression levels of nephrin, CD2AP, desmin, and α -SMA were assessed by real-time PCR (f–i). The data were expressed as mean ± SD and normalized to β -actin mRNA expression. ∗ P < 0.05 versus control group; # P < 0.05 compared to DM group; ▲ P < 0.05 compared to TZQ2 group.

Article Snippet: CD2 associated protein antibody (CD2AP) was purchased from Signalway Antibody LLC (MD, USA).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Control