Rabbit anti podocin

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Rabbit anti-podocin is a specific antibody produced in rabbits that targets the podocin protein. Podocin is a structural component of the glomerular filtration barrier in the kidney. This antibody can be used to detect and analyze podocin expression in various research applications.

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Podocin (15 citations) Anti-podocin (14 citations) Rabbit anti- (2 citations) Rabbit anti-podocin primary antibody (2 citations) Rabbit anti-podocin antibody (2 citations)

10 protocols using rabbit anti podocin

Immunoblot Analysis of Podocyte Signaling

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Commercial antibodies used were as follows: rabbit anti-Akt (C67E7, 4691, Cell Signaling Technology, Danvers, MA), rabbit anti-pAkt S473 (D9E, 4060, Cell Signaling Technology), rabbit anti-Akt1 (C73H10, 2938, Cell Signaling Technology), rabbit anti-Akt2 (5B5, 2964, Cell Signaling Technology), rabbit anti-Akt3 (4059, Cell Signaling Technology), rabbit anti-ERK1/2 (137F5, 4695, Cell Signaling Technology), rabbit anti-pERK1/2 T202/Y204 (D13.14.4E, 4370, Cell Signaling Technology), rabbit anti-p38 (9212, Cell Signaling Technology), rabbit anti-p-p38 T180/Y182 (D3F9, 4511, Cell Signaling Technology), rabbit anti-podocin (PO372, Sigma), mouse anti-β-actin (AC15, A1978, Sigma), rabbit anti-cadherin (ab16505, Abcam, Cambridge, UK), and rabbit anti-ERβ (H-150, SC-8974, Santa Cruz). Rabbit anti-nephrin was described previously.^{44 (link)} Phospho-specific anti-nephrin antibodies were generated and validated previously.^{45 (link)} All primary antibodies were used at 1:1000 except for podocin and β-actin, which were used at 1:5000. Secondary horseradish peroxidase-conjugated goat anti-mouse (170-6516, BioRad, Hercules, CA) and goat anti-rabbit (170-6515, BioRad) antibodies were used at 1:5000 for immunoblot detection.

Mahesaniya A., Williamson C.R., Keyvani Chahi A., Martin C.E., Mitro A.E., Lu P., New L.A., Watson K.L., Moorehead R.A, & Jones N. (2022). Sex Differences in Glomerular Protein Expression and Effects of Soy-Based Diet on Podocyte Signaling. Canadian Journal of Kidney Health and Disease, 9, 20543581221121636.

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Protein Expression Analysis in Podocytes

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Total proteins extracts from control and Ang-II and/or inhibitors treated podocytes were obtained using ice-cold RIPA buffer (BioRad, Sao Paulo, Brazil) with protease and phosphatase inhibitors (Sigma Aldrich St. Louis, MO, USA). Immunoblot analysis was performed on aliquots containing 30 μg/lane of proteins resolved in 10% SDS-PAGE as previously described [22 (link), 27 (link)]. The following primary antibodies were used in this study: rabbit anti-AT1 receptor (1:1000, Merck Millipore); rabbit anti-chaperone GRP 78 (1:8000, StressMarq, Victoria, BC, CA); rabbit anti-phospho-eIf2-α (eukaryotic initiation factor 2, an ER stress marker, 1:1000, Cell Signaling, Denver, MA, USA); rabbit anti-phospho-PKC-δ (1:3000, Abcam, Cambridge, MA, USA); rabbit anti-p38MAPK and anti-phospho-p38MAPK (1:1000, Cell Signaling); rabbit anti-Bcl-2 (1:1000, Cell Signaling); rabbit anti-Bax (1:4000, Santa Cruz); mouse anti-NHE1 (1:2000, Abcam); rabbit anti-podocin (1:1000, Sigma Aldrich); rabbit anti-GAPDH (1:2000, Cell Signaling); mouse anti-β-actin (1:5000, Abcam); and horseradish peroxidase-conjugated goat secondary antibodies against rabbit and mouse (Jackson ImmunoResearch Laboratories, Baltimore, MD, USA). Protein expressions are shown as the ratio to the endogenous control, namely GAPDH or β- actin.

Cardoso V.G., Gonçalves G.L., Costa-Pessoa J.M., Thieme K., Lins B.B., Casare F.A., de Ponte M.C., Camara N.O, & Oliveira-Souza M. (2018). Angiotensin II-induced podocyte apoptosis is mediated by endoplasmic reticulum stress/PKC-δ/p38 MAPK pathway activation and trough increased Na+/H+ exchanger isoform 1 activity. BMC Nephrology, 19, 179.

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Western Blotting for Protein Expression

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Western blotting was performed using established methodology [6 (link),28 (link)]. Briefly, cells were washed with PBS and lyzed in RIPA buffer. Proteins (20–30 μg) were separated by SDS/PAGE (12% gel) and then transferred on to an immunoblot PVDF membrane (Bio-Rad, Hercules, CA). After blocking in PBS/Tween (0.1%) with 5% non-fat milk, the membrane was incubated with primary antibodies overnight at 4°C followed by horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, 1:3000) and then developed using ECL solution (Pierce). Primary antibodies used were mouse anti-APOL1 (Proteintech, 66124-1-lg, 1:1000), rabbit anti-nephrin (Abcam, ab58968, 1:1000), rabbit anti-podocin (Sigma, P0372, 1:2000), rabbit anti-GRP78 binding Ig protein (BiP) (Abcam, ab21685, 1:1000), rabbit anti-p-eIF2α (Cell Signaling Technology, #9721, 1:1000), and mouse anti-actin (Santa Cruz Biotechnology, sc-8432, 1:3000). For protein expression quantitation, the films were scanned with a CanonScan 9950F scanner and the acquired images were then analyzed using the public domain NIH image program (http://rsb.info.nih.gov/nih-image/).

Wen H., Kumar V., Lan X., Shoshtari S.S., Eng J.M., Zhou X., Wang F., Wang H., Skorecki K., Xing G., Wu G., Luo H., Malhotra A, & Singhal P.C. (2018). APOL1 risk variants cause podocytes injury through enhancing endoplasmic reticulum stress. Bioscience Reports, 38(4), BSR20171713.

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Podocyte Stress Signaling Pathways

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Aldosterone was obtained from Sigma Aldrich (St. Louis, MO). FR167653, p38α MAP Kinase inhibitor, was kindly provided by Astellas Pharma Inc. (Tokyo, Japan). Primary antibodies used for immunohistochemical studies and Western blotting were goat anti-nephrin (R&D Systems, Minneapolis, MN), rabbit anti-podocin (Sigma Aldrich), rabbit anti-phospho-p38 MAPK (Cell Signaling Technology, Boston, MA), rabbit total p38 MAPK (Cell Signaling Technology), rabbit anti-WT1 (Santa Cruz, Dallas, TX), and rabbit anti-p53 (Vector Laboratories, Burlingame, CA), rabbit anti-phospho-MKP-1 (Cell Signaling Technology), rabbit anti-phospho-MKK3 (Cell Signaling Technology), and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Santa Cruz) antibodies. pRc/RSV Flag MKK350 (link) and pRc/RSV Flag MKK3 (glu)51 (link) were gifts from Professor Roger Davis (Addgene plasmid #14671 and #14670, respectively).

Kato Y., Mori K., Kasahara M., Osaki K., Ishii A., Mori K.P., Toda N., Ohno S., Kuwabara T., Tokudome T., Kishimoto I., Saleem M.A., Matsusaka T., Nakao K., Mukoyama M., Yanagita M, & Yokoi H. (2017). Natriuretic peptide receptor guanylyl cyclase-A pathway counteracts glomerular injury evoked by aldosterone through p38 mitogen-activated protein kinase inhibition. Scientific Reports, 7, 46624.

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Podocyte Injury Signaling Analysis

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Antibodies used in this study were rabbit anti-WT1 (Santa Cruz Biotechnology, Inc), rabbit anti-GR (Santa Cruz Biotechnology, Inc.), goat anti-nephrin (Santa Cruz Biotechnology, Inc.), and rabbit anti-podocin (Sigma). Anti-phospho FAK (Tyr 397) (EMD Millipore), anti-FAK, clone 77 (Fisher Scientific) and GAPDH (Affinity Bioreagents) were also used. Complete Freund’s adjuvant was purchased from Sigma. Rabbit IgG was purchased from Jackson Immunoresearch Laboratories. Alexa Fluor 594 goat anti-Rabbit IgG Ab and Alexa Fluor 488–conjugated phalloidin were purchased from Invitrogen. LPS serotype O55:B5 was purchased from Calbiochem and dexamethasone phosphate was purchased from MP Biomedicals.

Zhou H., Tian X., Tufro A., Moeckel G., Ishibe S, & Goodwin J. (2017). Loss of the podocyte glucocorticoid receptor exacerbates proteinuria after injury. Scientific Reports, 7, 9833.

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Kidney Histology and Imaging Techniques

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Human kidney biopsy and mouse kidneys were processed for histology, IF and TEM as described.^{31 (link),32 (link)} IF was performed in mouse kidney frozen sections and cultured podocytes. Primary antibodies were mouse anti-MYO9A (clone 4C11, Abnova), rabbit anti-podocin (P0372, Sigma), rabbit anti-laminin (L9393, Sigma), rabbit anti-aquaporin2 (PAS-78809, Invitrogen), rabbit anti-megalin^{33 (link)}; secondary antibodies were Alexa Fluor-488 and 594-conjugated. Images were acquired using Olympus IX71 or Leica-SP5-Spectral confocal microscope.

Li Q., Gulati A., Lemaire M., Nottoli T., Bale A, & Tufro A. (2021). Rho-GTPase Activating Protein myosin MYO9A identified as a novel candidate gene for monogenic focal segmental glomerulosclerosis.. Kidney international, 99(5), 1102-1117.

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Antibody Validation for Nephrin Signaling

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Primary antibodies used for these experiments were as follows: mouse anti-Myc (Cell Signalling, 9B11), mouse anti-phosphotyrosine 4G10 (#16–101; Upstate Biotechnology), rabbit anti-podocin (Sigma Aldrich, P0372) and mouse anti-β-actin (Sigma Aldrich, A1978). Rabbit anti-nephrin antibodies were provided by Dr. Tomoko Takano (McGill University) [10 (link)]. Phospho-specific anti-nephrin antibodies (Y1176/Y1193 and Y1217) were generated and validated previously [15 (link)]. Secondary antibodies (all Invitrogen) included horseradish peroxidase (HRP)-conjugated goat anti-rabbit (#A11008) and HRP-conjugated goat anti-mouse (#A11001), as well as goat anti-mouse Alexa Fluor-conjugated 594 (#A11005).

Cooper C.J., Dutta N.T., Martin C.E., Piscione T.D., Thorner P.S, & Jones N. (2018). Characterization of a novel disease-associated mutation within NPHS1 and its effects on nephrin phosphorylation and signaling. PLoS ONE, 13(9), e0203905.

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Immunofluorescence Staining of Kidney Markers

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The following primary antibodies were used: rat anti-Tjp1 (Santa Cruz Biotech), rabbit anti-Tjp1 (Invitrogen), rabbit anti-Tjp2 (Invitrogen), rabbit anti-Tjp3 (Invitrogen), rabbit anti-WT1 (Santa Cruz Biotech), goat anti-VE-cadherin (Santa Cruz Biotech), rabbit atni-Caludin2 (Invitrogen), guinea pig anti-Nephrin (Progen), rabbit anti-Podocin (Sigma-Aldrich), rabbit anti-Fyn (Sigma-Aldrich), rabbit anti-Synaptopodin (Sigma-Aldrich), rabbit anti-α-actinin-4 (Millipore), rabbit anti-CD2AP (Cell signaling), and rabbit anti-β-actin (Sigma-Aldrich).

Itoh M., Nakadate K., Horibata Y., Matsusaka T., Xu J., Hunziker W, & Sugimoto H. (2014). The Structural and Functional Organization of the Podocyte Filtration Slits Is Regulated by Tjp1/ZO-1. PLoS ONE, 9(9), e106621.

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Immunohistochemical Analysis of Podocyte Markers

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Cultured podocytes planted on cover slides in six-well plates or frozen cryostat sections were fixed with 4% paraformaldehyde at room temperature(RT) for 20 min, permeabilized with 0.1% Triton X-100 for 10 min, then blocked with 5% bovine serum albumin for 30 min at RT before further incubated overnight at 4 °C with the following primary antibodies: goat anti-synaptopodin (Santa Cruz, 1:100), rabbit anti-Flot2 (Cell Signaling Technology, 1:50), Rabbit anti-podocin (Sigma,1:200).Then cultured podocytes or sections were washed three times with PBS for 5 min before the secondary antibodies (FITC-donkey anti-goat IgG 488, Life Technologies, USA, 1:250; goat anti-rabbit Alexa Fluor 555,Cell Signaling Technology, 1:200 were applied for 1 hour at RT in the dark. Culture podocytes were stained with phalloidin (Cytoskeleton,1:500) and Cholera toxin subunit B (CTXB, Life Technologies, USA, 1:500) for 30 min and then with DAPI(Sigma, St. Louis, MO, USA) for 5 min at RT. All images were taken using laser confocal microscopy (LCSM, Zeiss KS 400, Postfach, Germany).

Yu C., Zhang H., Liu S., Li R., Zhao X., Chen Y., Li Z., Ma J., Wang W., Ye Z., Liang X., Zhang L, & Shi W. (2023). Flot2 acts as a novel mediator of podocyte injury in proteinuric kidney disease. International Journal of Biological Sciences, 19(2), 502-520.

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Proteomic Analysis of Cultured Podocytes

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Cultured podocytes under different experimental conditions were conducted as previously described 27 (link),29 (link). Twelve protein fractions collected from the sucrose density gradient centrifugation mentioned above were stored at -20 °C and then analyzed by western blotting. The protein concentration was evaluated using a BCA protein assay reagent kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA). Equal amount of proteins was separated on 9% sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% nonfat dry milk in TBS-Tween for 1h at RT followed by overnight at 4°C incubation with the following primary antibodies: rabbit polyclonal anti-Flot2 (1:1000, Cell Signaling Technology, USA), rabbit anti-GAPDH (1:5000, Bioworld Technology,Nanjing, China), rabbit anti-nephrin (Abcam), rabbit anti-Podocin (Sigma), anti-CD71(TfR, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA). After washing in TBS-Tween (3 times for 10 min), horseradish peroxidase conjugated goat anti-rabbit or goat anti-mouse IgG at a concentration of 1:3000 was applied for 1h at RT. Bands were visualized using enhanced chemiluminescence (ECL) reagent (Advansta, Menio Park, CA, USA).

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