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Ab3421

Manufactured by Abcam
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Sourced in Israel, United States, United Kingdom

Ab3421 is a rabbit polyclonal antibody that recognizes human Myc proto-oncogene protein. It can be used for the detection of Myc protein in various applications, such as Western blotting, immunohistochemistry, and immunocytochemistry.

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Lab products found in correlation

Sab4200181, by Merck Group (2 mentions) α tubulin, by Merck Group (1 mentions) Mab348, by Merck Group (1 mentions) T5168, by Merck Group (1 mentions) Sc 11184, by Santa Cruz Biotechnology (1 mentions) C0979, by Merck Group (1 mentions) Precision cover glasses, by Marienfeld (1 mentions) Ab125689, by Abcam (1 mentions) Ab16771, by Abcam (1 mentions) Vectashield without dapi, by Vector Laboratories (1 mentions) 20r pp004, by Biosynth (1 mentions) Cryostat, by Leica (1 mentions) Ab1530, by Abcam (1 mentions) Z0334, by Agilent Technologies (1 mentions) Ab104224, by Abcam (1 mentions) 4 6 diamidine 2 phenylindole dihydrochloride dapi, by Merck Group (1 mentions) D9542, by Merck Group (1 mentions) A1rs confocal microscope, by Nikon (1 mentions) Alexa fluor 488, by Thermo Fisher Scientific (1 mentions) A21422, by Thermo Fisher Scientific (1 mentions)

12 protocols using ab3421

1

Immunostaining of Neuronal Markers

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4.1G (1:100; kindly provided by E. Peles) AnkG (1:100; kindly provided by M. Rasband), APP (1:1000; MAB348, Merck Millipore, Massachusetts, USA), ATG5 (1:100; pab50264 Covalab, France), CASPR (mk, m; 1:1000; clone K65/35 NeuroMab, California, USA), CASPR2 (1:500; kindly provided by E. Peles), Catalase (1:200; C0979 Sigma-Aldrich, Missouri, USA), CD3 (1:150; MCA1477 Serotec, MorphoSys, Germany), EEA1 (1:300; ab2900 Abcam, Cambridge, United Kingdom), Kv1.1 (1:50; sc11184 Santa Cruz Biotechnology, Texas, USA), Kv1.1 (1:50; clone 20/78 NeuroMab), Kv7.2 (1:2000; PA1-929 ThermoSicentific), LAMP1 (WB, 1:400; IF, 1:200; IEM, 1:200; 553792 BD Bioscience, New Jersey, USA), LIMP-2 (WB, 1:250; IF, 1:2000; kindly provided by J. Blanz), MAC-3 (1:400; 553322 BD Bioscience), Nav1.6 (1:500; ASC-009 Alomone labs, Israel), NF155 (1:1000; kindly provided by P. Brophy), P0 (1:1000 [Archelos et al., 1993 (link)]); P2 (1:500; sc-49304 Santa Cruz), PLP (1:5000 [Jung et al., 1996 (link)]), PMP22 (1:1000; SAB4502217 Sigma), PMP70 (1:600; ab3421 Abcam), RAB7 (1:300; R4779 Sigma), TAG-1 (1:500; kindly provided by E. Peles), ßiv spectrin (1:400; kindly provided by M. Rasband), α-Tubulin (1:1000; T 5168 Sigma), III β-Tubulin (1:1000; Covance, New Jersey, USA).
Kleinecke S., Richert S., de Hoz L., Brügger B., Kungl T., Asadollahi E., Quintes S., Blanz J., McGonigal R., Naseri K., Sereda M.W., Sachsenheimer T., Lüchtenborg C., Möbius W., Willison H., Baes M., Nave K.A, & Kassmann C.M. (2017). Peroxisomal dysfunctions cause lysosomal storage and axonal Kv1 channel redistribution in peripheral neuropathy. eLife, 6, e23332.
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2

Immunofluorescence Imaging of Lipid Droplets and Peroxisomes

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Cells were plated on coverslips (Marienfeld precision cover glasses 1.5 H) fixed in 4% paraformaldehyde for 10 min, and permeabilized with 0.5% TritonX-100 in PBS for 5 min. In the case of digitonin treatment to decrease cytosolic staining, cells were treated with PBS containing 0.01% digitonin for 5 min on ice, followed by fixation with 4% paraformaldehyde, as described previously45 (link). Fixed cells were incubated with primary antibodies against PLIN1 (20R-pp004; Fitzgerald Industries, 1:300), ATGL (2138S; Cell Signaling, 1:300), PMP70 (ab3421; Abcam, 1:300 (all fluorescence images of immunostained PMP70 except for Supplemetary Fig. 4d), SAB4200181; Sigma, 1:300 (fluorescence images of immunostained PMP70 for Supplementary Fig. 4d), PEX5 (ab125689; Abcam, 1:300 (fluorescence images of immunostained PMP70 for Fig. 6a), sc-23188; Santacruz, 1:200 (fluorescence images of immunostained PMP70 for Supplementary Fig. 4d)), Catalase (ab16771, Abcam, 1:300) overnight, washed three times for 5 min each, incubated in secondary antibody for 1–2 h, washed three times for 5 min each, and mounted on glass slides with mounting medium (Vectashield without DAPI; Vector Laboratories). Cells were observed and imaged using an LSM 700 confocal microscope, and an OMX SIM microscope.
Kong J., Ji Y., Jeon Y.G., Han J.S., Han K.H., Lee J.H., Lee G., Jang H., Choe S.S., Baes M, & Kim J.B. (2020). Spatiotemporal contact between peroxisomes and lipid droplets regulates fasting-induced lipolysis via PEX5. Nature Communications, 11, 578.
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3

Immunostaining of Dorsal Root Ganglia

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Sections of DRG tissue (14 μm) were cut at −23 °C using cryostat (Leica). Sections were stained with anti-ABCD1 (Abcam, ab197013, 1:200), anti-GFAP (Dako, Z0334, 1:500), anti-Neurofilament (SMI32) (Biolegend, 801,701, 1:1000), peripherin (Abcam, ab1530, 1:500, Cambridge, UK), CGRP (Abcam, ab42072, 1:1000), C-caspase3 (Cell signaling, 9661, 1:400), and anti-NeuN (Abcam, ab104224, 1:1000) antibodies separately or in combination. We used an anti-ABCD3 antibody to visualize peroxisomes (Abcam, ab3421, 1:1000). Isolectin GS-IB4 Alexa Fluor™ 488 Conjugate from Griffonia simplicifolia (Invitrogen) was prepared at 1mg/mL stock solution and applied to slides at 1:500 dilution when needed. The slides were imaged by confocal laser microscope. Fluorescence intensity was quantified by Image J (Wayne Rasband, NIH).
Gong Y., Laheji F., Berenson A., Qian A., Park S.O., Kok R., Selig M., Hahn R., Sadjadi R., Kemp S, & Eichler F. (2022). Peroxisome Metabolism Contributes to PIEZO2-Mediated Mechanical Allodynia. Cells, 11(11), 1842.
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4

Peroxisome Localization and Quantification

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Tissues were fixed in 4% paraformaldehyde overnight at 4 °C, processed for cryoprotection, and embedded in Tissue-Tek O.C.T. Compound. Sections were cut at 8-micron thickness on a cryostat. We labeled peroxisomes with rabbit anti-PEX14 (#10594-1-AP, Proteintech, 1:200 dilution) or anti-PMP70 (#ab3421, Abcam, 1:200 dilution) and stained nuclei with 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI) (#D9542, Sigma Aldrich, 1:1000 dilution). Secondary donkey anti-rabbit IgG antibodies either conjugated with an Alexafluor 488 (#A21206, Invitrogen) or 568 tag (#A10042, Invitrogen) were used for this study. The sections were imaged with a Nikon A1RS confocal microscope at the University of Wisconsin-Madison Optical Imaging Core. For each sample in this study, we collected seven images and analyzed these images using the ‘Analyze Particles’ application in NIH’s ImageJ program as detailed in Darwisch et al.36 (link).
Landowski M., Bhute V.J., Grindel S., Haugstad Z., Gyening Y.K., Tytanic M., Brush R.S., Moyer L.J., Nelson D.W., Davis C.R., Yen C.L., Ikeda S., Agbaga M.P, & Ikeda A. (2023). Transmembrane protein 135 regulates lipid homeostasis through its role in peroxisomal DHA metabolism. Communications Biology, 6, 8.
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5

Constructing and Characterizing Hsd17b4 Domains

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All plasmids in this study were constructed through polymerase chain reaction (PCR)-base strategy and sequenced to confirm their identity. Full cDNA sequence of mouse Hsd17b4 was purchased from Darmacon (USA) (MMM4769-202765308). Full sequence Hsd17b4 was constructed in the pEBB-Flag and pEBB-GFP vector (Min et al., 2020 (link)). The untagged protein expressing construct was generated as well. In order to identify PS binding region of Hsd17b4, the three domains of Hsd17b4 were constructed in the pEBG-GST vector. GST-Hsd17b41-305, GST-Hsd17b4321-621, and GST-Hsd17b4633-730 contain the hydroxyacyl-CoA dehydrogenase domain, Enoyl-CoA hydratase 2 domain, and the SCP2-like domain of Hsd17b4. HA-Tim-4IgV was previously reported (Lee et al., 2019 (link)).
The antibodies used in this study were anti-Hsd17b4 (15116-1-AP [Proteintech, USA] and NBP2-46005 [Novus, USA]), anti-GST (SC-138; Santa Cruz Biotechnology, USA), anti-Catalase (ab209211; Abcam, UK), anti-Actin (SC-47778; Santa Cruz Biotechnology), anti-Pex5 (GTX109798; GeneTex, USA), anti-Pmp70 (ab3421; Abcam), anti-HA (sc-7392; Santa Cruz Biotechnology), Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488, and Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (A11008 and A21422; Thermo Fisher Scientific, USA).
Lee S.A., Lee J., Kim K., Moon H., Min C., Moon B., Kim D., Yang S., Park H., Lee G., Park R, & Park D. (2021). The Peroxisomal Localization of Hsd17b4 Is Regulated by Its Interaction with Phosphatidylserine. Molecules and Cells, 44(4), 214-222.
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6

Antibody Panel for Peroxisomal Protein Detection

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Rabbit FLAG antibody (#14793, Cell Signaling Technology), mouse CAT antibody (YIF-LF-MA0003, Biomol International), mouse GAPDH antibody (AM4300, Invitrogen), rabbit PGD antibody (#13389, Cell Signaling Technology), rabbit TKT antibody (#8616, Cell Signaling Technology), rabbit NRF2 antibody (#12721, Cell Signaling Technology), rabbit CAT antibody (ab16731, Abcam), rabbit PMP70 antibody (ab3421, Abcam), rabbit PEX12 antibody (ab103456, Abcam), rabbit PGLS antibody (ab127560, Abcam), rabbit β-actin antibody (ab8227, Abcam), rabbit IREB2 antibody (ab181153, Abcam), mouse PMP70 antibodies (SAB4200181, Sigma). Rabbit antibodies to PEX5 (Otera et al., 2000 (link)), PEX13 (Mukai and Fujiki, 2006 (link)), PEX14 (Shimizu et al., 1999 ), ACOX1 (Tsukamoto et al., 1990 (link)), LONP2 (Okumoto et al., 2011 ) were used as described in corresponding references.
Dubreuil M.M., Morgens D.W., Okumoto K., Honsho M., Contrepois K., Lee-McMullen B., Traber G.M., Sood R.S., Dixon S.J., Snyder M.P., Fujiki Y, & Bassik M.C. (2020). Systematic Identification of Regulators of Oxidative Stress Reveals Non-canonical Roles for Peroxisomal Import and the Pentose Phosphate Pathway. Cell reports, 30(5), 1417-1433.e7.
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7

Western Blot Analysis of Protein Expression

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RIPA buffer (cat. no. R0010; Solarbio) was used to lyse cells, and protein concentrations were determined with a BCA protein assay kit (Beijing Leagene Biotech Co., Ltd.). Protein samples (25 µg/lane) were separated by SDS-PAGE on 10% gels and transferred onto PVDF membranes. The membranes were blocked by protein-free rapid block buffer (Epizyme Pharmaceutical Biotechnology Co, LTD) for 15 min at room temperature and were incubated overnight at 4˚C with primary antibodies against AGPS (1:500, sc-374201, Santacruze), MDM2 (1:500,sc-965, Santacruze), PMP70(1: 1000, ab3421, Abcam), Flag (1: 1000, 201,126-3A6, ZENBIO), c-Myc (1: 1000, sc-40, Santacruze), TrkA (1:1000,2510,CST), p-TrkA (1:1000,PA5-104,674, ThermoFisher), and β-tubulin (1:1000, ABL1030,Abbkine). Following primary antibody incubation, the membranes were further incubated with Dylight 800-Goat Anti-Rabbit IgG (1:100, A23910 Abbkine) or Dylight 800-Goat Anti-Mouse IgG secondary antibodies (1:100, A23920 Abbkine) at 25˚C for 1 h. The membranes were then scanned by an imaging system (ODYSSEY ® CLx, Gene Company limited), and the optical density was measured using Image Studio Lite (LI-COR Biosciences).
Zhang Y., Huang Z., Li K., Xie G., Feng Y., Wang Z., Li N., Liu R., Ding Y., Wang J., Yang J, & Jia Z. (2024). TrkA promotes MDM2-mediated AGPS ubiquitination and degradation to trigger prostate cancer progression. Journal of Experimental & Clinical Cancer Research : CR, 43, 16.
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8

Immunofluorescence staining of cell markers

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After PFA fixation, cells were permeabilized with 0.1% Triton-X-100 in PBS during 5 min. For blocking, cells were incubated with 10% goat serum in PBS for 1h at room temperature. Primary antibodies, diluted in 3% goat serum in PBS, against MB (FL-154, cs-25607, Santa Cruz Biotechnology, 1:500), CD36 (ab133625, Abcam, 1:50), or PMP70 (ab3421, Abcam, 1:500), were incubated with cells at 4°C overnight. Upon PBS washing (3 x 10 min), dye-coupled secondary antibodies, diluted 1:1000 in 3% goat serum in PBS, were incubated with the cells for 45 min at room temperature.
Armbruster J., Aboouf M.A., Gassmann M., Egert A., Schorle H., Hornung V., Schmidt T., Schmid-Burgk J.L., Kristiansen G., Bicker A., Hankeln T., Zhu H, & Gorr T.A. (2022). Myoglobin regulates fatty acid trafficking and lipid metabolism in mammary epithelial cells. PLoS ONE, 17(10), e0275725.
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9

Quantifying Diaphragm Protein Dynamics

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Diaphragm protein extracts were assayed as previously described [29 (link)]. Briefly, diaphragm tissues were homogenized 1:10 (wt/vol) in 5 mM Tris (pH 7.5) and 5 mM EDTA (pH 8.0) with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) and centrifuged at 1500 g for 10 min at 4°C. Supernatant was collected and protein content was assessed by the Bradford method (Sigma-Aldrich). Proteins were separated via polyacrylamide gel electrophoresis. Membranes were probed for Beclin 1 (#3495), Atg12 (#4180), LC3B (#2775) (Cell Signaling Technology, Danvers, MA), Atg7 (NBP2-24682) (Novus Biologicals, Minneapolis, MN), p62 (ab56416), catalase (ab16731), PMP70 (ab3421) (Abcam, Cambridge, UK) and cathepsin L (sc-6498), spectrin (sc-48382). α-tubulin (sc-58667) (Santa Cruz Biotechnology, Dallas, TX) was also probed for as a loading control. In addition, mitochondrial protein extracts were also assayed via western blot. Membranes were probed for 4-HNE (ab46545), Parkin (ab15954), PINK1 (ab23707), Mul1 (ab84067), p62 (ab56416) (Abcam), OPA1 (612606), DLP1 (611112) (BD Biosciences, San Jose, CA), Mfn2 (M6444) (Sigma-Aldrich) and Fis1 (alx-210-907) (Enzo Life Sciences, Farmingdale, NY). VDAC (sc-8829) (Santa Cruz) was also probed as a loading control for all mitochondrial proteins.
Smuder A.J., Sollanek K.J., Nelson W.B., Min K., Talbert E.E., Kavazis A.N., Hudson M.B., Sandri M., Szeto H.H, & Powers S.K. (2017). Crosstalk between autophagy and oxidative stress regulates proteolysis in the diaphragm during mechanical ventilation. Free radical biology & medicine, 115, 179-190.
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10

Colocalization of ABCD3 and Ubiquitin

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To assess the colocalization of ABCD3 and UB, HeLa cells were cultured on a glass cover slip and treated with VH298 for 48 h. The cells were then washed with PBS (LB 001–02; WELGENE, Republic of Korea), fixed with 4% paraformaldehyde (P2031; Biosesang, Korea) for 20 min, and stained with the antibodies (anti-ABCD3 (ab3421; Abcam, Cambridge, UK) and anti-Ubiquitin (sc-8017; Santa Cruz Biotechnology, Dallas, TX, USA)). Fluorescent images of the morphology of peroxisomes and UB were obtained by using a confocal laser scanning microscope (LSM 800; Carl Zeiss). For the analysis of the colocalization between ABCD3 and UB, the Coloc2 plugin in the free software ImageJ Fiji 64-bit (NIH) was utilized. The calculation of double-fluorescence correlation coefficients was performed through the application of Pearson’s correlation coefficient in the Coloc2 plugin as a measure of the association between the two signals (ABCD3-UB). The mean Pearson’s correlation coefficient (R) values for the level of colocalization between ABCD3 and UB are represented graphically.
Kim Y.H., Park N.Y., Jo D.S., Bae J.E., Kim J.B., Park K., Jeong K., Kim P., Yeom E, & Cho D.H. (2024). Inhibition of VHL by VH298 Accelerates Pexophagy by Activation of HIF-1α in HeLa Cells. Molecules, 29(2), 482.
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