- 38-1000 - Invitrogen Antibodies UCHL1 antibody

Submitted references Chemical phenotypes of intrinsic cardiac neurons in the newborn pig (Sus scrofa domesticus Erxleben, 1777).

Ragauskas T, Rysevaite-Kyguoliene K, Pauziene N, Inokaitis H, Pauza DH
Journal of morphology 2022 Jan;283(1):51-65

Sensory neuron dysfunction in orthotopic mouse models of colon cancer.

Balogh M, Zhang J, Gaffney CM, Kalakuntla N, Nguyen NT, Trinh RT, Aguilar C, Pham HV, Milutinovic B, Nichols JM, Mahalingam R, Shepherd AJ
Journal of neuroinflammation 2022 Aug 12;19(1):204

Inhibition of UCH-L1 Deubiquitinating Activity with Two Forms of LDN-57444 Has Anti-Invasive Effects in Metastatic Carcinoma Cells.

Kobayashi E, Hwang D, Bheda-Malge A, Whitehurst CB, Kabanov AV, Kondo S, Aga M, Yoshizaki T, Pagano JS, Sokolsky M, Shakelford J
International journal of molecular sciences 2019 Jul 31;20(15)

Bladder decompensation and reduction in nerve density in a rat model of chronic bladder outlet obstruction are attenuated with the NLRP3 inhibitor glyburide.

Hughes FM Jr, Sexton SJ, Ledig PD, Yun CE, Jin H, Purves JT
American journal of physiology. Renal physiology 2019 Jan 1;316(1):F113-F120

NLRP3 Promotes Diabetic Bladder Dysfunction and Changes in Symptom-Specific Bladder Innervation.

Hughes FM Jr, Hirshman NA, Inouye BM, Jin H, Stanton EW, Yun CE, Davis LG, Routh JC, Purves JT
Diabetes 2019 Feb;68(2):430-440

NLRP3/IL-1β mediates denervation during bladder outlet obstruction in rats.

Lütolf R, Hughes FM Jr, Inouye BM, Jin H, McMains JC, Pak ES, Hannan JL, Purves JT
Neurourology and urodynamics 2018 Mar;37(3):952-959

Sympathetic Neuronal Activation Triggers Myeloid Progenitor Proliferation and Differentiation.

Vasamsetti SB, Florentin J, Coppin E, Stiekema LCA, Zheng KH, Nisar MU, Sembrat J, Levinthal DJ, Rojas M, Stroes ESG, Kim K, Dutta P
Immunity 2018 Jul 17;49(1):93-106.e7

C-Terminal Farnesylation of UCH-L1 Plays a Role in Transport of Epstein-Barr Virus Primary Oncoprotein LMP1 to Exosomes.

Kobayashi E, Aga M, Kondo S, Whitehurst C, Yoshizaki T, Pagano JS, Shackelford J
mSphere 2018 Jan-Feb;3(1)

Specific deletion of Cdh2 in Sertoli cells leads to altered meiotic progression and subfertility of mice.

Jiang X, Ma T, Zhang Y, Zhang H, Yin S, Zheng W, Wang L, Wang Z, Khan M, Sheikh SW, Bukhari I, Iqbal F, Cooke HJ, Shi Q
Biology of reproduction 2015 Mar;92(3):79

Identification of a novel developmental mechanism in the generation of mesothelia.

Winters NI, Thomason RT, Bader DM
Development (Cambridge, England) 2012 Aug;139(16):2926-34

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of (A) mouse brain, (B) rat brain homogenates, (C) NGP, (D) A549, and (E) NCI-H69 cell lysates using Rb anti-UCH-L1 (C-term) (Product # 38-1000).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of (A) mouse brain, (B) rat brain homogenates, (C) NGP, (D) A549, and (E) NCI-H69 cell lysates using Rb anti-UCH-L1 (C-term) (Product # 38-1000).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on membrane enriched extracts (30 µg lysate) of Neuro-2a (Lane 1), SH-SY5Y (Lane 2), U-87 MG (Lane 3), PC-12 (Lane 4), IMR-32 (Lane 5), A549 (Lane 6), DU 145 (Lane 7), tissue extracts of Mouse Brain (Lane 8) and Rat Brain (Lane 9). The blot was probed with Anti-PGP9.5 Rabbit Polyclonal Antibody (Product # 38-1000, 2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 24 kDa band corresponding to PGP9.5 was observed across cell lines and tissues tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody using iBind Flex Western Starter Kit (Product # SLF2000S). Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of PGP9.5/UCHL1 was performed by loading 20 µg of SH-SY5Y wild type (Lane 1), SH-SY5Y Cas9 control (Lane 2), SH-SY5Y PGP9.5/UCHL1 knockout (Lane 3) whole cell extracts. The blot was probed with Anti-PGP9.5/ UCHL1 Polyclonal Antibody (Product # 38-1000) (1 µg/mL) and Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036) (1:4000 dilution). Loss of signal upon CRISPR mediated knockout (KO) confirms that antibody is specific to PGP9.5/UCHL1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical staining of Alzheimer's disease brain tissue using Rb anti-UCH-L1 (C-term) (Product # 38-1000).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Representative histological sections of bladders stained for PGP9.5 that illustrate the quantitation method. A, Representative staining of Sham and BOO + Veh sections that illustrate both the dramatic increase in size (upper panels) as well as the decrease in neuronal numbers (brown spots in the lower panels, several of which are marked with arrows) of the BOO + Veh bladder compared to Sham. B, Representative quantitation of a Sham bladder. The left panel demonstrates the actual staining (brown). There is strong staining of both neurons and the urothelia. The middle panel demonstrates how the bladder wall was chosen to exclude adjacent non-bladder tissue, the lumen, and the urothelia. The chosen ROI is indicated in red and this is the ROI used to calculate bladder cross-sectional area by the NIS-Elements image analysis software. The right panel illustrates how stained neurons in the ROI were identified by the software and false colored blue. The number of neurons (blue spots) was then calculated by the Elements software

Supportive validation

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Experimental details: Figure 1 Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) de-ubiquitinating activity is involved in regulation of membrane trafficking pathways. ( A ) UCH-L1 is physically associated with the vesicles of the cellular endolysosomal pathway. Electron microscopy of 293 cells immune gold-labeled for UCH-L1 (10 nm gold particles, black dots). Two hundred and ninety-three cells transfected with UCH-L1 expression vector were subjected to ultrastructural examination. Fixed ultra-sections were stained with UCH-L1 primary and colloidal gold-labeled secondary antibodies. Arrows show the intra-cellular compartments of the endo-lysosomal pathway. ( B ) UCH-L1 de-ubiquitinating (DUB) activity is involved in regulation of CD63 secretion in exosomes. Two hundred and ninety-three cells were transfected with WT and C90S UCH-L1 expression vectors. After 48 h of incubation, exosomes were purified by sequential ultracentrifugation as described in Materials and Methods section. Western blot analysis demonstrates that CD63 levels in the exosome fraction were reduced when the cells were transfected with DUB-dead UCH-L1 mutant compared to WT-expressing control. Western blot for CD63 in total lysates of the same cells confirms the results in exosomal fractions showing reverse correlation in CD63 levels between WT- and C90S-expressing cells (normalization to GAPDH). ( C ) Inhibition of UCH-L1 DUB activity with the selective small-molecule inhibitor LDN-57444 decreases the levels pro-metastaticHIF-1alp

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Experimental details: Figure 3 Both forms of UCH-L1 inhibitor, LDN and LDN-POx reduce motility of LMP1-positive nasopharyngeal cells. ( A ) De novo expression of UCH-L1 in LMP1-positive NP cells. Total lysates from LMP1-negative and LMP1-positive cells NP69 cells were separated in 12% polyacrylamide gel electrophoresis (PAGE), and expression of endogenous UCH-L1 protein was determined in by Western blot analysis as described in Materials and Methods. GAPDH levels served as loading control. ( B ) Determination of non-toxic concentrations of LDN and LDN-POx forms of the UCH-L1 inhibitor. NP69 and NP69-LMP1 cells were seeded in culture plates at a density of 2-5 x 10 3 cells/well and either DMSO (control) or indicated concentrations of free LDN-57444, or micellated LDN-POx forms of the LDN-57444 inhibitor were added to each well. 72 h later 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assays were performed as described in Materials and Methods. The results demonstrate that concentrations of the inhibitor less than 5 muM did not affect the viability of either cell line. ( C ) Inhibition of UCH-L1 DUB activity reduces motility of only LMP1-positive NP69 cells, but not those that are LMP1-negative. Sub-confluent LMP1-negative and -positive NP69 cells had been incubated with or without 3 muM LDN or LDN-POx for 48 h. Then the cells were scratched and cultured for additional 24 h. The widths of the 'wound' (scratched areas) were measured by ImageJ soft

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Both LDN and LDN-POx decrease expression of pro-metastatic factors as well as motility of oral squamous carcinoma (OSC) cells. ( A ) Determination of non-toxic concentrations of LDN and LDN-POx forms of the UCH-L1 inhibitor in metastatic human squamous carcinoma cells (HSC). HSC-2, -3 and -4 cells were treated with LDN or micellated LDN-POx forms of the UCH-L1 inhibitor and MTS assays were performed as described in Figure 3 B. The results demonstrate that concentrations of the inhibitor less than 5 muM did not affect the viability of all three cell lines. ( B ) Inhibition of UCH-L1 DUB activity reduces motility of highly metastatic HSC-3 cells. The cells were treated with either LDN or LDN-POx (3 muM each) (DMSO as control). After 48 h incubation a scratch assay was performed as described in Figure 3 C. The results of statistical analysis on the bottom indicate that both LDN and LDN-POx had inhibitory effect on HSC-3 cells motility. ( C ) Inhibition of UCH-L1 DUB activity reduces the expression of pro-metastatic factors in HSC-3 cells. Total lysates from HSC-3 cells treated for 72 h with both, free and micellated forms of UCH-L1 inhibitor (DMSO and PBS served as control respectively) were separated in 4-20 gradient PAGE, and Western blots with indicated antibodies (N-cadherin, beta-catenin and CD44) were performed. Protein levels were normalized on GAPDH and UCH-L1. ( D ) Inhibition of UCH-L1 DUB activity reduces invasive capacity of HSC-3 cells. The cells treated wi

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 8 NLRP3 controls changes in the densities of nerves related to specific DBD symptoms. A : Representative micrographs of PGP9.5 staining (i.e., all neurons) in the bladder used to quantify nerves. The left micrograph depicts the entire transverse cross section stained and scanned into a TIFF file used for quantitation, as described in the Research Design and Methods section. The yellow box indicates the area zoomed in on the right micrograph to allow better visualization. Block arrow points at urothelia that stain nonspecifically for PGP9.5, or at least are of nonneuronal origin. Arrows indicate the brown staining in the bladder wall considered to stain positive for this antigen. B : The number of PGP9.5 + nerves in bladder wall of 15 week mice from nondiabetic (non-diab) and diabetic (diab) mice that express NLRP3 ( NLRP3 +/+ ). C : Same analysis as B in mice that have the NLRP3 gene deleted ( NLRP3 -/- ). D and E : The size of the bladder wall in the same sections and groups quantitated in B and C , respectively. F and G : Density of PGP9.5 + neurons in the same sections and groups quantitated in B and C , respectively. H : Representative micrographs of NF-200 staining (Adelta-fibers) in the bladder used to quantify nerves. The left micrograph depicts the entire transverse cross section, whereas the yellow box indicates the area zoomed in on the right, and arrows point at staining in the bladder wall considered to be positive for this antigen. I

38-1000

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