Citation
Vaildating Sigma One Receptor Antibodies To Investigate The Knockout Phenotype

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Title:
Vaildating Sigma One Receptor Antibodies To Investigate The Knockout Phenotype
Series Title:
19th Annual Undergraduate Research Symposium
Creator:
DiVita, Keeley
Language:
English
Physical Description:
Undetermined

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Subjects / Keywords:
Center for Undergraduate Research
Center for Undergraduate Research
Genre:
Conference papers and proceedings
Poster

Notes

Abstract:
Recent reports have called into question the specificity of multiple antibodies targeting the Sigma-1 receptor (SIG1R), a cellular chaperone protein being studied as a therapeutic target for numerous neurological and non-neurological disorder. Thus, we established a standardized battery to test the specificity of multiple commercial antibodies and two novel antibodies generated in collaboration with encore Biotechnology. To analyze the antibodies using western blotting, we trialed different protocols specifically designed to detect SIG1R in tissue samples. We ultimately were able to reproducibly detect SIG1R in tissue at the expected molecular weight, with no band present in the samples from transgenic SIG1 knockout mice using a single commercially available antibody (Invitrogen). As our next step, we chose to verify proper subcellular localization of SIG1R in cultured dopamine neurons using this antibody. Consistent with the published literature on SIG1R, the staining revealed a distribution of staining that was peri-nuclear and formed puncta near the plasma membrane. Immunolabeling of cultured dopamine neurons with other available antibodies failed to reproduce this localization pattern. Future directions include verification of the Invitrogen antibody’s specificity in SIG1R knockout cultured dopamine neurons, and examination of SIG1R labeling in tissue of both wild-type and knockout mice. ( en )
General Note:
Research authors: Keeley DiVita, Joe Lebowitz, Danielle Sambo Ph.D., Habibeh Khoshbouei Ph. D. - University of Florida
General Note:
University Scholars Program
General Note:
Faculty Mentor: Recent reports have called into question the specificity of multiple antibodies targeting the Sigma-1 receptor (SIG1R), a cellular chaperone protein being studied as a therapeutic target for numerous neurological and non-neurological disorder. Thus, we established a standardized battery to test the specificity of multiple commercial antibodies and two novel antibodies generated in collaboration with encore Biotechnology. To analyze the antibodies using western blotting, we trialed different protocols specifically designed to detect SIG1R in tissue samples. We ultimately were able to reproducibly detect SIG1R in tissue at the expected molecular weight, with no band present in the samples from transgenic SIG1 knockout mice using a single commercially available antibody (Invitrogen). As our next step, we chose to verify proper subcellular localization of SIG1R in cultured dopamine neurons using this antibody. Consistent with the published literature on SIG1R, the staining revealed a distribution of staining that was peri-nuclear and formed puncta near the plasma membrane. Immunolabeling of cultured dopamine neurons with other available antibodies failed to reproduce this localization pattern. Future directions include verification of the Invitrogen antibody’s specificity in SIG1R knockout cultured dopamine neurons, and examination of SIG1R labeling in tissue of both wild-type and knockout mice. - Center for Undergraduate Research, University Scholars Program

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University of Florida
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Copyright Keeley DiVita. Permission granted to University of Florida to digitize and display this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.

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Keeley DiVita, 1 Joe Lebowitz, 1 Danielle Sambo, 1 Habibeh Khoshbouei 1 College of Medicine, 1 Department of Neuroscience, University of Florida, Gainesville, FL Future Directions Verification KO cultured dopamine neurons Utilization of the KO specific labeling protocol to examine SIG1 R expression in models of Neurodegenerative disease Conclusions Generation of a novel antibody targeted against SIG1 R showed intense nuclear staining, but recapitulated nonspecific staining seen in all commercially available antibodies. Of all commercial antibodies tested, only two (Santa Cruz B 5 & Invitrogen 42 3300) were consistent with labeling for YFP using tagged SIG1 R. The Invitrogen, but not Santa Cruz, antibody yielded labeling in cultured neurons that matched the expected localization pattern of SIG1 R Denaturing samples at 95 deg and transferring to a nitrocellulose membrane was able to show KO specificity using the rb SIG R antibody Background The Sigma 1 receptor (SIG 1R) is a small chaperon protein that resides in the membrane of the endoplasmic reticulum (ER ) and is highly expressed in dopamine neurons. SIG 1R has been implicated in numerous neurodegenerative disease, including SIG 1R activation has been shown to be neuroprotective in multiple animal models of disease. Multiple commercial SIG 1R antibodies have recently been shown to non specifically label SIG1 R KO mice, complicating both the use of the KO model and the antibodies themselves. In this study, we investigate multiple commercial and one novel SIG1 R antibodies with immunolabeling and western blotting to probe for KO specific labeling Methods Westerns blots Lysates were created from HEK293 cells and HEK293 cells overexpressing a YFP tagged sigma receptor to be utilized as a positive control. Lysates were also made prepared from either whole mouse brains, striatal tissue from mouse brains, or whole from wild type and KO mice. Samples were heated either to 95 degrees for five minutes or 65 degrees for fifteen minutes for denaturing before being prepared for western blot loading. Samples were loaded into a 10% SDS PAGE gel and ran at 120V for 2 hours before being transferred at 400 miliamps for 1 hour and 15 minutes to either PVDF or nitrocellulose membranes. Membranes were blocked for 40 minutes in 5% milk at room temperature and then went into an overnight incubation with the primary antibody. Membranes were washed three times with TBST and then incubated with Licor anti rat 800 secondary antibody for 1h at room temperature in the dark. Following three brief washes, membranes were imaged on a LiCor Odyssey imaging system. Immunolabeling Brains from one month old wild type or SIG1 R KO mice were transcardially perfused with 4% PFA, and cultured neurons were fixed for 20 min. at RT with 4% PFA For blocking in permeabilization brain slices were incubaed in 0.3% TritonX 100 with 10% goat serum for 1 hr at 37 deg and cultured cells were incubated with 0.5% TritonX 100 with 10% goat serum for 25 min at RT. Both sample types were incubated overnight with the specified primary antibody in 0.1% TritonX 100 with 5 % goat serum before being washed three times and incubated in the appropriate fluorescent secondary antibodies ( AlexaFluor 488 or 561) in the same solution for one hr at RT. Samples were then washed overnight and mounted using Fluoromount G before being imaged in a Nikon A1R confocal microscope. All images in a single panel were treated identically for presentation. A Wt liver WT Brain KO brain WT STR 75 50 10 KO Liver KO Liver WT Brain WT Brain KO Brain WT Brain KO Brain 150 100 75 50 37 25 20 15 A. Western blot of tissue samples from wild type and SIG1 R KO mice labeled with rb SIG1 R. Samples were denatured at 95 deg for 5 minutes, and the gel was transferred to a PVDF membrane. In the KO brain lysates, a strong band is visible ~75 kDa corresponding to the 75 kDa band in WT samples that is likely the detection of SIG1 R trimers. No samples labeled for SIG1 R monomers (25 kDa ), but a faint band ~ 50kDa is visible suggesting detection of SIG1 R dimers. B. Western blot of tissue samples from wild type mice, SIG1 R KO mice, and YFP SIG1 R cells labeled with rb SIG1 R. Samples were denatured at 65 deg for 15 min, and the gel was transferred to a nitrocellulose membrane. Both lanes with KO tissue exhibit a strong band ~75 kDa suggesting non specificity of this method. B. Western blot of tissue samples from wild type mice & SIG1 R KO mice labeled with rb SIG1 R. Samples were denatured at 95 deg for 5 minutes, and the gel was transferred to a nitrocellulose membrane. Bands at 75 kDa are present in WT samples which show detection of SIG1 R trimers T hese bands are absent in KO tissue samples, which suggests that this tissue preparation and membrane type is able to reveal the KO specificity of the rb SIG1 R antibody. A. A novel SIG1 R antibody reveals nuclear staining, but recapitulates nonspecific labeling seen in commercially available antibodies in KO Mice A. Immunolabeling in the striatum of a 1 month old mouse for the dopamine transporter (DAT) and SIG1 R using a novel antibody generated in collaboration with EnCore Biotechnology (Gainesville, FL; clone #233). Nuclei of neurons (red) surrounded by dopamine terminals (green) intensely label for SIG1 R. B. Same immunostaining as A but in a location where dopamine neuron fibers can be seen travelling through a field of SIG1 R positive nuclei. C. Immunolabeling of the striatum in SIG1 R KO mice reveals similar nuclear staining to that seen in the wild type, recapitulating the non specific pattern of all commercially available SIG1 R antibodies. A. C. B. SIG 1R DAT Striatum 20X MidBrain fibers 20X Striatum SIG1 R KO 20X 250 150 100 75 50 37 25 20 15 HEK parental YFP sig1r HEK HEK parental YFP sig1r HEK HEK parental YFP SIG1R labeling is consistent with two commercially available SIG1 R antibodies HEK Parental HEK parental YFP sig1r HEK YFP sig1r HEK 250 150 100 75 50 37 25 20 15 15 25 75 100 150 50 mW ( kDa ) A. B. C. A. labeling of parental and overexpressing HEK lysates probing for YFPwith a highly specific antibody ( Abcam cat#: ab6556). Signal is present only in lysates containg the taggedSIG1 R. B. labeling of parental and overexpressing HEK lysates probing for SIG1 R using a commercially available antibody raised in mouse (Santa Crz Biotech cat#: sc 137075 B5). Bands at the expected molecular weights for SIG1 R (25 kDa 75 kDa ) are present in parental cells, with a bright band corresponding to the overexpressed tagged SIG1R present at 50 kDa consistent with YFP labeling. C. labeling of parental and overexpressing HEK lysates probing for SIG1 R using a commercially available antibody raised in rabbit(Invitrogen cat #: 42 3300). Bands at the expected molecular weights for SIG1 R (25 kDa 75 kDa ) are present in parental cells though this signal is weaker than that seen with the mouse antibody. A bright band corresponding to the overexpressed tagged SIG1R present at 50 kDa remains present, consistent with YFP labeling. YFP ms SIG1 R rb SIG1 R rb SIG1 R but not ms SIG R, yields the expected localization pattern in cultured dopamine neurons SIG 1R DAT 6 0X A. B. ms SIG1 R rb SIG1 R A. Immunolabeling of cultured dopamine neurons identified by DAT staining using the ms SIG1 R primary antibody as above (Santa Cruz B 5). Examination of the isolated SIG1 R signal (rightmost panel) shows a weak signal relative to background noise that is present all throughout the cell body and processes, with only the nucleus being identifiable, suggesting a nonspecific labeling. B. Immunolabeling of cultured dopamine neurons identified by DAT staining using the rb SIG1 R primary antibody as above (Invitrogen 42 3300). Consistent with previous reports, SIG1 R labeling presents as distinct punctae throughout the cell body and processes, but not in the nucleus (rightmost panel). These punctae are thought to be endoplasmic reticulum structures known as subsurface cisternae, which is consistent with the known localization of SIG1 R localization in cells. Altering tissue prep and membrane type can reveal KO specificity of rb SIG1 R B 25 37 YFP SIG1 R HEK 10 100 150 C. 25 37 50 75 100 150 Acknowledgements and funding We would like to thank Dr. J.A. Pino and Adithya Gopinath for assistance with western blot optimization. Funding was provided by the UF Center for undergraduate research via the UScholars program, and the NIH via R01DA026947 08S1/NIDA Establishing a Reliable P rotocol for Highly Specific L abeling of the Sigma 1 Receptor in multiple applications