Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72
- McGill University, Canada
- University of Toronto, Canada
- University of Oxford, United Kingdom
Figures
Figure 1
Summary of the antibody validation pipeline.
IB = immunoblot; IP = immunoprecipitation; IF = immunofluorescence; KO = knockout.
Figure 2 with 1 supplement
Analysis of 16 C9ORF72 antibodies by immunoblot.
Cell lysates from HEK-293 parental, heterozygous (+/-) or two individual C9ORF72 KO clones (KOA, KOB) were prepared and processed for immunoblot with the indicated C9ORF72 antibodies. The arrows …
Figure 2—figure supplement 1
Analysis of 16 C9ORF72 antibodies by immunoblot.
(A) Selected immunoblots on HEK-293 cell lysates from Figure 2 or on mouse brain samples from part (B) of this supplement are shown with longer exposure. (B) Brain lysates from WT and C9ORF72 KO …
Figure 3 with 1 supplement
Analysis of 16 C9ORF72 antibodies by immunoprecipitation.
(A) HEK-293 cell lysates were prepared and immunoprecipitation was performed using the indicated C9ORF72 antibodies pre-coupled to protein G-Sepharose (prot. G). Controls included the Protein …
Figure 3—figure supplement 1
Comparison of the amount of C9ORF72 remaining in the unbound fraction after immunoprecipitation.
HEK-293 cell lysates were prepared and immunoprecipitation was performed using the indicated C9ORF72 antibodies and the appropriate protein A/G-Sepharose (prot. A/G) bead controls. After the O/N …
Figure 4 with 3 supplements
Analysis of 16 C9ORF72 antibodies by immunofluorescence.
(A) Parental and KO cells were transfected with LAMP1-YFP or LAMP1-RFP, respectively. Parental and KO cells were combined and incubated with buffer only or stained with the indicated GeneTex C9ORF72 …
Figure 4—figure supplement 1
Testing of C9ORF72 antibodies by immunofluorescence analysis in HEK-293 cells fixed with PFA HEK-293 parental and KO cells were fixed with PFA, permeabilized and incubated with the indicated C9ORF72 antibodies.
Representative images are shown. Cells are outlined with yellow dashed lines.
Bars = 16 µm.
Figure 4—figure supplement 2
Testing of C9ORF72 antibodies by immunofluorescence analysis in HEK-293 cells fixed with −20°C methanol HEK-293 parental and KO cells were fixed with −20°C methanol and incubated with the indicated C9ORF72 antibodies.
Representative images are shown. Cells are outlined with yellow dashed lines.
Bars = 16 µm.
Figure 4—figure supplement 3
immunofluorescence strategy in U2OS cells.
(A) Lysates were prepared from selective human cell lines and processed for quantitative immunoblot using antibody GTX634482 (HEK, human embryonic kidney; HeLa, human cervical cancer; U2OS, human …
Figure 5
Analysis of C9ORF72 antibodies by immunohistochemistry.
(A) DAB immunohistochemistry of C9ORF72 WT and C9ORF72 KO mouse brains in the sagittal plane using C9ORF72 antibody GTX634482. Micrographs are ordered from the rostral to caudal aspect of the mouse …
Figure 6 with 2 supplements
C9ORF72 localizes to lysosomes.
(A) Immunofluorescence of endogenous C9ORF72 stained with GTX632041 in U2OS cells expressing LAMP1-RFP. The large images on the left show a maximum projection to highlight intracellular structures. …
Figure 6—figure supplement 1
C9ORF72 co-staining with Rab.
GTPases Immunofluorescence of endogenous C9ORF72 stained with GTX632041 in U2OS cells expressing GFP-Rab5a, GFP-Rab7a, GFP-Rab9 and GFP-Rab11a as indicated. Insets are higher magnifications of the …
Figure 6—figure supplement 2
C9ORF72 subcellular fractionation.
(A) Lysates were prepared from HEK-293 cells non-transfected or expressing 3xHA-eGFP-OMP25 (MitoIP). Lysates were incubated with anti-HA magnetic beads to immunoprecipitate mitochondria. …
Figure 7
Macrophages express C9ORF72 at high levels.
(A) Lysates were prepared from rat cortical neurons, rat hippocampal neurons, mouse bone marrow-derived macrophages (BMDMs) and mouse whole brain, and processed for quantitative immunoblot using …
Figure 8
C9ORF72 localizes in part to late phagosomes and phagolysosomes.
(A) Immunofluorescence of C9ORF72 (GTX632041, magenta) and F-actin staining (cyan) in human MDMs. The inset is a higher magnification of the region indicated by the arrow and shows a grayscale image …
Additional files
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Supplementary file 1
Summary of C9ORF72 antibodies and their properties.
- https://doi.org/10.7554/eLife.48363.017
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Supplementary file 2
Mass spectrometry data from immunoprecipitation of C9ORF72 from parental and KO cell lines.
- https://doi.org/10.7554/eLife.48363.018
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Transparent reporting form
- https://doi.org/10.7554/eLife.48363.019
