To confirm the expression of targetable integrins on the MC-26 cells, cells were harvested with Cell Dissociation Buffer, enzyme-free (ThermoFisher Scientific). Cells were stained for αv integrin (550024 BD Pharmingen; 1:100), NRP-1 (AF566 R&D Biosystems; 1:100), and p32 (AB 2991 Millipore; 1:100) at 4 °C for 1 hour and probed with secondary antibodies conjugated with fluorophores using standard protocols. Cells were analyzed by flow cytometry on a BD LSR II Flow Cytometer.
Af566
Manufactured by R&D Systems
Sourced in United States
AF566 is a laboratory reagent produced by R&D Systems. It functions as a recombinant human protein.
Automatically generated - may contain errors
Lab products found in correlation
Ab 2991, by Merck Group (4 mentions)
Lsr 2 flow cytometer, by BD (2 mentions)
Cell dissociation buffer, by Thermo Fisher Scientific (2 mentions)
Recombinant human sema3a, by Thermo Fisher Scientific (2 mentions)
Sema3a, by R&D Systems (2 mentions)
F7425, by Merck Group (2 mentions)
Ab13970, by R&D Systems (2 mentions)
Fluorescence tagged secondary antibodies, by Jackson ImmunoResearch (2 mentions)
Af1835, by Merck Group (2 mentions)
Sc 22458, by R&D Systems (2 mentions)
Af2414, by Santa Cruz Biotechnology (2 mentions)
Sc 206, by Abcam (2 mentions)
Af5375, by R&D Systems (2 mentions)
Ab1930, by Merck Group (2 mentions)
Custom antibody, by R&D Systems (2 mentions)
Af3870, by R&D Systems (2 mentions)
Chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
T9026, by Merck Group (1 mentions)
Ab23393, by Abcam (1 mentions)
Mab5272, by Merck Group (1 mentions)
19 protocols using af566
1
Integrin Expression on MC-26 Cells
2
Sema3a Modulation of Motor Neuron Processes
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MN were plated at 1000 cells per well as above. 12 hours after plating, they were assessed by light microscopy, at which point most could be seen to have budded polar processes. 24 hours after plating, recombinant human Sema3a was added (Peprotech) at indicated concentrations. For Nrp1 blockade, a Nrp1- blocking antibody validated for this purpose (R&D systems AF566) was added at a concentration of 10 µg/ml and incubated with MN for 30 minutes prior to Sema3a addition. Goat IgG control added separately to control for nonspecific effects (not shown.) Cells were cultured an additional 36 hours before fixation and immunolabeling.
3
Western Blot Protein Detection
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All samples were resolved on a 7% SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked in 4% milk in TBS-T (TBS, pH 7.4, and 0.1% Tween 20) for 1 h at room temperature. Primary antibodies [α-tubulin (1:30,000, T9026, Sigma-Aldrich), actin (1:1000, SC-1616-G, Santa Cruz Biotechnologies), Npn1 (1:1000, AF566, R&D Systems), or Sema3A (1:1000, ab23393, Abcam)] were diluted in 3% bovine serum albumin (BSA) and incubated overnight at 4°C. The following day, membranes were washed and incubated with appropriate horseradish peroxidase (HRP)-linked secondary antibodies (1:10,000) in 3% BSA (Jackson ImmunoResearch) followed by visualization with a chemiluminescent substrate (Thermo Fisher Scientific).
4
Immunofluorescence Characterization of Taste Receptors
5
Integrin Expression on MC-26 Cells
6
Immunohistochemical Analysis of Tumor Samples
7
Antibody Characterization for Neuronal Proteins
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Primary antibodies used for immunohistochemistory and western blot were as follows; goat anti-NOVA2 (C-16) (sc-10546, Santa Cruz), rabbit anti-NOVA1 [EPR13847] (ab183024, abcam), human anti-pan NOVA (anti-Nova paraneoplastic human serum), rabbit anti-PTBP2 (Polydorides et al., 2000 (link)), rat anti-L1 (MAB5272, Millipore), goat anti-TAG1/Contactin-2 (AF4439, R&D systems), goat anti-NetrinG1a (AF1166, R&D systems), rabbit anti-NURR1 (M-196) (sc-5568, Santa Cruz), rabbit anti-neurofilament (AB1981, Chemicon), mouse anti-NF200 (N5389, SIGMA), mouse anti-ctbp2 (612044, BD), rabbit anti-myoVI (sc-50461, Santa Cruz), and goat anti-neuropilin-1 (AF566, R&D systems). Anti-NOVA1 and anit-NOVA2 antibody specificity for immunohistochemistory and immunoprecipitation was confirmed (Figure 1—figure supplement 2 ).
8
Immunohistochemical Analysis of Tumor Samples
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All histology sectioning and staining was performed at the KI Histology Core. Organs were fixed using formalin immediately after necropsy for 24 hours and stored at 4 °C prior to embedding into paraffin, sectioning, and staining. OVCAR-8 tumour sections were stained with p32 (Genscript custom antibody; 1:100) and NRP-1 (R&D Biosystems AF3870; 1:100). MC-26 tumour sections were stained with αv integrin (Millipore AB1930; 1:100), p32 (AB 2991 Millipore; 1:100), and NRP-1 (AF566 R&D Biosystems; 1:100).
9
Protein Extraction and Western Blotting
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Cells were lysed with lysis buffer (50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 0.1% NP-40, and 250 mM NaCl) in the presence of protease inhibitor cocktail 2 and phosphatase inhibitor cocktail (Sigma-Aldrich). Lungs from P7 mice were lysed in GL35 (Cytoskeleton, Inc). 25 µg of protein was resuspended in Laemmli sample buffer, denatured for 5 min, separated by SDS-PAGE, and transferred to nitrocellulose membrane (Whatman).
Protein extracts were analyzed by SDS-PAGE and transferred to nitrocellulose membrane (Whatman) for immunoblotting with the following primary antibodies: rabbit anti-GAPDH (Abcam), goat anti-NRP1 C-19 (Santa Cruz Biotechnology, Inc.) or AF566 (R&D Systems); mouse anti-ABL1 (BD); rabbit anti-pCRKL (Y207), rabbit anti-pKDR (Y1175) or KDR, rabbit anti-pPXN (Y118), rabbit anti-pAKT (S473) or AKT, or rabbit anti-pERK1/2 or ERK1/2 (Cell Signaling Technology).
Protein extracts were analyzed by SDS-PAGE and transferred to nitrocellulose membrane (Whatman) for immunoblotting with the following primary antibodies: rabbit anti-GAPDH (Abcam), goat anti-NRP1 C-19 (Santa Cruz Biotechnology, Inc.) or AF566 (R&D Systems); mouse anti-ABL1 (BD); rabbit anti-pCRKL (Y207), rabbit anti-pKDR (Y1175) or KDR, rabbit anti-pPXN (Y118), rabbit anti-pAKT (S473) or AKT, or rabbit anti-pERK1/2 or ERK1/2 (Cell Signaling Technology).
10
Visualizing Microtubule-Focal Adhesion Interactions
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A total of 20,000 ECs were seeded onto FN‐coated/BSA‐blocked coverslips and adhered for 90 min before being washed with PBS and immersed in −20°C methanol for 20 min. Alternatively, cells were prepared as per the microtubule stability assay protocol described above. Coverslips were then washed with PBS, blocked for 10 min at room temperature with 0.5% BSA, 1% goat serum in PBS with 0.25% Triton X‐100 and incubated with primary antibody diluted 1:250 in PBS for 1 h at room temperature. After subsequent PBS washes, the coverslips were incubated with Alexa Fluor®‐conjugated secondary antibodies raised in donkey (Fisher Scientific) diluted 1:500 in PBS. Coverslips were washed again in PBS before being mounted onto slides using Prolong Gold® with DAPI (Fisher Scientific). Primaries used were anti‐alpha‐tubulin (Abcam 52866), anti‐paxillin (Abcam 32084) anti‐talin (Sigma T3287) and anti‐Nrp1 (R&D Systems AF566).
To quantify microtubule targeting focal adhesions, images were taken of stained cells using an epifluorescent microtubule, and then, the number of microtubules with an end overlapping with a focal adhesion was counted for each cell.
Simultaneous phalloidin (Thermo Fisher A12380) and alpha‐tubulin staining were carried out using PHEMO fixation46 .
To quantify microtubule targeting focal adhesions, images were taken of stained cells using an epifluorescent microtubule, and then, the number of microtubules with an end overlapping with a focal adhesion was counted for each cell.
Simultaneous phalloidin (Thermo Fisher A12380) and alpha‐tubulin staining were carried out using PHEMO fixation
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