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21 protocols using anti v5

1

Antibody Validation Protocols for Western Blotting

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Anti-ataxin-3: rabbit polyclonal, 1:10,000 [52 (link)]; mouse monoclonal 1H9, 1:1000, Millipore. Anti-HA: rabbit monoclonal, 1:1000, Cell Signaling Technology. Anti-MYC: mouse monoclonal 9E10, 1:1000, Santa Cruz Biotechnology; mouse monoclonal 9B11, 1:1000, Cell Signaling Technology. Anti-V5: rabbit monoclonal D3H8Q, 1:1000, Cell Signaling Technology.
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2

Antibody Detection of Epitope-Tagged Proteins

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The HA epitope was detected with mouse monoclonal antibody (mAb) HA.11 (BioLegend; 901515) and rabbit polyclonal antibody (pAb) anti-HA (Invitrogen; PI715500). The Ty1 epitope was detected with mouse mAb BB2 [61 (link)]. The c-Myc epitope was detected with mouse mAb 9E10 [62 (link)]. The V5 epitope was detected with mouse mAb anti-V5 (Invitrogen; R96025) and rabbit mAb anti-V5 (Cell Signaling Technology; 13202S). The OLLAS epitope was detected with rat mAb anti-OLLAS [63 (link)]. Toxoplasma-specific antibodies include rabbit pAb anti-IMC6 [64 (link)], mouse mAb anti-IMC1 [65 (link)], mouse mAb anti-ISP1 [32 (link)], rabbit pAb anti-Centrin1 (Kerafast; EBC004) [66 (link)], and rabbit anti-Catalase [67 (link)].
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3

Isolation and Western Blot Analysis of ARC

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Ad libitum-fed or 24 h-fasted mice were deeply anesthetized with isoflurane before removing the brain. ARC was isolated by microdissection and solubilized in lysis buffer (38.5 mM Tris-HCl, 1 mM EGTA, 1 mM EDTA, 1% Triton-X 100, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, and 250 mM sucrose) with protease inhibitor on ice. Protein concentration was determined by bicinchoninic acid assay and the same amount of protein was loaded on polyacrylamide gels for SDS separation. Western blot was performed using either anti-V5 (Cell Signaling Technology, Rabbit mAb #13202) or anti-pAMPK (Cell Signaling Technology Rabbit mAb #2535).
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In Situ Hybridization and Immunohistochemistry

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The brain frozen sections or cultured neurons fixed in 4% paraformaldehyde for 15 min were incubated with pre-hybridization buffer (50% formamide [Wako], 5 × saline-sodium citrate buffer [SSC], 0.05% heparin sodium [Wako], 0.02% ribonucleic acid from torula yeast [Sigma]) for 60 min at 65 °C and then hybridized with a fluorescently labeled locked nucleic acid (LNA) probes, PolyT(25)Vn 5′-fluorescein (Exiqon 300510-04), Scramble-ISH 5′-fluorescein (Exiqon 300,514–04), and 5′-FAM-AGGCCAGGTCTTCTTCAGAAATCA-3′ (for exogenous FUS mRNA) (Exiqon) diluted to final concentration of 80 nM, for 24 h at 65 °C in a dark, humidified chamber. Next, sections were washed with 2 × SSC/formamide for 1 h at 65 °C and washed with TTBS (0.5 M NaCl, 0.02 M Tris HCl [pH 8.0], 0.1% tween 20) three times. Then immunohistochemistry was performed using anti-c-Myc 9E10 (1:1000), and anti-V5 (Cell Signaling, #06-549, 1:1000). Fluorescence intensity was determined by densitometry using ImageJ.
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5

Immunohistochemistry for c-Myc and V5 Proteins

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The brain frozen sections or cultured neurons fixed with − 20 °C methanol for 10 min were incubated with 500 nM SYTO RNASelect (Life technologies) for 20 min at room temperature as per the manufacturer's instruction. Then, immunohistochemistry was performed using anti-c-Myc 9E10 (1:1000) and anti-V5 (Cell Signaling, #06-549, 1:1000).
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6

Characterizing KDM5A Ubiquitination in TNBC

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The indicated plasmids (Flag-Fbxo22, HA-KDM5A, and V5-Ubiquitin) were transfected into HEK293T cells. Prior to collection, cells were treated as previously described (Zhang et al. 2019 (link)). The obtained lysates were assayed and immunoprecipitated with protein A/G agarose (Sigma) pre-conjugated with the indicated antibody anti-HA (1:50, #3724, Cell Signaling). In addition, to prevent detection of ubiquitination of the E3 ligase itself and proteins associated with KDM5A, ubiquitination assays were also performed under denaturing conditions. Transfected cells were lysed in lysis buffer and incubated with SDS-PAGE sample buffer at 100 °C for 8 min, followed by incubation with HA antibody for IP and Immunoblotting. Antibodies used for immunoblotting were anti-flag (1:50, F3165, Sigma), anti-HA (1:50, #3724, Cell Signaling), and anti-V5 (1:50, 13,202, Cell Signaling).
Additionally, the ubiquitination level of KDM5A in TNBC cell lines was also examined. The obtained lysates were immunoprecipitated with protein A/G agarose (Sigma) pre-conjugated with the antibody of anti-KDM5A (1:100, ab70892, Abcam) or anti-IgG (1:50, #3900, Cell Signaling). The expression of the relevant proteins was then detected by immunoblotting using the antibodies of mouse anti-KDM5A (1:1000, ab78322, Abcam) and anti-ubiquitin (1:1000, 04–263, Millipore).
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7

Western Blot Analysis of HUVEC Protein Extracts

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Protein extracts from HUVECs were collected by lysis in RIPA buffer (Sigma; 150 mM NaCl, 1.0% (v/v) IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0) supplemented with 1 × EDTA-Free Complete Protease Inhibitor Cocktail (Roche) and 1 mM phenylmethylsulfonyl fluoride (Carl Roth). Proteins were separated by SDS–PAGE (Tris-glycine gels with Tris/glycine/SDS buffer, Bio-Rad) and transferred onto nitrocellulose membranes using the Trans Turbo Blot system (Bio-Rad). The following primary antibodies were used: anti-c-MYC (Cell Signaling Technology, 9402, 1:1,000), anti-N1ICD (Cell Signaling Technology, 4147, 1:1,000), anti-V5 (Cell Signaling Technology, 13202, 1:2,500) and anti-TUBULIN (Cell Signaling Technology, 2148, 1:5,000). Peroxidase-conjugated Goat IgGs (1:5,000) secondary antibodies were purchased from Jackson Immuno Research Labs. Target proteins were visualized by chemiluminescence using the ECL detection kit (Clarity Western ECL Substrate, Bio-Rad) and the ChemiDoc MP Imaging System (Bio-Rad).
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8

Comprehensive Antibody Staining Protocol

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Anti JMJD6 (Santa Cruz, sc28349), anti UBF (Bethyl, A301-859A), anti histone H3 (Abcam, Ab1791), anti BrdU (Sigma, B2531), anti gamma H2AX (Cell signaling, 9718(20E3)), anti GAPDH (Chemicon, MAb374), anti myc (Santa Cruz, sc-40), anti Treacle (Santa Cruz, sc374536), anti V5 (Cell Signaling, 12032), anti NBS1 (Sigma, PLA0179), anti phospho ATM (S1981) (Cell signaling, 10H11.E12).Anti Rad51 (Millipore, PC130), Anti RPA S33 (Bethyl, A306-246A-T)
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9

Western Blot Antibody Validation

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Anti-Akt (#4685), anti-phospho-Akt S473 (#4060), anti-phospho-Akt T308 (#9275), anti-β Actin (#4970), anti-GSK3β (#9315), anti-phospho-GSK3β (#9336), anti-GST (#2625), and anti-V5 (#13202) were purchased from Cell Signaling Technologies (product numbers listed for each). Horseradish peroxidase-conjugated anti-rabbit and anti-mouse immunoglobulin G (IgG) antibodies were purchased from Millipore.
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10

Antibodies and Reagents for Protein Analysis

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The following antibodies were used in western blot, co-immunoprecipitation, immunofluorescence or IHC: anti-NAC1 (NB110-77345, Novus, Littleton, CO, USA), anti-HDAC4 (ABE262, Millpore, Billerica, MA, USA), anti-HIF-1α (#3716), anti-p-HDAC4 Ser246 (#3443), anti-Flag (#8146), anti-V5 (#13202), anti-GLUT1 (#12939), anti-lactate dehydrogenase-A (#2012), anti-14-3-3 (#8312), anti-von Hippel-Lindau (#68547), anti-GST (#2622), and anti-Myc (#2278) (Cell Signaling Technology, Danvers, MA, USA), anti-β-actin (sc-47778), anti-ubiquitin (sc-8017), anti-CD31(sc-376764) and anti-pan-Ace (Ace-K) (sc-8649) (Santa Cruz, Dallas, TX, USA), anti-HDAC6 (YT2118, Immunoway, Plano, TX, USA), mouse IgG (A7028) and rabbit IgG (A7016, Beyotime, Shanghai, China). MG132, CoCl2 and cycloheximide were purchased from Sigma-Aldrich Corporation (Sigma-Aldrich, St Louis, MI, USA). The primers and oligo DNA used in this study are listed in Supplementary Table 1.
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