Cell lysates were electrophoresed on a 10 or 12% SDS-PAGE gel and transferred onto polyvinylidene difluoride membranes, which were then blocked with 5% BSA in TBST [50 mmol/L Tris-HCl (pH 7.4), 150 mmol/l NaCl, and 0.1% Tween 20] for 1 hr, incubated with various primary antibodies overnight and detected with either anti-rabbit or anti-mouse secondary antibodies for 1 hr using SuperSignal West Dura Extended Duration Substrate (Thermo Scientific). The blots were re-probed with anti-Actin or anti-GAPDH antibody for protein loading control. Primary antibodies used in the study are: anti-HOXC5 antibody (1:1000, Sigma, #HPA026794), anti-Flag (M2) antibody (1:5000, Sigma-Aldrich, #F1804) and anti–V5 antibody (1:5000, Cell Signaling, #13202). The blots were re-probed with anti-Actin (1:5000, Santa Cruz Biotechnology, #sc1616) or anti-GAPDH antibody (1:1000, Cell Signaling, #2118) for protein loading control. The uncropped western blot scans were shown in Supplementary Fig. 18 .
Anti v5 antibody
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
The Anti-V5 antibody is a monoclonal antibody that specifically recognizes the V5 tag, a common epitope tag used for protein detection and purification. It can be used in various immunological techniques, such as Western blotting, immunoprecipitation, and immunofluorescence, to detect and localize V5-tagged proteins.
Automatically generated - may contain errors
Lab products found in correlation
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Anti gapdh antibody, by Cell Signaling Technology (1 mentions)
Anti flag m2 antibody, by Merck Group (1 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Microm hm 340e, by Thermo Fisher Scientific (1 mentions)
Lsm 700 confocal microscope, by Zeiss (1 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Pierce protein a plus agarose, by Thermo Fisher Scientific (1 mentions)
5 protocols using anti v5 antibody
1
Western Blot Analysis of Protein Targets
2
Validating Epitope-Tagged Constructs in DU145 Cells
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To positively ascertain the transfection of α5.pcDNA3.1/V5 and α5D398N.pcDNA3.1/V5 epitope-tagged constructs (a generous gift from Dr. Larry S. Barak) [40 (link)] into DU145 cells, specific immunofluorescence staining was performed on fixed cells. In brief, 24 h after nucleofection, the cells were fixed using 4% PFA in PBS (ThermoFisher, Waltham, MA, USA) for 15 min, and subsequently incubated with anti-V5 antibody (1:500, Cell Signaling, Danvers, MA, USA) overnight at 4 °C. Afterwards, cells were incubated with AlexaFluor 488 (1:1000, Invitrogen, Waltham, MA, USA) for 1 h. Samples were visualized using a Nikon Eclipse Ti2-E Widefield microscope with a Photometrics BSI camera and SpectraX laser as light source, with either a 40× or 100× objective lens.
3
Immunofluorescence Staining of Mouse Dorsal Tissue
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For the Immunofluorescence staining of mouse dorsal tissue, sample tissue embedded in paraffin block was sectioned using a microtome (Microm HM340E, Thermo Scientific, Waltham, MA, USA) longitudinally. HORS cells and tissue sections were stained with anti-FGF12 antibody (Abcam, Cambridge, UK; 1:200) and anti-V5 antibody (Cell signaling; 1:200) overnight. Alexa Fluor 488 goat anti-rabbit IgG was used as a secondary antibody and nuclei of cells were stained with 4′6-diamidino-2-phenylindole (DAPI). All images of immunofluorescence staining were visualized using a Zeiss LSM700 confocal microscope (Carl Zeiss, Jena, Germany).
4
GPR37 Binding Assay with Hydrophobic Compounds
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DMSO-soluble chemicals (honokiol, artemisinin, artesunate, or DMSO without drug) were directly loaded on hydrophobic NC membranes (Bio-Rad). Compound-coated membranes were dried and blocked with 1% BSA. Membranes were incubated with lysates obtained from GPR37-expressing HEK293 cell or GFP-expressing cell (mock) lysates for 2 h, followed by detection using an anti-V5 antibody (Cell Signaling, mouse, 1:1000, #80076). Blots were further incubated with an HRP-conjugated secondary antibody (Jackson Immunoresearch, raised in donkey, 1:5000), developed in ECL solution (Pierce), and Bio-Rad ChemiDoc XRS revealed the chemiluminescence signal. The intensity of the dots was analyzed using NIH Image J software.
5
Immunoprecipitation Assay of c-JUN in MDA-MB-231 Cells
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For immunoprecipitation assay, MDA-MB-231 cells were transfected with an expression plasmid encoding c-JUN (pcDNA3.1/V5-His-c-JUN) or GFP as a control (pMAX/GFP). After 48 h, the cells were collected, and total proteins were lysed in a lysis buffer (50 mM Tris-HCl, pH 7.9, 10 mM KCl, 400 mM NaCl, 1 mM EDTA, 0.2 % NP-40, 10 % glycerol, 1 mM phenylmethylsul-phonyl fluoride and a cocktail of protease inhibitors). The cell lysates were incubated with the anti-V5 antibody (Cell Signaling Technology) overnight at 4 °C. Antibody-bound protein complexes were precipitated using Pierce Protein A Plus agarose (Thermo Fisher Scientific). Agarose beads were washed thrice with the lysis buffer and heated in a sodium dodecyl sulphate (SDS) sample loading buffer at 95 °C for 5 min. The eluted samples were separated by SDS-poly acrylamide gel electrophoresis, and immunoblotting analysis was performed using anti-V5 and anti-HDAC1 antibodies. Blots were visualized using an enhanced chemiluminescence detection system (Amersham Phar macia Biotech).
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