Product Image Gallery
Western blot analysis of V5 Epitope Tag was performed by loading various amounts of E. coli lysate containing a multi-epitope tagged protein per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a low fluorescence PVDF membrane and blocked with Sea Block blocking buffer for at least 1 hour. The membrane was probed with a AlexaFluor488-conjugated V5 Epitope Tag monoclonal antibody (Product # 37-7500-A488) at a dilution of 1:500 over night at 4C on a rocking platform and washed in TBS-0.1% Tween-20. Detection was performed using a fluorescence imaging system.
Figure 5. LAP2 depletion exacerbates the progerin-induced proliferation defect whereas specific overexpression of LAP2alpha rescues it. ( A ) Growth curve of normal (ctrl shRNA) and LAP2-depleted (shLAP2) primary fibroblasts expressing progerin or lamin A. Dotted lines indicate SEM (n = 3). Inset: growth rate after 8 days, error bras indicate SEM (*p < 0.05, Student and apos;s t -test). ( B ) Western blot of primary and TERT+ cells expressing v5-tagged lamin A (v5-LA) or progerin (v5-PG) with or without LAP2 silencing by shRNA (shLAP2). V5-tag, lamin A, lamin C, progerin (PG), LAP2alpha, LAP2beta and GAPDH are indicated. ( C ) Growth curve of fibroblasts expressing control vector or progerin in the presence or absence of doxycycline-inducible LAP2alpha. LAP2alpha expression was induced by addition of 0.25 ug/ml doxycycline. Dotted lines indicate SEM (n = 3). Inset: growth rate after 5 days, error bras indicate SEM (**p < 0.01, Student and apos;s t -test). ( D ) Western blot of primary fibroblasts carrying doxycycline-inducible v5-LAP2alpha, expressing control vector or progerin. LAP2alpha expression was induced by addition of 0.25 ug/ml doxycycline. LAP2alpha, progerin and actin are indicated. ( E ) Western blot showing doxycycline-dependent induction of v5-LAP2alpha in TERT+HGPS cells. ( F ) Box plot of H3K27me3 levels in human fibroblasts expressing progerin in the presence (red) or absence (blue) of ectopically expressed LAP2alpha (Student and apos;s t -test, p < 0.05, n > 7500 cell a
Figure 1--figure supplement 1. Progerin induced senescence, lamin B1 loss, DNA damage, and telomere shortening are prevented by TERT in primary and HGPS fibroblasts, control experiments. ( A ) Western blotting showing inducible expression of progerin or wild type lamin A in primary and TERT+ human fibroblasts +- doxycyclin (DOX) as indicated. ( B ) Immunofluorescence microscopy using V5-tag antibody (top panels) showing doxycyclin-dependent expression of v5-lamin A and v5-progerin in TERT+ human fibroblasts and localization to the nuclear periphery. Inset: higher magnification image of different field. DAPI staining is shown in bottom panels. Scale bar: 50 mum; scale bar inset: 20 mum. ( C ) Growth curve of TERT+ and primary cells in the presence or absence of progerin (+-DOX). Dotted lines indicate SEM (n = 5). Inset: growth rate after 6 days, error bars indicate SEM (**p < 0.01, one-way ANOVA with Tukey and apos;s post-test). ( D ) Quantification of lamin B1 levels upon progerin or lamin A expression in primary and TERT+ cells. Error bars indicate standard deviation (n = 4, *p = 0.05, Student and apos;s t -test). Values were normalized to no DOX control. ( E ) Quantification of progerin protein levels upon induction with doxycycline (+-DOX) in primary and TERT+ human fibroblasts. Levels were normalized to GAPDH loading control (n = 3, error bars indicate standard deviation). ( F ) Growth rate of primary and TERT+ fibroblasts in the presence (+DOX) or absence (-DOX) of exogenous lam
Figure 5--figure supplement 1. Depletion and expression of LAP2 in wild type and progerin expressing fibroblasts, control experiments. ( A ) Growth curve of primary and TERT+ control and LAP2 shRNA expressing fibroblasts. Dotted lines indicate SEM (n = 3). Inset: growth rate after 7 days. ( B ) Immunofluorescence microscopy analysis of lamin A/C and LAP2 levels in cells expressing scrambled (ctrl) or LAP2-specific shRNA (shLAP2). Scale bar: 20 mum. ( C ) Western blot of wild type cells expressing scrambled control shRNA (ctrl) or LAP2 shRNA (shLAP2). The LAP2 antibody recognizes both alpha and beta-isoforms of LAP2. Lamin A, lamin C and actin loading control are indicated. ( D ) Growth curve of normal (ctrl) and LAP2-depleted (shLAP2) TERT+ fibroblasts expressing progerin or lamin A. Dotted lines indicated SEM (n = 3). Inset: growth rate after 7 days. ( E ) Immunofluorescence microscopy showing dose-dependent induction of v5-LAP2alpha and its nucleoplasmic localization. Antibodies: v5, lamin B1 (LB1), merged + DAPI. Doxycyclin concentrations are indicated on the left. Scale bar: 50 mum. ( F ) Growth curve of primary fibroblasts expressing varying amounts of v5-LAP2alpha (doxycyclin concentration: 0, 0.25, 0.5 and 1 ug/ml). Dotted lines indicate SEM (n = 3). Inset: growth rate after 7 days (*p < 0.05, **p < 0.01, errors bars indicate SEM). ( G ) Western blot showing dose (doxycyclin)-dependent induction of v5-tagged LAP2alpha in normal dermal fibroblasts. Antibodies recognizin
Figure 1. Telomerase rescues dose dependent progerin-induced proliferation defects, DNA damage and gene expression changes without alleviating chromatin changes. ( A ) Immunofluorescence microscopy using v5-tag antibody showing doxycycline-dependent inducible expression of v5-progerin and its localization to the nuclear periphery. DAPI staining is shown on the bottom panels. Scale bar: 100 mum. ( B ) Western blotting showing doxycycline-dependent progerin expression in primary (left panel) and TERT+ (right panel) fibroblasts. Progerin migrates between lamin A and C as indicated (red arrowhead). Doxycycline concentrations (0-2000 ng/ml) are indicated under each lane. ( C ) Quantification of progerin-induced proliferation defects. Relative growth rates of primary (left panel) and TERT+ cells (right panel) according to progerin expression levels (*p < 0.05; ***p < 0.001 compared to control 0 ng/ml doxycycline, error bars represent SEM, 2-way ANOVA with Bonferroni and apos;s post-test). ( D ) Quantification of progerin-induced 53BP1 DNA damage foci in response to progerin expression levels, in primary (left panel, p < 0.01, chi 2 test) and TERT+ cells (right panel). 350-500 cells were counted for each condition. ( E ) Scatter plot analysis of primary (blue, red) and TERT+ (cyan, orange) cells showing an inverse correlation between H3K27me3 and progerin expression in each cell nucleus using immunofluorescence microscopy (Pearson r = -0.43 and -0.24 for TERT negative and TERT+ cells ex
Figure 3. BioID analysis reveals differential interaction of lamin A and progerin with lamina-associated polypeptide 2 (LAP2). ( A ) Western blot showing doxycycline-dependent expression of myc-BirA*-progerin (BirA-PG) and myc-BirA*-lamin A (BirA-LA) fusion constructs in primary and TERT+ cells. Antibodies are indicated: myc; lamin A, lamin C, LAP2alpha, actin, GAPDH. ( B ) Immunofluorescence microscopy confirms doxycycline-dependent induction and localization of BirA*-lamin A/BirA*-progerin fusion constructs to the nuclear periphery (green, myc tag; blue, DAPI staining). Scale bar: 20 mum. ( C ) Impaired interaction of LAP2 with progerin. Quantitative interactome of lamin A (black bars) or progerin (striped bars) with nuclear proteins lamin A, LAP2, lamin B1 and B2. Control: non-induced BirA*-lamin A (grey bars). BioID (emPAI) index: quantification based on the number of peptides for each protein detected by mass spectrometry error bars represent SEM (n = 3, ***p < 0.001, one-way ANOVA with Tukey and apos;s post-test). ( D ) Interaction of lamin A or progerin with LAP2alpha or emerin by co-immunoprecipitation. In vitro transcribed and translated v5-tagged lamin A, v5-tagged progerin, LAP2alpha and emerin (antibodies: v5-tag, LAP2alpha, emerin are indicated). Top panel: recombinant v5-tagged progerin and lamin A, myc-LAP2alpha and HA-emerin were efficiently immunoprecipitated using anti-v5-tag or anti-myc antibodies, respectively (input lanes two, three and four). Bottom panel: L
Published figure using V5 Tag monoclonal antibody (Product # 37-7500-A488)
Published figure using V5 Tag monoclonal antibody (Product # 37-7500-A488)
Western Blot (WB)
Host / Isotype
V5 synthetic peptide: Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr
Alexa Fluor® 488
PBS with 1mg/mL BSA
0.05% sodium azide
4° C, do not freeze
Epitope tags provide a method to localize gene products in a variety of cell types, study the topology of proteins and protein complexes, identify associated proteins, and characterize newly identified, low abundance or poorly immunogenic proteins when protein specific antibodies are not available. The V5 epitope tag is derived from a small epitope (Pk) present on the P and V proteins of the paramyxovirus of simian virus (SV5). The V5 tag is usually used with all 14 amino acids (GKPIPNPLLGLDST), although it has also been used with a shorter 9 amino acid sequence (IPNPLLGLD).
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Have you cited this antibody in a publication?
so we can reference it in this datasheet.
Protein Aliases: V5 Epitope Tag
Material safety data sheets (MSDS)