V5 tagged protein detected with Mouse anti V5-Tag (MCA1360)

V5 tagged protein detected with Mouse anti V5-tag (MCA1360)

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Mouse anti V5-TAG antibody, clone SV5-Pk1 used for immunofluorecence of tranfected RAW264.7 cells.
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Subcellular localisation of Hsa_aTRACP. Immunofluoresence staining of RAW264.7 cells transfected with a Hsa_aTRACP-V5 using the Alexa488 goat anti-mouse antibody (Invitrogen) in combination with the mouse anti-V5 IgG2a (Bio-Rad) and visualised using Alexa594 Phalloidin stain (Invitrogen). DNA was stained with DAPI (Roche). Evident is the diffuse cytoplasmic distribution of Hsa_aTRACP.

From: Hadler KS, Huber T, Cassady AI, Weber J, Robinson J, Burrows A, Kelly G, Guddat LW, Hume DA, Schenk G, Flanagan JU. Identification of a non-purple tartrate-resistant acid phosphatase: an evolutionary link to Ser/Thr protein phosphatases? BMC Res Notes. 2008 Sep 4;1:78.

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Mouse anti V5 Tag antibody, clone SV5-Pk1used for immunoprecipitation of V5 tagged RNF11protein
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Dynamic associations of RNF11 with both A20 and Itch in primary neurons. N2A cells transduced with V5-RNF11 lentivirus (N2A V5-RNF11) and transfected with FLAG-A20 were harvested for immunoprecipitation (IP) with V5 antibody (A) or harvested for IP with FLAG antibody (B). Proteins were resolved by SDS-PAGE and immunoblotted with anti-A20 and RNF11. In parallel, pull-down assays with V5 antibody (C) or with Itch antibody (D) from N2A V5-RNF11 cell lysates were resolved by SDS-PAGE. Immunoprecipitates and lysates were immunoblotted with anti-Itch and RNF11. (E) and (H) Murine primary cortical neurons were stimulated with 10?ng/ml TNF-a for 0 or 30 minutes and harvested for IP with RNF11 antibody. Control IP experiments were performed with antibody omitted. Proteins were resolved by SDS-PAGE and immunoblotted with anti-A20, Itch, RNF11 and actin. (F), (G), (I) and (J) ImageJ software was used to quantify the densitometry of the immunoprecipitated bands relative to the 0-minutes time point. Each input sample’s immunoreactivity was used as a loading control. All IPs are representative of at least three independent experiments. *P?
From: Pranski EL, Dalal NV, Herskowitz JH, Orr AL, Roesch LA, Fritz JJ, Heilman C, Lah JJ, Levey AI, Betarbet RS. Neuronal RING finger protein 11 (RNF11) regulates canonical NF-?B signaling. J Neuroinflammation. 2012 Apr 16;9:67. doi: 10.1186/1742-2094-9-67.

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Mouse anti V5 Tag antibody, clone SV5-Pk1used for immunoprecipitation of V5 tagged RNF11protein
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Myristoylation mutant of RNF11 is unable to associate with Itch. SH-SY5Y shRNA-RNF11 cells were transfected with shRNA-resistant RNF11 constructs or vector. Coimmunoprecipitation experiments using V5 antibody were performed 24 hours after transfection. Immunoprecipitates and lysates were resolved by SDS-PAGE and immunoblotted with anti-A20, Itch, RNF11 or actin. Blots are representative of three independent experiments.

From: Pranski EL, Dalal NV, Herskowitz JH, Orr AL, Roesch LA, Fritz JJ, Heilman C, Lah JJ, Levey AI, Betarbet RS. Neuronal RING finger protein 11 (RNF11) regulates canonical NF-?B signaling. J Neuroinflammation. 2012 Apr 16;9:67. doi: 10.1186/1742-2094-9-67.

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Mouse anti V5 tag antibody, clone SV5-Pk1 used for detection of V5 tagged REDD1 protein by western blotting.
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Negative feedback regulation of REDD1. (A) REDD1-V5 pcDNA3 (0.4 µg) was transfected in HEK293 cells for 3 days followed by cell lysis and detection of endogenous REDD1 proteins by Western blotting. (B) HEK293 cells with and without the transfection of REDD1-V5 pcDNA3 (0.15 µg) were treated with 20 nM rapamycin for 4 or 36 hours followed by cell lysis. (C) MEF TSC2+/+ and TSC2-/- cells were treated with rapamycin (40 nM) or PP242 (2 µM) for 24 hours before cell lysis. Detection of V5 tag, REDD1, a-tubulin, phosphorylated and total S6 Kinase 1 was performed by Western blotting as described in Materials and methods.

From: Tan CY, Hagen T (2013) mTORC1 Dependent Regulation of REDD1 Protein Stability. PLoS ONE 8(5): e63970.

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Mouse anti V5 tag antibody, clone SV5-Pk1 used for detection of V5 tagged REDD1 protein by western blotting.
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REDD1 is not regulated by Cullin E3 Ubiquitin ligases. (A,B) Untransfected (A) or REDD1-V5 pcDNA3 (0.3 µg) transfected (B) HEK293 cells stably expressing tetracycline inducible dnUbc12-HA were induced with 1 µg/ml tetracycline for 24 hours (A,B) or treated with 3 µM MLN4924 (A) or 20 µM MG-132 (A,B) for 8 hours followed by cell lysis. (C,D) Untransfected HEK293 (C) or HEK293 transfected with REDD1-V5 pcDNA3 (0.3 µg) (D) were pre-treated with 3 µM MLN4924 followed by cycloheximide (40 µM) treatment and cell lysis at the indicated time points. (E) HEK293 cells stably expressing tetracycline inducible dnUbc12-HA were induced with 1 µg/ml tetracycline for 24 hours followed by cycloheximide (40 µM) treatment and cell lysis at the indicated time points.

From: Tan CY, Hagen T (2013) mTORC1 Dependent Regulation of REDD1 Protein Stability. PLoS ONE 8(5): e63970.

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Mouse anti V5 tag antibody, clone SV5-Pk1 used for detection of V5 tagged REDD1 protein by western blotting.
Image caption:
REDD1 is not degraded by Cul4a or phosphorylated by GSK3ß at Thr 23 and Thr25. (A) HEK293 cells were transfected with empty vector or tetracycline inducible dnCul4a-V5 pcDNA4/TO (1 µg) and Cul4A-V5 pcDNA3 (0.03 µg) followed by tetracycline (1 µg/ml) induction for 24 hours and cell lysis. (B) HEK293 cells were transfected with 0.5 µg Cul4a-V5 pcDNA3 for 15 hours followed by transfection of 20 nM control or Cul4a siRNAs to determine siRNAs efficiency. (C) HEK293 cells were transfected with 20 nM control or Cul4a siRNAs for 3 days followed by cell lysis. (D) REDD1-V5 pcDNA3 wild type, T23A T25A or T23D T25D plasmids (0.4 µg) were transfected in HEK293 cells for 3 days and treated with 20 µM MG-132 for 6 hours followed by cell lysis. (E) HEK293 cells were treated with 30 mM LiCl or GSK3 inhibitor IX (5 µM or 10 µM) for 20 hours followed by cell lysis. (F) HEK293 cells were co-transfected with 0.2 µg REDD1-V5 pcDNA3 and 0.3 µg GSK3ß pcDNA3 or empty pcDNA3 for 3 days followed by MG-132 (20 µM) treatment for 6 hours followed by cell lysis. (G) HEK293 cells were transfected with 3 µg FLAG-REDD1 or FLAG-FRAT1 for 3 days followed by cell lysis and FLAG immunoprecipitation. In vitro phosphorylation of REDD1 and FRAT1 was carried out as described in Materials and Methods..

From: Tan CY, Hagen T (2013) mTORC1 Dependent Regulation of REDD1 Protein Stability. PLoS ONE 8(5): e63970.

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Mouse anti V5 tag antibody, clone SV5-Pk1 used for detection of V5 tagged podocin protein by western blotting
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The podocin short isoform is retained in the ER and is found in the detergent resistant membrane fraction. A) PCR with podocin specific primers yields products for both isoforms from a human kidney cDNA library. B) Immunofluorescence staining of the canonical and the short isoform in Hela cells transfected with the corresponding constructs show that only the canonical isoform reaches the plasma membrane (arrowhead in the enlarged part of the image). The short isoform is retained in the endoplasmic reticulum. Scale bar?=?10 µm. C) DRM association of both podocin isoforms. HEK293T cells expressing the respective V5-tagged podocin isoform were lysed in 1% TX-100 on ice and subjected to sucrose density gradient centrifugation. Fractions 1–7 were collected from the top and analyzed by Western blot. Both isoforms fractionate into the DRMs (fractions 1 and 2, as identified by flotillin staining). Antibodies against the transferrin receptor (TfR) and Flotillin-2 were used as markers for the Triton soluble and insoluble fractions, respectively.

From: Völker LA, Schurek EM, Rinschen MM, Tax J, Schutte BA, Lamkemeyer T, Ungrue D, Schermer B, Benzing T, Höhne M. Characterization of a short isoform of the kidney protein podocin in human kidney. BMC Nephrol. 2013 May 6;14:102.

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Mouse anti V5 tag antibody, clone SV5-Pk1 used for detection of V5 tagged podocin protein by western blotting
Image caption:
The podocin short isoform interacts with known podocyte proteins. CD2AP, TRPC6, neprin and NEPH1 co-precipitate with both podocin isoforms (FL, full length (canoncical isoform); SI, short isoform). FLAG- and V5-tagged proteins were expressed in HEK293T cells and precipitated with anti-FLAG antibody as indicated. Western blot analysis was performed with a V5 specific antibody. Expression levels of FLAG.podocin constructs in the lysates are shown below.

From: Völker LA, Schurek EM, Rinschen MM, Tax J, Schutte BA, Lamkemeyer T, Ungrue D, Schermer B, Benzing T, Höhne M. Characterization of a short isoform of the kidney protein podocin in human kidney. BMC Nephrol. 2013 May 6;14:102.

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Mouse anti V5 tag antibody, clone SV5-Pk1 used for detection of V5 tagged podocin protein by western blotting
Image caption:
The podocin short isoform is N-glycosylated. A) PNGase-F treatment removes the double band from the short isoform in DRM fraction 7. Lysates from Figure 3 were subjected to treatment with PNGase-F and immunoblotted and detected with anti-V5 antibody. B) N to S mutation of the N-glycosylation consensus motif completely abrogates the formation of a double band. The asparagine at position 287 corresponds to amino acid 355 in the full-length protein. HEK293T cells were transfected with V5-tagged Podocin (short isoform or short isoform N287S, respectively) and lysates were immunoblotted and detected with anti-V5 antibody.

From: Völker LA, Schurek EM, Rinschen MM, Tax J, Schutte BA, Lamkemeyer T, Ungrue D, Schermer B, Benzing T, Höhne M. Characterization of a short isoform of the kidney protein podocin in human kidney. BMC Nephrol. 2013 May 6;14:102.

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Mouse anti V5 tag antibody, clone SV5-Pk1 used for the detection of V5 tagged NFAT5 protein by western blotting
Image caption:
Effect of knock down of 14-3-3-ß and/or -e on protein abundance of NFAT5. (A) Knock down of 14-3-3-ß and/or -e reduces NFAT5 protein abundance. siRNAs against 14-3-3-ß and/or -e were transfected for 3 days into HEK293 cells incubated at 300 mosmol/kg, then the medium was changed, keeping the osmolality at 300 or increasing it to 500 mosmol/kg for 8 h by adding NaCl. Upper panel is a representative Western blot. The siRNAs singly and in combination reduce NFAT5 protein abundance (mean ± SEM, n = 3, *P < 0.05). (B) Time course of the effect of 14-3-3-ß and -e in combination. As in (A) except that NFAT5 protein was measured just before NaCl was increased (zero time) and after that at the intervals that are shown. Upper panel is a representative Western blot (Mean ± SEM, *P < 0.05, n = 3). (C) Lack of effect of 14-3-3-ß and/or -e separately or combined on NFAT5 mRNA abundance. As in (A) except that NFAT5 mRNA was measured 16 h after NaCl was increased (P > 0.05, n = 3). (D–F) siRNA-mediated knockdown of 14-3-3-ß and -e in combination increases degradation of NFAT5 protein. siRNAs against 14-3-3-ß and -e were transfected for 3 days into HEK293 cells incubated at 300 mosmol/kg. (D) One hundred µg/mL cycloheximide (CHX) or vehicle (DMSO control) was added for 1 h before changing to otherwise identical media kept at 300 mosmol/kg or increased to 500 mosmol/kg by adding NaCl. Media contained CHX or DMSO. NFAT5 protein was measured by Western blot at the start and end of the 8 h increase in NaCl, and the difference (?) was calculated. (*P < 0.05, n = 3). (E and F) Measurement of the rate of NFAT5 degradation by cycloheximide chase. HEK293 cells were preincubated for 7 h at 300 or 500 mosmol/kg, then 100 µg/mL cycloheximide was added for 1 h. Cells maintained in cycloheximide were harvested at the intervals shown. (*P < 0.05, n = 3–4). (G) Lack of coimmunoprecipitation of 14-3-3-ß and -e with NFAT5. NFAT5-V5 was immunoprecipitated from HEK293 cells, then the immunoprecipitates were immunoblotted for the proteins shown. CDK5 and HSP90 coimmunoprecipitate with NFAT5-V5, but 14-3-3-ß and -e do not.

From: Izumi Y, Burg MB, Ferraris JD. 14-3-3-ß and -{varepsilon} contribute to activation of the osmoprotective transcription factor NFAT5 by increasing its protein abundance and its transactivating activity. Physiol Rep. 2014 Apr 22;2(4):e12000.

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Mouse anti V5 tag antibody, clone SV5.Pk1 used for the detection of V5 tagged podocin by immunofluorescence
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Podocin localizes to endosomal vesicles. Immunofluorescence for transiently expressed, V5- (a–c, e) or Flag-tagged (d) podocin using anti-V5 or anti-Flag antibody in HeLa cells. Costainings of endogenous calnexin (a), endogenous golgin-97 (b), endogenous EEA1 (c), Lysotracker Red (d) and eGfp-tagged and transiently expressed CD63 (e) as markers for the endoplasmatic reticulum, Golgi apparatus, early endosomes, acidic organelles and late endosomes respectively, displayed that podocin localizes to the endosomal compartment. Analogously to HeLa cells, transiently expressed V5-tagged podocin colocalizes with eGFP tagged CD63/LAMP3 in cultured human podocytes (f).

From: Gödel M, Ostendorf BN, Baumer J, Weber K, Huber TB (2013) A Novel Domain Regulating Degradation of the Glomerular Slit Diaphragm Protein Podocin in Cell Culture Systems. PLoS ONE 8(2): e57078.

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Mouse anti V5 tag antibody, clone SV5-Pk1 used for the detection of V5 tagged p53-binding protein-1 by western blotting
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hMSL2 mediates modification of 53BP1, hMSL1 and hMOF. (A) Alignment of the two peptide sequences in 53BP1 (subject) that are reported as similar to the sequence containing H2BK34 (query), and schematic showing the M-domain of 53BP1. Lysine 1568 (white circle) lies within the second Tudor domain and lysine 1690 (grey circle) lies within the nuclear localization sequence (NLS). OD represents the oligomerization domain. Also shown is lysine 1273 (black circle) reported to be ubiquitylated by RAD18. (B) Immunoblot analysis of U2OS cells that were transfected with both His-ubiquitin and HA2F-hMSL2, together with either V5-53BP1-M-domain wild-type or point mutant constructs. Mock cells were not transfected with any of the plasmids. (C and D) Immunoblot analysis of U2OS cells, with transfection of His-ubiquitin and with/without transfection of HA2F-hMSL2. Cells were treated with 10 Gy of IR as indicated and harvested 15 minutes after IR. V5-Mdomain, endogenous hMSL1 and endogenous hMOF are indicated with an arrowhead. Modified proteins are indicated with an open arrowhead.

From: Lai Z, Moravcová S, Canitrot Y, Andrzejewski LP, Walshe DM, et al. (2013) Msl2 Is a Novel Component of the Vertebrate DNA Damage Response. PLoS ONE 8(7): e68549.

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Mouse anti V5 tag antibody, clone SV5-Pk1 used for the detection of V5 tagged Rho GTPase-activating protein 28 protein by western blotting and immunofluorescence
Image caption:
Arhgap28-V5 inhibits RhoA activation and stress fiber formation in SaOS-2 cells. SaOS-2 cells were transfected with empty vector or Arhgap28-V5. A. The expression of Arhgap28-V5 was confirmed by western blotting using an antibody to V5. B. Effect of Arhgap28-V5 expression on the basal activity of RhoA (n = 5), Rac1 (n = 3) and Cdc42 (n = 3). Bars show SEM. * indicates significant difference found, p<0.05. C–D. F-actin in cells expressing Arhgap28-V5 (C) and Arhgap28R425A-V5 (D) was visualized by fluorescence microscopy using anti-V5 antibodies and Atto 488-conjugated phalloidin (representative images from 3 independent transfections). Arrows point to membrane ruffling and F-actin protrusions. Bars = 25 µm.

From: Yeung C-YC, Taylor SH, Garva R, Holmes DF, Zeef LA, et al. (2014) Arhgap28 Is a RhoGAP that Inactivates RhoA and Downregulates Stress Fibers. PLoS ONE 9(9): e107036.