v5 tag  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Dredd-mediated cleavage of Kenny uncouples the IKK complex from selective autophagy to enable innate immunity

doi: 10.64898/2026.04.24.720600

Figure Lengend Snippet: (A) Adult Canton S flies were fed with 5 % sucrose and 100 µM chloroquine for 16 h. Endogenous Ref(2)P, Kenny and Atg8a levels in lysates from adult intestines were analysed with Western blotting with anti-Ref(2)P, anti-Kenny, anti-Atg8a and anti-Tubulin antibodies, n=3. (B) Adult Canton S flies were fed with 5% sucrose and Ecc15 for 16 h, Ref(2)P and Kenny levels were analysed in lysates from dissected intestines by Western blotting with anti-Ref(2)P, anti-Kenny, and anti-Actin antibodies. The relative protein levels of endogenous Ref(2)P and Kenny levels were quantified, n>4. (C) Adult Canton S and Atg8a-GFP flies were fed with 5% sucrose and Ecc15 for 16 h before whole flies were lysed and GFP-immunoprecipitations were performed and the samples were analysed by Western blotting with anti-Ref(2)P, anti-Kenny, anti-Atg8a, and anti-Actin antibodies. The ratio of immunoprecipitated Ref(2)P and Kenny to total Ref(2)P and Kenny was quantified, n=3. (D) Adult wildtype Canton S and GFP-Kenny expressing flies were fed with 5% sucrose and Ecc15 for 16 h before lysis. GFP-immunoprecipitations were performed and the samples were analysed by Western blotting with anti-Ref(2)P, anti-GFP, and anti-Actin antibodies. The ratio of immunoprecipitated Ref(2)P to total Ref(2)P was quantified, n=3.

Article Snippet: The following antibodies were used: anti-GFP (ab6556, Abcam), anti-HA (clone 3F10, #11867423001, Roche), anti-V5 (Clone SV5-Pk1, #MCA1360, Bio-Rad), anti-Ref(2)P (ab178440, Abcam), anti-Ref(2)P, and anti-Actin (C-11, sc-1615, Santa Cruz).

Techniques: Western Blot, Immunoprecipitation, Expressing, Lysis

Journal: bioRxiv

Article Title: Dredd-mediated cleavage of Kenny uncouples the IKK complex from selective autophagy to enable innate immunity

doi: 10.64898/2026.04.24.720600

Figure Lengend Snippet: (A) Dissected intestines from adult flies expressing mCherry-Atg8a (magenta) crossed with flies expressing HA-tagged wildtype or D21E mutant Kenny were stained for HA (yellow) and Ref(2)P (cyan). Co-localisation of Atg8a, Kenny and Ref(2)P is indicated as white puncta in the merged image. Midguts were imaged by confocal microscopy using at least 3 intestines per repeat, n=3. Scale bar 100 µm. (B) Adult control UbiGal4 flies and flies expressing HA-tagged wildtype or D21E mutant Kenny by control of UbiGal4 were fed with 5% sucrose and Ecc15 for 16 h before lysis. HA-immunoprecipitations were performed and the samples were analysed by Western blotting with anti-Ref(2)P, anti-HA and anti-Actin antibodies, n=5. The relative interaction between Ref(2)P and Kenny were quantified. (C) Adult control DaGal4 flies and flies expressing V5-tagged wildtype or Δ21 mutant Kenny by control of DaGal4 were lysed and Kenny expression levels were analysed with Western Blotting with anti-Kenny (upper panel), anti-V5 (middle panel), and anti-Actin antibodies, n>3.

Article Snippet: The following antibodies were used: anti-GFP (ab6556, Abcam), anti-HA (clone 3F10, #11867423001, Roche), anti-V5 (Clone SV5-Pk1, #MCA1360, Bio-Rad), anti-Ref(2)P (ab178440, Abcam), anti-Ref(2)P, and anti-Actin (C-11, sc-1615, Santa Cruz).

Techniques: Expressing, Mutagenesis, Staining, Confocal Microscopy, Control, Lysis, Western Blot

Journal: bioRxiv

Article Title: Dredd-mediated cleavage of Kenny uncouples the IKK complex from selective autophagy to enable innate immunity

doi: 10.64898/2026.04.24.720600

Figure Lengend Snippet: (A) Drosophila S2 cells were transfected with empty vector, V5-tagged Dredd, and HA-tagged Kenny. The cell lysates were analysed by Western blotting using anti-HA, anti-V5, and anti-Actin antibodies, n>3. (B) Drosophila S2 cells were transfected with empty vector, HA-tagged Dredd and V5-tagged Kenny. V5-immunoprecipitations were performed and the samples were analysed by Western blotting with anti-HA, anti-V5, and anti-Actin antibodies, n=3. (C) Schematic representation of Dredd. (D) Drosophila S2 cells were transfected with empty vector, V5-tagged Kenny, and HA-tagged pro-domain or caspase domain of Dredd. V5-immunoprecipitations were performed and the samples were analysed by Western blotting with anti-HA, anti-V5, and anti-Actin antibodies, n=3. ( E ) Drosophila S2 cells were transfected with V5-tagged Kenny, and HA-tagged DED1 of Dredd. V5-immunoprecipitations were performed and the samples were analysed by Western blotting with anti-HA, anti-V5, and anti-Actin antibodies, n=3. (F) Schematic representation of the HA-tagged Kenny construct showing the positions of D21E, D27E, D67E and D88E point mutations. (G) Drosophila S2 cells were transfected with empty vector, V5-tagged Dredd, and HA-tagged Kenny WT and HA-tagged Kenny mutants D21E/D27E/D67E/D88E. The cell lysates were analysed by Western blotting using anti-HA, anti-V5, and anti-Actin antibodies, n>3.

Article Snippet: The following antibodies were used: anti-GFP (ab6556, Abcam), anti-HA (clone 3F10, #11867423001, Roche), anti-V5 (Clone SV5-Pk1, #MCA1360, Bio-Rad), anti-Ref(2)P (ab178440, Abcam), anti-Ref(2)P, and anti-Actin (C-11, sc-1615, Santa Cruz).

Techniques: Transfection, Plasmid Preparation, Western Blot, Construct

Journal: bioRxiv

Article Title: Dredd-mediated cleavage of Kenny uncouples the IKK complex from selective autophagy to enable innate immunity

doi: 10.64898/2026.04.24.720600

Figure Lengend Snippet: (A) Structural modelling of the Dredd-Kenny interaction. Dredd (blue, AlphaFold: Q8IRY7) is modelled on the complex of two KSHV-FLIP (grey) proteins associated with a NEMO (pink) dimer (PDB: 3CL3). The molecular graphics and analyses were performed with the UCSF Chimera package . ( B ) Drosophila S2 cells were transfected with empty vector, HA-tagged wildtype, G98R, or C386A point mutant of Dredd and V5-tagged Kenny. V5-immunoprecipitations were performed and the samples were analysed by Western blotting with anti-HA, anti-V5, and anti-Actin antibodies. The ratio of immunoprecipitated Dredd to total Dredd was quantified, n=4. (C) Drosophila S2 cells were transfected with empty vector, V5-tagged wildtype, G98R, or C386A point mutant of Dredd and HA-tagged Kenny. Kenny cleavage was analysed from transfected S2 cells by Western blotting with anti-HA, anti-V5, and anti-Actin antibodies. The ratio of cleaved to total Kenny was quantified, n=5. (D) Adult male guts from Canton S flies or flies expressing GFP-Kenny ( NP1Gal4>UAS-GFP-Kenny ) in a wildtype, dredd D44 , or dredd L23 mutant background, fed with 5% sucrose and Ecc15 , were dissected. Kenny cleavage was analysed from dissected guts lysed in lysis buffer and analysed by Western blotting with anti-GFP and anti-Actin antibodies, n=3.

Article Snippet: The following antibodies were used: anti-GFP (ab6556, Abcam), anti-HA (clone 3F10, #11867423001, Roche), anti-V5 (Clone SV5-Pk1, #MCA1360, Bio-Rad), anti-Ref(2)P (ab178440, Abcam), anti-Ref(2)P, and anti-Actin (C-11, sc-1615, Santa Cruz).

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Immunoprecipitation, Expressing, Lysis

Journal: bioRxiv

Article Title: Dredd-mediated cleavage of Kenny uncouples the IKK complex from selective autophagy to enable innate immunity

doi: 10.64898/2026.04.24.720600

Figure Lengend Snippet: (A) Dissected intestines from adult flies expressing mCherry-Atg8a (magenta) crossed with flies expressing HA-tagged wildtype or D21E mutant Kenny were stained for HA (yellow) and Ref(2)P (cyan). Co-localisation of Atg8a, Kenny and Ref(2)P is indicated as white puncta in the merged image. Midguts were imaged by confocal microscopy using at least 3 intestines per repeat, n=3. Scale bar 100 µm. (B) Adult control UbiGal4 flies and flies expressing HA-tagged wildtype or D21E mutant Kenny by control of UbiGal4 were fed with 5% sucrose and Ecc15 for 16 h before lysis. HA-immunoprecipitations were performed and the samples were analysed by Western blotting with anti-Ref(2)P, anti-HA and anti-Actin antibodies, n=5. The relative interaction between Ref(2)P and Kenny were quantified. (C) Adult control DaGal4 flies and flies expressing V5-tagged wildtype or Δ21 mutant Kenny by control of DaGal4 were lysed and Kenny expression levels were analysed with Western Blotting with anti-Kenny (upper panel), anti-V5 (middle panel), and anti-Actin antibodies, n>3.

Article Snippet: The following antibodies were used: anti-GFP (ab6556, Abcam), anti-HA (clone 3F10, #11867423001, Roche), anti-V5 (Clone SV5-Pk1, #MCA1360, Bio-Rad), anti-Ref(2)P (ab178440, Abcam), anti-Ref(2)P, and anti-Actin (C-11, sc-1615, Santa Cruz).

Techniques: Expressing, Mutagenesis, Staining, Confocal Microscopy, Control, Lysis, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The protein phosphatase 2A-B56α complex regulates N-Myc degradation in neuroblastoma

doi: 10.1016/j.jbc.2026.111298

Figure Lengend Snippet: PP2A activity is dependent on the N-Myc phosphorylation sites S62 and T58. A , Kelly cells were transduced with pLenti6.3 expressing V5-tagged WT N-Myc and mutant S62D or T58A N-Myc. Cells were treated with 25 μg/ml cycloheximide in a chase experiment to assess protein stability over 60 min by immunoblot of whole cell lysate for V5-tagged N-Myc. B , quantification of V5-tagged N-Myc plotted after normalization to Vinculin and the untreated control. Data plotted as mean ± S.D. are from four independent experiments ( n = 4), and half-lives were determined from exponential decay curve fitting. C , Kelly cells expressing the WT, S62D, and T58A N-Myc are treated with DMSO or 20 μM DT-061 for 3 h and immunoblotted for V5-tagged N-Myc, representative images are presented. D , quantification of V5-tagged N-Myc after normalization to Vinculin and DMSO controls from three independent experiments ( n = 3). Statistical significance was determined by two-way ANOVA; ∗ p < 0.05; ns, not significant. E , representative images of clonogenic assays of Kelly WT and S62D N-Myc plated at low density and treated with DT-061 for 14 days. F , colony formation assays from panel E were quantified by absorbance of dissolved crystal violet stained colonies, and plotted data represent the mean ± S.D. of three independent biological replicates (two technical replicates for each biological replicate).

Article Snippet: Membranes were blocked in 5% non-fat milk/TBST for 1 hour and incubated overnight at 4 °C with primary antibodies: c-Myc (ABclonal, A19032, 1:1000), phospho S62 c-Myc (Abcam, ab185656, 1:1000), N-Myc (Abcam, ab16898, 1:1000), Vinculin (Santa Cruz Biotech, sc-73614, 1:5,000), GAPDH (Santa Cruz Biotech, sc-32233, 1:2000), V5-tag (Cell Signaling Technology, 13,202, 1:1000), AP2β (Cell Signaling Technology, 2509, 1:1000), GATA3 (Cell Signaling Technology, 5852, 1:1,000), PHOX2B (Santa Cruz Biotech, sc-376997, 1:1,000), PARP (Cell Signaling Technology, 9542L, 1:1000), Cleaved caspase-3 (Cell Signaling Technology, 9664L, 1:1,000), and PPP2R2A (Santa Cruz Biotech, sc-81606, 1:1000).

Techniques: Activity Assay, Phospho-proteomics, Transduction, Expressing, Mutagenesis, Western Blot, Control, Staining