anti pk  (Bio-Rad)

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: Frontiers in Microbiology

Article Title: Importin α1 contributes to Venezuelan equine encephalitis virus-induced cell death, but is not required for the capsid-mediated blockage of nucleocytoplasmic trafficking

doi: 10.3389/fmicb.2026.1784611

Figure Lengend Snippet: Insertion of V5 tag at the N-terminus of capsid enables successful detection of the capsid. (A) Sequence alignment between a region of VEEV TC-83 and VEEV Cm showing key mutation sites [created in BioRender. Kehn-hall, K. (2026), https://BioRender.com/d8zhltb ]. (B,C) Schematic of VEEV and VEEV Cm with V5 tag inserted at the N-terminus of the capsid protein [created in BioRender. Kehn-hall, K. (2026), https://BioRender.com/nk52hc0 ]. (D,E) Graphs depicting the replication kinetics of V5-C and V5-Cm relative to their respective parental viruses. Vero cells were infected at an MOI 1, and viral titers were determined by plaque assay at 4, 6, 8, 12, and 24 hpi. ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Data are represented as mean ± SD. N = 3. (F–I) Western blots for V5-C and V5-Cm expression at various time points (MOI 5) in HMC3 and Vero cells. E1-V5 sample was used as a positive control. Mock-infected cells were used as a negative control, and actin was probed as a loading control.

Article Snippet: Blots were probed with mouse anti-V5 primary antibody (Bio-Rad, MCA1360) and goat anti-mouse secondary antibody (Invitrogen, 32430).

Techniques: Sequencing, Mutagenesis, Infection, Plaque Assay, Western Blot, Expressing, Positive Control, Negative Control, Control

Journal: Frontiers in Microbiology

Article Title: Importin α1 contributes to Venezuelan equine encephalitis virus-induced cell death, but is not required for the capsid-mediated blockage of nucleocytoplasmic trafficking

doi: 10.3389/fmicb.2026.1784611

Figure Lengend Snippet: Compounds 1564 and I2 reduce capsid-importin α1 interaction. (A) Co-immunoprecipitation was performed using HMC3 cells infected with VEEV V5-C at an MOI of 1. Mock uninfected cells were included as controls. Cells were collected at 9 hpi, protein lysate was quantified and normalized, and subjected to co-immunoprecipitation assay. Pull-down of V5-tagged capsid was done using V5 tag antibody, then western blot was done by probing for importin α1 (KPNA2), CRM1, or V5. A hemagglutinin (HA) antibody pulldown was included as a co-immunoprecipitation control. (B) HMC3 cells were pre-treated with respective inhibitor (50 μM) or DMSO for 1 h, then transfected with capsid-V5 plasmid for 6 h, and post-treated with the inhibitor or DMSO for 18 h. Following protein lysates collection and normalization, capsid was pulled down using V5 antibody, then probed for KPNA2 or V5. Pulldown with HA was included as a co-immunoprecipitation control.

Article Snippet: Blots were probed with mouse anti-V5 primary antibody (Bio-Rad, MCA1360) and goat anti-mouse secondary antibody (Invitrogen, 32430).

Techniques: Immunoprecipitation, Infection, Co-Immunoprecipitation Assay, Western Blot, Control, Transfection, Plasmid Preparation

Journal: Frontiers in Microbiology

Article Title: Importin α1 contributes to Venezuelan equine encephalitis virus-induced cell death, but is not required for the capsid-mediated blockage of nucleocytoplasmic trafficking

doi: 10.3389/fmicb.2026.1784611

Figure Lengend Snippet: Loss of KPNA2 reduces capsid nuclear localization. (A) 293 WT cells on PLL-coated coverslips were infected with VEEV V5-C or VEEV V5-Cm at an MOI of 10 for 9 h, then prepared for immunofluorescence imaging. Images were acquired using confocal microscope. (B) The capsid nuclear-to-cytoplasmic ratio in 293 WT cells infected with either VEEV V5-C or V5-Cm was determined using the cellular analysis tool on Gen5 image software. At least 20 cells per condition were utilized in the quantification. Statistics were determined using an unpaired t -test. ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. (C) KPNA2 KO or 293 WT cells on PLL-coated coverslips were transfected with either pcDNA3.1 or KPNA2 plasmid for 24 h, then infected with VEEV V5-C at MOI 10 for 9 h and processed for immunofluorescence microscopy. (D) Western blot confirmation for successful transfections of KO cells with pcDNA3.1 or KPNA2 plasmid. (E) The capsid nuclear-to-cytoplasmic ratio was determined using the cellular analysis tool on Gen5 mage software. At least 60 cells per condition were utilized in the quantification. ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Slides were stained with DAPI and probed for V5 tag. Blue indicates the nucleus (DAPI) and red indicates V5 tagged capsid protein. Both A, E are presented in log 10 scale.

Article Snippet: Blots were probed with mouse anti-V5 primary antibody (Bio-Rad, MCA1360) and goat anti-mouse secondary antibody (Invitrogen, 32430).

Techniques: Infection, Immunofluorescence, Imaging, Microscopy, Software, Transfection, Plasmid Preparation, Western Blot, Staining

Journal: Frontiers in Microbiology

Article Title: Importin α1 contributes to Venezuelan equine encephalitis virus-induced cell death, but is not required for the capsid-mediated blockage of nucleocytoplasmic trafficking

doi: 10.3389/fmicb.2026.1784611

Figure Lengend Snippet: Compounds 1564 and I2 significantly reduce nuclear import of VEEV capsid. (A) HMC3 cells on PLL-coated coverslips were pre-treated with 1564 (50 μM), I2 (50 μM), or DMSO, then infected with V5-C at MOI 1, and post-treated with 1564, I2, or DMSO. The slides were then probed, stained, and prepared for immunofluorescence microscopy. Images were acquired using confocal microscope. Blue indicates the nucleus (DAPI) and red indicates V5-tagged capsid protein. (B) The capsid nuclear-to-cytoplasmic ratio was determined using the cellular analysis tool on Gen5 Image software. At least 60 cells per condition were utilized in the quantification. Y-axis is in log 10 scale. ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: Blots were probed with mouse anti-V5 primary antibody (Bio-Rad, MCA1360) and goat anti-mouse secondary antibody (Invitrogen, 32430).

Techniques: Infection, Staining, Immunofluorescence, Microscopy, Software

Journal: Frontiers in Microbiology

Article Title: Importin α1 contributes to Venezuelan equine encephalitis virus-induced cell death, but is not required for the capsid-mediated blockage of nucleocytoplasmic trafficking

doi: 10.3389/fmicb.2026.1784611

Figure Lengend Snippet: Capsid interacts with other importin α family members. Following infection with VEEV V5-C (MOI 1), HMC3 cells were collected, protein lysates were quantified, and subjected to co-immunoprecipitation assays. A hemagglutinin (HA) antibody pulldown was included as a co-immunoprecipitation control. (A) Immunoprecipitation of V5-tagged capsid was done using a V5 tag antibody and the blot was probed with either a KPNA3 (importin α4) antibody or V5 antibody. (B) Immunoprecipitation of V5-tagged capsid was done using a V5 tag antibody and the blot was probed with either a KPNA4 (importin α3) or V5 antibody.

Article Snippet: Blots were probed with mouse anti-V5 primary antibody (Bio-Rad, MCA1360) and goat anti-mouse secondary antibody (Invitrogen, 32430).

Techniques: Infection, Immunoprecipitation, Control