control anti gfp antibodies  (Developmental Studies Hybridoma Bank)

Journal: Journal of Virology

Article Title: Design of antibody structure-guided epitope vaccines in silico to induce potent immune responses against emerging viruses

doi: 10.1128/jvi.00689-25

Figure Lengend Snippet: Analysis of protein interactions of epitope peptides with scFv1. ( A ) Analysis of protein interaction between P30 peptide and scFv1. Plasmids pEGFP-P30 and pCMV-Flag-scFv1 were constructed and co-transfected into HEK293T cells. After 48 h, the proteins were collected and incubated with magnetic beads treated with mouse anti-GFP mAb. The samples were eluted with SDS-PAGE loading buffer and verified by Western blot. Plasmids pEGFP and pCMV empty vector were used as controls. ( B ) Analysis of protein interaction between S390F peptide and scFv1. ( C ) Analysis of protein interaction between G394K peptide and scFv1. ( D ) The self-activation verification of the bait plasmid PGBKT-scFv1 on SD-deficient medium. ( E ) Negative and positive controls in yeast two-hybrid assays. The pGADT-T plasmid was co-transformed with pGBKT-lam and pGBKT-53 plasmids in Y 2 Hgold competent cells as negative and positive controls, respectively. ( F ) The protein interactions between scFv1 and the P30, S390F, and G394K peptides were analyzed by yeast two-hybrid assay. After dilution, the bait plasmid pGBKT-scFv1 was co-transformed with the pGADT-P30, S390F, and G394K plasmids in Y 2 Hgold competent cells and cultured in SD-Leu-Trp and SD-Leu-Trp-His-Ade deficient medium for 48–96 h. ( G ) After a 10-fold dilution, the number of plaques on SD-LTHA deficient medium of competent cells co-transformed with pGADT-P30, S390F, and G394K with pGBKT-scFv1 plasmids was counted. * P < 0.05. ** P < 0.01. ( H ) After 100-fold dilution, the number of plaques on SD-LTHA deficient medium. * P < 0.05. ** P < 0.01. ( I ) After 1,000-fold dilution, the number of plaques on SD-LTHA deficient medium. * P < 0.05. ** P < 0.01. ( J ) The co-localization of P30, S390F, and G394K peptides with scFv1 was observed using laser confocal microscopy. To achieve this, pEGFP-P30, S390F, and G394K were co-transfected with pCMV-DsRed-scFv1 into HEK293T cells and cultured for 48 h. The fluorescence expression of the proteins was observed and analyzed under a 63× confocal microscope after DAPI staining with an anti-fluorescence quencher.

Article Snippet: In brief, TBS-washed protein A + G magnetic beads were incubated with GFP-tagged IgG2a mAb (1:3,000, Proteintech) for 1 h at room temperature.

Techniques: Construct, Transfection, Incubation, Magnetic Beads, SDS Page, Western Blot, Plasmid Preparation, Activation Assay, Transformation Assay, Y2H Assay, Cell Culture, Confocal Microscopy, Fluorescence, Expressing, Microscopy, Staining

Journal: Autophagy

Article Title: Evidence of an unprecedented cytoplasmic function of DDX11, the Warsaw breakage syndrome DNA helicase, in regulating autophagy

doi: 10.1080/15548627.2025.2507617

Figure Lengend Snippet: DDX11 interacts with SQSTM1 to regulate the autophagic flux. (A,B,C) Immunoblotting using an antibody against SQSTM1 shows reduction in SQSTM1 level after siRNA treatment, while LC3-II protein level was not affected. (D,E,F) Control (CTRL) and DDX11 KO ( DDX11 KO) RPE-1 cells were transfected with siRNA against SQSTM1 or control scrambled siRNA for 72 h. At 48 h post -siRNA transfection, DDX11 KO cells were transfected with a vector expressing DDX11-Flag wild-type protein. Cells were serum starved for 16 h and collected. LC3-II protein level was analyzed by western blot. TUBA/tubulin was used as loading control. In the absence of SQSTM1, re-expression of DDX11 was not able to rescue the LC3 lipidation. Graphs show the quantifications of LC3-II:TUBA. (G) Proximity ligation assays (PLAs) in RPE-1 cells using antibodies against DDX11 and SQSTM1. Cells were left in full medium or serum starved for 16 h. Red spots indicate the proximity of the two proteins in the cytoplasm compartment. CTRL refers to a negative control experiment, where only the anti-DDX11 antibody was used in the PLA protocol. (H) Quantification of PLA dots in full medium (FM) and starvation condition (NO FBS). Number of cells counted, n = 50 (experiment repeated three times, in triplicates). (I) Co-immunoprecipitation experiments. RPE-1 cells were grown in full medium (FM) or serum starved for 16 h (NO FBS). After cell lysis, SQSTM1 was immunoprecipitated using a specific antibody. DDX11 co-immuno-precipitates with SQSTM1 in both conditions. (J) I n vitro co-affinity-isolation assay using purified DDX11 and SQSTM1 with anti-DDX11 antibody conjugated protein a sepharose beads. (K) Schematic diagram of the SQSTM1 full-length and truncated forms, fused to GFP, that were used to map the polypeptide chain portion required for the association with DDX11. (L) Co-immunoprecipitation experiments of SQSTM1 full-length and truncated forms, fused to GFP. RPE-1 cells were transfected with vectors expressing the indicated GFP chimeric proteins. After 24 h, co-immuno-precipitation was performed using anti-GFP antibody conjugated beads. Western blot analysis shows that DDX11 associates with the full-length SQSTM1 and the ΔUBA truncated form but not with the UBA domain alone.

Article Snippet: The following antibodies were used: mouse monoclonal anti DDX11 (Santa Cruz Biotechnology, sc271711); mouse monoclonal anti-Flag antibody (Merck, F1804); rabbit polyclonal anti-LC3 (Novus Biologicals, NB100–2220); mouse monoclonal anti-LAMP1 (Cell Signaling Technology, 15665); rabbit polyclonal anti-SQSTM1 (MBL International, PM045); mouse monoclonal anti-TUBA/α-tubulin antibody (Merck, T6199); rabbit polyclonal anti-ATG16L1 antibody (MBL Life Sciences, PM040); rabbit monoclonal anti-ATG5 antibody (Cell Signaling Technology, 129945); rabbit polyclonal anti-BECN1/BECLIN1 antibody (Cell Signaling Technology, 3738S); rabbit polyclonal anti-AKT antibody (Cell Signaling Technology, 9272S); rabbit polyclonal anti-phospho-AKT (Ser473) antibody (Cell Signaling Technology, 9271 L); rabbit polyclonal anti-BRIP1/FANCJ antibody (Novus Biologicals, NBP1–31883); mouse monoclonal anti-histone H3 antibody (Abcam, ab24834); rabbit monoclonal anti-GFP antibody (Cell Signaling Technology, 2956); mouse and rabbit HRP-conjugated secondary antibody (Merck, A9044 and 12–348, respectively); Alexa Fluor 488 anti-rabbit antibody, Alexa Fluor 555 anti-mouse antibody and Alexa Fluor 633 anti-mouse antibody (Thermo Fisher Scientific, A-11008, A-21137, A-21146, respectively).

Techniques: Western Blot, Control, Transfection, Plasmid Preparation, Expressing, Ligation, Negative Control, Immunoprecipitation, Lysis, Isolation, Purification

Journal: Autophagy

Article Title: Evidence of an unprecedented cytoplasmic function of DDX11, the Warsaw breakage syndrome DNA helicase, in regulating autophagy

doi: 10.1080/15548627.2025.2507617

Figure Lengend Snippet: DDX11 regulates ATG16L1 localization. (A,B) Control (CTRL), DDX11 KO ( DDX11 KO) and DDX11-complemented DDX11 KO ( DDX11 KO + WT DDX11 ) RPE-1 cells were transfected with ATG16L1-GFP for 24 h. Confocal imaging shows accumulation of ATG16L1 in the perinuclear area that was reverted by re-expressing the DDX11-Flag wild-type protein. Total cells analyzed: n = 50 per experiment, performed in triplicates. Scale bar: 10 µm. (C) Co-immunoprecipitation experiment using an anti-ATG16 antibody in control (CTRL) and DDX11 KO RPE-1 cells. Western blot analysis shows co-immunoprecipitation of ATG5-ATG12 in both control and DDX11 KO ( DDX11 KO) RPE-1 cells indicating that, in the absence of DDX11, the ATG12–ATG5-ATG16L1 complex formation is not affected. Asterisks denote the IgG heavy chains. (D) Control (CTRL), DDX11 KO ( DDX11 KO) and DDX11-complemented DDX11 KO ( DDX11 KO + WT DDX11 ) RPE-1 cells were transfected with a vector expressing GFP-ATG16L1 for 48 h. Co-immunoprecipitation experiment was performed using anti-GFP antibody conjugated beads. The indicated proteins were detected by western blot of the pulled-down sample using specific antibodies. (E) Control (CTRL), DDX11 KO ( DDX11 KO) and DDX11-complemented DDX11 KO ( DDX11 KO + WT DDX11 ) RPE-1 cells were transfected with GFP-ATG16L1 and mCherry-LC3 for 24 h. Then, cells were processed as described for panel A. Confocal imaging reveals that ATG16L1 and LC3 do not colocalize in DDX11 KO cells. This phenotype is reversed by re-expressing the DDX11-Flag wild-type protein. Scale bar: 10 µm.

Article Snippet: The following antibodies were used: mouse monoclonal anti DDX11 (Santa Cruz Biotechnology, sc271711); mouse monoclonal anti-Flag antibody (Merck, F1804); rabbit polyclonal anti-LC3 (Novus Biologicals, NB100–2220); mouse monoclonal anti-LAMP1 (Cell Signaling Technology, 15665); rabbit polyclonal anti-SQSTM1 (MBL International, PM045); mouse monoclonal anti-TUBA/α-tubulin antibody (Merck, T6199); rabbit polyclonal anti-ATG16L1 antibody (MBL Life Sciences, PM040); rabbit monoclonal anti-ATG5 antibody (Cell Signaling Technology, 129945); rabbit polyclonal anti-BECN1/BECLIN1 antibody (Cell Signaling Technology, 3738S); rabbit polyclonal anti-AKT antibody (Cell Signaling Technology, 9272S); rabbit polyclonal anti-phospho-AKT (Ser473) antibody (Cell Signaling Technology, 9271 L); rabbit polyclonal anti-BRIP1/FANCJ antibody (Novus Biologicals, NBP1–31883); mouse monoclonal anti-histone H3 antibody (Abcam, ab24834); rabbit monoclonal anti-GFP antibody (Cell Signaling Technology, 2956); mouse and rabbit HRP-conjugated secondary antibody (Merck, A9044 and 12–348, respectively); Alexa Fluor 488 anti-rabbit antibody, Alexa Fluor 555 anti-mouse antibody and Alexa Fluor 633 anti-mouse antibody (Thermo Fisher Scientific, A-11008, A-21137, A-21146, respectively).

Techniques: Control, Transfection, Imaging, Expressing, Immunoprecipitation, Western Blot, Plasmid Preparation