Rabbit anti ha mab

The following antibodies were used in this study: mouse anti-Flag mAb (used 1:1000 in WB, F1804), rabbit anti-GAPDH mAb (used 1:10000 in WB) (G9545), mouse anti-α-tubulin mAb (used 1:10000 in WB, 05–829) and mouse anti-vinculin (used 1:500 in WB, V9131) from Sigma-Aldrich (St. Louis, MO, USA); mouse anti-DLC1 mAb (used 1:500 in WB and IF, 612021) and mouse anti-FAK mAb (used 1:1000 in WB, 610088) from BD Biosciences (Heidelberg, Germany); rabbit anti-paxillin pAb (used 1:500 in WB and IF, sc-5574) and mouse anti-His mAb (used 1:200 in WB, sc-8036) from Santa Cruz Biotechnology (Dallas, TX, USA); rabbit anti-USP7 mAb (WB 1:1000, 4833), rabbit anti-GFP mAb (WB 1:1000, 2956) and rabbit anti-HA mAb (WB 1:1000, 3724) were from Cell Signaling (Danvers, MA, USA); rabbit anti-HECTD1 pAb (WB 1:1000, 20605–1-AP) was from Proteintech (Manchester, UK). HRP-labeled secondary anti-mouse and anti-rabbit IgG antibodies were purchased from and Dianova (Hamburg, Germany), Alexa-Fluor®-labeled secondary IgG antibodies were from Invitrogen (Carlsbad, CA, United States). Inhibitors used were: Bortezomib from UBPBio (Dallas, TX), MG-132 from Selleck Chemicals (Houston, TX); PR-619, HBX 41108 and P5091 from Cayman Chemicals (Ann Arbor, MI); cycloheximide from Santa Cruz (Dallas, TX).

Polyclonal antibody to B. cenocepacia Hcp was raised in rats at the University of Sheffield Biological Services unit using N-terminal His-tagged Hcp which was purified following its overproduction in E. coli as described below. Rabbit anti-FLAG mAb and anti-VSVg pAb were obtained from Sigma-Aldrich (cat. no. F7425 and V4888, respectively), whereas rabbit anti-HA mAb was purchased from Cell Signaling Technology (cat. no. C29F4). HRP-conjugated mouse anti-MBP polyclonal antibodies were obtained from New England Biolabs (cat. no. E8038S). The mouse mAb to the β subunit of E. coli RNA polymerase was obtained from Neoclone (cat. no. W0023). Proteins harbouring a polyhistidine tag were detected using the HisProbe-HRP conjugate (Thermo Fisher, cat. no. 15165) or mouse anti-KLH-conjugated His-tag peptide (BioLegend, 652501). HRP-linked goat-derived (secondary) antibodies that cross-react to rat and rabbit IgG were obtained from SouthernBiotech (cat. no. 3050–05) and Vector Laboratories Ltd (cat. no. PI-1000), respectively. HRP-conjugated rabbit anti-mouse IgG secondary antibody were purchased from Thermo Fisher (cat. no. 31450).

FITC- and APC-CD19, CD11b, GR-1, FITC-TER119, FITC-CD44, PE-Dll1, Dll4, APC-c-kit, CD11c, B220, ST2, PE/Cy7-CD4, APC/Cy7-Thy1.2 and CD19 mAbs were all purchased from BioLegend (San Diego, CA). PE-CD19, PerCP/Cy5.5-CD25, APC-CD8, DX5, PDGFRα and PE/Cy7-CD45 mAbs were purchased from Thermo Fisher Scientific (Tokyo, Japan). Anti-NotchLs mAb, HRJ1-5, was established by immunization of Armenian hamsters with a recombinant rat Jagged1-human Fc chimeric protein (34) (R and D Systems Inc, Minneapolis, MN). Expression of NotchLs assessed by HRJ1-5 was detected by biotinylated goat anti-hamster IgG Ab (Thermo Fisher Scientific) and PE-conjugated streptavidin (BD Bioscience). Intracellular staining of HA-labeled Dll1 or Dll4 was detected by rabbit anti-HA mAb (Cell Signaling Technology) and DyLight649-conjugated donkey anti-rabbit IgG Ab (BioLegend). Expression of cell surface markers was analyzed by flow cytometry with a FACSVerse (BD Biosciences).