Fitc conjugated anti mouse antibody

MCF-7 cells were fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100 for 5 min and blocked with 5% BSA in PBS for 1 h. Rabbit anti-TRIM3 (HAP043396, Sigma, 1:200) and mouse anti-ERα monoclonal antibodies (SC-56833, 1:200) were used, followed by an Alexa Fluor 647 (Invitrogen, 1:400) anti-rabbit antibody and a FITC-conjugated anti-mouse antibody (Jackson ImmunoResearch, West Grove, PA, 1:400). As negative controls, samples were incubated with secondary antibodies without primary antibodies. Images were acquired under conditions fulfilling the Nyquist criterion using a Nikon A+ laser scanning confocal system with a 60X oil NA1.4 objective and pinhole size of 1.0 Airy units. The acquired images were further processed and assembled using ImageJ.

The 5 × 10⁴ cells were seeded in cover slips and cultured for 24 h before siRNA and drug treatment (see above). Cells were then fixed with 3.7% paraformaldehyde (PFA) for 15 min, and permeabilized with 0.5% Triton X-100 in PBS for 10 min. The cells were then blocked with 3% BSA for 1 h, prior to incubation at 4°C overnight with primary antibodies, including γH₂AX (Cat. 07-164, Millipore: dilution 1: 400) and 53BP1 (Cat. Sc-22760, Santa Cruz, Dallas, TX; dilution 1:400). On the following day, the cells were washed with PBS and incubated for 1 h with the secondary antibody, including FITC-conjugated anti-mouse antibody (Cat. 715-095-150, dilution 1:400, Jackson ImmunoResearch Labs, West Grove, PA) and TRITC-conjugated anti-rabbit antibody (Cat. 711-025-152, Jackson ImmunoResearch Labs; dilution 1:400). Finally, the cells were mounted with cover slips using mounting media containing DAPI stain before visualization.

Size determination of MVs_MUC1 was performed by Nanoparticle Tracking Analysis (NTA) technology (30 (link)). MVs were thawed on ice and diluted in PBS between 1:500 and 1:20,000 to achieve the optimal number of MVs/mL. Three videos (30 s each) were recorded for each sample loading, employing the NanoSight NS300 instrument (Malvern Instruments Ltd, Malvern, UK). Measurements were performed employing the NTA 2.3 analytical software. Results were shown as the average of the three recordings.
MUC1 expression on MVs_MUC1 was evaluated by flow cytometry. MVs_MUC1 (5 μg/sample) were incubated with the anti-MUC1 MoAb Ma552 (Monosan) (1:100 for 30 min, 50 μL/sample, RT). After washing in PBS w/o Mg⁺⁺ and Ca⁺⁺(1 mL/sample, 30 min at 13,000 rpm, RT), MVs_MUC1 were incubated with FITC-conjugated anti-mouse antibody (1:600; Jackson-Immunoresearch Laboratories, 50 μL/sample). MoAb MOPC21 (1:100; Sigma-Aldrich) was employed as isotype control. To exclude background noise, flow cytometry analysis was performed setting the lowest Forward Scatter Threshold [300] and the highest FSC/SSC voltage. A total of 30,000 events were acquired with low flow rate, using a FACSCanto II flow cytometer running FACSDiva data acquisition and analysis software (Becton Dickinson).

DC phenotype staining was performed using the following antibodies directly conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE): IgG₁-FITC and IgG₁-PE as isotype controls (both from Biolegend); anti-HLAII-DR-FITC, anti-CD86-FITC, anti-CD83-PE (all from BD Biosciences), anti-CD40-PE, anti-CD14-PE, and anti-CCR7-FITC (all from Biolegend). DCs (2 × 10⁵ cells/50μL sample) were incubated with conjugated MoAb (according to the manufacturer's recommendation) for 30 min at room temperature (RT). After washing (in 2 mL of PBS w/o Mg⁺⁺ and Ca⁺⁺, centrifuged at 250 × g for 5 min), cell pellet was resuspended in PBS (100 μL); at least 1 × 10⁴ events were evaluated using a FACSCanto II flow cytometer running FACSDiva data acquisition and analysis software (Becton Dickinson).
To evaluate MUC1 expressed by MUC1-DG75 cells, 1 × 10⁵ cells were incubated with MoAb Ma552 (1:40; Monosan, Netherlands, 50 μL/sample) for 30 min at RT and binding revealed with FITC-conjugated anti-mouse antibody (1:600; Jackson-Immunoresearch Laboratories, PA, USA). MoAb MOPC21 (1:100; Sigma-Aldrich, 50 μL/sample) was employed as isotype control.

17-AAG (17-(Allylamino)-17- demethoxygeldanamycin) and MG132 were purchased from Sigma and Calbiochem, respectively. The antibodies against HA, FLAG and endogenous CHIP were obtained from Sigma, while those against HSP90 and Myc were from Cell Signaling and those against GFP, ubiquitin and actin from Santa Cruz. The anti-USP19 antibody (A301-587A) was purchased from Bethyl Laboratories. The goat anti-mouse IgG-HRP antibody, goat anti-rabbit IgG-HRP, rabbit anti-goat IgG-HRP secondary antibodies and FITC-conjugated anti-mouse antibody, and Cyanine 3 conjugated anti-rabbit secondary antibody were purchased from Jackson Immuno-Research. The proteins were visualized using an ECL detection kit (Amersham Pharmacia Biotech). CytoTox-ONE^™ reagent was a product of Promega. Human USP19_a, USP19_b, USP5 and all the mutants were cloned into HA-pcDNA3 vector. The mutants (C506S, CS1M, CS2M, CS12M, ΔN393) of USP19_b, C335A mutant of USP5 and the Atx3_100Q-UIM^mut (S236A/S256A) mutant were generated using site-directed mutagenesis via PCR technique. The expression plasmid for human HSP90 was cloned into pcDNA3.1-Myc/His, and the pEGFP-N1 vector was used to express EGFP. The polyQ proteins (including Atx3_22Q, Atx3_100Q, Atx3_100Q-UIM^mut, Htt-N552_18Q, Htt-N552_100Q and Htt-N171_100Q) and TDP-35 were cloned into pcDNA3-FLAG plasmid.