Rabbit anti lamp1

Mouse anti-VAPA (MAB5820) and VAPB (MAB58551) antibodies were from R&D Systems (diluted 1:250 and 1:500 respectively), rabbit anti-LAMP1 was purchased from Cell Signalling (D2D11, diluted 1:100), rabbit anti-NPC1 (immunoEM, diluted 1:50) was a kind gift from Elina Ikonen (University of Helisinki), rabbit anti-NPC1 (WB, diluted 1:2000) and rabbit anti-Gramd1b (diluted 1:800 for WB, 1:10 for immunno-EM) were from Abcam (EPR5209 and ab154934 respectively), and rabbit anti-STARD3 (diluted 1:1000) was a kind gift from Catherine Tomasetto (IGBMC, France). Antibody to the extracellular domain of EGFR used for gold conjugation was isolated from the Mab 108 hybridoma (ATCC, HB-9764).

For flow cytometry and ImageStream analysis: phycoerythrin (PE)-conjugated LAG3 (eBioscience), PD1-botin (RMP1–14, Biolegend), PD1-BV421 (Biolegend), (perCP-conjugated CD8a (eBioscience), and FITC-conjugated Thy1.1. For immunoprecipitation: rat-anti-mLAG3 (BD, Pharmingen monoclonal), rat-anti-mPD-1 (a gift from T. Honjo, or eBioscience, Rat IgG2), control rat-IgG1 and IgG2 (Biolegend), control rabbit IgG (Santa-Cruz or Biolegend), rabbit anti-SHP2 (Santa-Cruz or AbCam). For Western blot analysis: goat anti-LAG-3 mAb (C9B7W, R&D Systems), goat anti-PD1 mAb (R&D Systems), anti-SHP1 (AbCam), anti-SHP2 (Santa-Cruz). All the secondary antibodies were from KPL. For confocal microscopy: rat-anti-mPD-1 (RMP1–14, from Tasuku Honjo, Osaka University), goat-anti-LAG3 (R&D Systems), rabbit-anti-EEA1 (Cell Signaling), rabbit-anti-Rab11b (Cell Signaling), rabbit-anti-LAMP1 (Cell Signaling), mouse-anti-TGN38 (BD Biosciences), rabbit-anti-tubulin-r (Biolegend), pLck (Cell Signaling). The following secondary antibodies, absorbed against cross-reactive species, were used: goat-Cy3 and mouse-DyLight-649 were from Jackson ImmunoResearch and rat-Alexafluo-488 and rabbit-Alexafluo-647 antibodies were from Molecular Probe.

Cerebrospinal fluid (CSF, 270 μl) proteins were precipitated with 10% trichloroacetic acid (TCA) for ~18 h at 4°C, centrifuged for 10 min at 10,000 x g, and the pellet reconstituted in 30 μl 1X SDS-sample buffer. An aliquot of this was analyzed by Coomassie staining after separation on a 4–12% NuPage gel (Invitrogen), and total protein per lane was quantified by the Odyssey infra-red (IR) scanner in the 700 nm channel. In parallel, an aliquot was analyzed by standard Western blotting procedures for LAMP1 detection, using rabbit-anti-LAMP1 (1: 2,000, Cell Signaling) and the Odyssey IR dye labeled secondary anti-rabbit-800 antibodies (1: 10,000, LI-COR). Integrated signals for both the Coomassie stained proteins (700 nm channel) and the immuno-detected LAMP1 (800 nm channel) were used to calculate the relative levels of LAMP1/protein per CSF sample.

HeLa cells were seeded (50,000 cells/well) on 13 mm cover glasses in 24 wells plate and incubated for 24 h. The cells were treated with 100 nM siRNA-equivalent concentration alone or complexed with Short/Long PGAamine for 1.5, 4, and 24 h. The cells were washed several times with PBS, fixed with 4% paraformaldehyde for 30 min at room temperature and washed with PBS again. Cells were then permeabilized by incubation in Triton X-100 0.2% solution in PBS for 10 min, followed by blocking in a solution of 1:100 normal mouse IgG1 and normal rabbit IgG (Santa Cruz, Heidelberg, Germany). Cells were stained with mouse anti-EEA1 (BD, East Rutherford, NJ, USA) and with rabbit anti-LAMP1 (Cell Signaling Technology, Danvers, MA, USA) primary antibodies, and then with Goat anti mouse IgG-FITC and Goat anti rabbit IgG-Rhodamine secondary antibodies (Santa Cruz, Heidelberg, Germany). Intracellular trafficking was followed using Leica SP8 confocal imaging systems (×60 Magnification) (Leica Microsystems, Wetzlar, Germany), and colocalization was assessed using the Imaris software (Bitplane, Zurich, Switzerland). Averages and standard deviations (SD) were calculated based on at least 5 fields.

MiaPaCa2 cells (2 × 10⁵ per well) were seeded in 24-well plate on cover slips for 24 h. Polyplexes containing APA (3.5 μg ml⁻¹) and Cy5-labeled siRNA (100 nM, Syntezza Bioscience) were added to cells and incubated for 4, 24, and 48 h. Slides were washed twice with PBS, fixed in 4% paraformaldehyde (PFA, Electron Microscopy Sciences) at room temperature for 20 min and washed again with PBS. Next, 0.1% Triton X-100 in PBS was added for 2 min and slides were washed twice with PBS. Actin filaments were stained with fluorescein isothiocyanate (FITC)-labeled phalloidin (Sigma-Aldrich) for 40 min and slides were washed and mounted with Prolong Gold antifade reagent with DAPI (Invitrogen). For immunofluorescence staining, fixed permeabilized cells were blocked with normal mouse/rabbit IgG (Santa Cruz Biotechnology, Inc.) in blocking solution (PBS, 2% BSA) for 30 min. Cells were then incubated with mouse anti-EEA1 (BD Biosciences) and rabbit anti-LAMP1 (Cell Signaling) primary antibodies for 1 h and washed three times. Finally, anti-mouse FITC and anti-rabbit Rhodamine (BD Biosciences) secondary antibodies were applied for 30 min at room temperature and slides were washed and mounted as described above. Cellular internalization and trafficking were monitored using Leica SP5 Confocal Imaging system (Leica Microsystems).

Following compound incubation, cells in 96-well plates were fixed in 3.7% formaldehyde (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 10 min at room temperature. The cells were then washed three times with PBS and blocked with PBS + 2% BSA (Sigma-Aldrich) + 0.05% saponin (FLUKA) for 2 h at 4 °C. Primary antibody incubations were performed with anti-GLP1R (Mab7F38 from Developmental Studies Hybridoma Bank, described in [24 (link)]), rabbit anti-early endosome-associated antigen 1 (EEA1) (Cell Signaling, Danvers, MA, USA) #C45B10) or rabbit anti-LAMP1 (Cell Signaling, Danvers, MA, USA) antibodies diluted in PBS + 2% BSA + 0.05% saponin overnight at 4 °C. Cells were then washed three times with PBS + 0.05% saponin and incubated with Alexa Fluor conjugated secondary antibodies (Life Technologies, Carlsbad, CA, USA), diluted in PBS + 2% BSA + 0.05% saponin, for 2 h at room temperature. The cells were then washed three times with PBS + 0.05% saponin and incubated with DAPI (Sigma-Aldrich, St. Louis, MO, USA) to counterstain the nuclei and, in some experiments, CellMask Deep Red (Life Technologies, Carlsbad, CA, USA) was used as a counterstain for the whole cell. Images were acquired on a Yokogawa CV7000 spinning disk confocal microscope with a 60× water objective, with at least 8 fields of view acquired from duplicate wells for each condition acquired.