Ab2733

Cells were cultured overnight on poly-D-lysine-coated coverslips in 24 well plates and treated with 200 ng/ml of DOX, encapsulation-DOX, covalent conjugation-DOX, PEG coat-DOX for the indicated time. Cells were fixed with 4% paraformaldehyde in medium overnight at 4 °C. Permeabilization was performed in PBS with 0.1% Triton X-100 for 15 min at RT. After a blocking period of 2 hrs with 1% bovine serum albumin (BSA, Generay Biotech Co., Shanghai, China) in PBS, cells were incubated with EEA-1 (polyclonal, ab2900, Abcam) and mannose 6-phosphate receptor (M6PR, a late endosome marker, 1:1000, ab2733, Abcam) antibodies overnight at 4 °C in the absence of light. After washing twice times with PBS, cells were incubated with Alexa Fluor 405 Goat Anti-Rabbit IgG (H + L) and Alexa Fluor 405 Goat Anti-Mouse IgG (H + L) (1:200 dilution factor, A11008, A21057, Molecular Probes) for 2 hrs at RT. Lastly, cells were mounted, visualized using confocal microscopy LSM700 (Carl Zeiss, Germany) and analyzed using the ZEN software. The number of co-localized (yellow color: co-localized section of DOX and intracellular vesicles) regions was counted in each cells (where a total 10 cells were analyzed for determining the number of EE or LE at a given time).

Cells were seeded into 12-well plates on the day before treatment with siRNAs or drugs. After treatment, the cells were washed with phosphate-buffered saline (PBS) and then fixed with 4% paraformaldehyde for 15 min at room temperature. The cells were then blocked with 5% goat serum and incubated with a mouse anti-IGF-IIR antibody (ab2733, Abcam) overnight at 4 °C. After staining with the primary antibody, the cells were incubated with a rabbit anti-mouse HRP-conjugated antibody for 1.5 h at room temperature. Finally, the cells were washed and incubated with the HRP substrate (1-Step Ultra TMB solution, Thermo Scientific Pierce, Rockford, IL, USA) for 30 min. The reaction was stopped using 1 M sulfuric acid. The sample was measured at 550 mm.

Antibodies used in this study were mouse CD-MPR (1/100, 22d4, Developmental Studies Hybridoma Bank (DSHB)), mouse GalNAc-T2 (neat, UH-4, gift from U. Mandel and H. Clausen), rabbit GCC88 (1/300, HPA021323, Sigma), mouse CI-MPR (1/100, ab2733, Abcam), rabbit giantin (1/300, HPA011008, Sigma), mouse GM130 (1/300, 610823, BD Biosciences), mouse golgin-245 (1/200, 611281, BD Biosciences), rabbit golgin-84 (1/300, HPA000992, Sigma), rat HA (1/300, 3 F10, Roche), sheep TGN46 (1/300, AHP500, AbD serotec), rabbit TMF (1/300 HPA008729, Sigma), and rabbit ZFPL1 (1/500, HPA014909, Sigma).

The human monoclonal antibody 860-55D against intact capsids was purchased from Mikrogen (Neuried, Germany). The monoclonal mouse antibody 3113-81C (US Biological, Boston, MA) was used for the detection of viral proteins by Western blot as well as for the detection of progeny virus by immunofluorescence. A polyclonal chicken anti-Gb4 IgY antibody was a gift from J. Müthing (University of Münster). Antibodies against late endosomes (M6PR, ab2733), lysosomes (Lamp1, ab25630), cis-Golgi (GM130, ab52649) and GAPDH (ab9485) were obtained from abcam (Cambridge, UK). A rat anti-FLAG monoclonal antibody (200474) was purchased from Agilent (Santa Clara, CA). MS2 capsid proteins were detected with a polyclonal rabbit antibody (ABE76-I, Merck Millipore, France).