Alexa fluor 647 donkey anti rabbit igg

Primary antibodies: Mouse monoclonal antibody PY72 (Glenney et al., 1988 (link)) (InVivo Biotech Services, Henningsdorf, Germany), rabbit anti EGFR pY₁₀₆₈ (Cell Signaling; 1:400), goat anti EGFR (R&D Systems; 1:300). Secondary antibodies: Alexa Fluor® 568 donkey anti-rabbit IgG (Life Technologies, 1:200), Alexa Fluor® 568 donkey anti-mouse IgG (Life Technologies, 1:200), Alexa Fluor® 647 donkey anti-goat IgG (Life Technologies, 1:200), Alexa Fluor® 647 chicken anti-mouse IgG (Life Technologies, 1:200), Alexa Fluor® 647 donkey anti-rabbit IgG (Life Technologies, 1:200), IRDye® 800CW Donkey anti-Rabbit IgG (Licor, 1:10000), IRDye® 680RD Donkey anti-Mouse IgG (Licor, 1:10000).

Following cell treatment with AF488- or AF647-labelled Aβ fibrils, cells were washed with PBS, pulse vortexed and fixed for 30 min at RT in 100 µl of 1x fix/perm buffer (# 00-5523-00, Life Technologies). Cells were washed twice with addition of 200 µl of 1x permeabilization buffer (# 00-5523-00, Life Technologies) and centrifugation at 400g for 5 min at RT. Cells were stained for 30 min at RT in 30 µl of rabbit anti-HIF1A antibody (1:100, NB100-479, Novus Biologicals, diluted in permeabilization buffer), washed twice and incubated with AlexaFluor 647 donkey anti-rabbit IgG (#A31573, Life Technologies) or Pacific Blue goat anti-rabbit IgG (#P10994, Life Technologies) for 30 min at RT. Following 2 washes, stained cells were resuspended in 200 µl FACS buffer and analysed using the BD™ LSR II analyser.

BLTK-1 cells 1–2 × 10⁴ cells/well were seeded onto microscope slide coverslips and after 16 h treated with vehicle (0), MF (5 μM, 17.5 μM), vehicle (0), MF (3 μM), DXM (200 nM), MF (3 μM) + HSP90i (50 nM), HSP90i (50 nM) + DXM (200 nM) or vehicle (0), MF (3 μM), P4 (0.3 μM), AG-205 (1 μM), MF (3 μM) + AG-205 (1 μM), and P4 (0.3 μM) + AG-205 (1 μM) in stimulation medium. Cells were fixed in 4% PFA in PBS pH 7.4 for 15 min at room temperature and permeabilized for 10 min in 0.1% Triton X-100. To reduce autofluorescence cells were incubated with 100 mM NH4CL for 10 min. After blocking unspecific binding sites with 3% BSA in PBS with 0.05% Tween 20 for 30 min cells were incubated for 1 h with primary antibodies anti-GR (SC-56851, Santa Cruz Biotechnology, Dallas, TX, USA; dilution 1:400), anti-PGRMC1 (PAB20135, Abnova Corporation; dilution 1:1000) or anti-HMGB1 (ab79823, Abcam, Cambridge, UK; dilution 1:350) diluted in blocking solution. Next, cells were incubated with secondary fluorescent antibody Alexa Fluor 488 goat anti-mouse IgG (ab150113, Abcam, Cambridge, UK; dilution 1:400) or Alexa Fluor 647 donkey anti-rabbit IgG (Life Technologies, Carlsbad, CA, USA; dilution 1:600) for 45 min. To detect cell nuclei, cells were incubated with DAPI for 1 min.

Chemicals, unless indicated otherwise, were from Sigma Aldrich (St. Louis, MO, USA). Synthetic androgen R1881 was from Sigma Aldrich, extracellular matrices fibronectin, vitronectin, laminin, collagen I, collagen IV and Src inhibitor PP2 were from Millipore (Billerica, MA, USA). Cycloheximide (Sigma-Aldrich) and MG132 (EMD-Millipore, Billerica, MA, USA) were used at indicated concentrations. Antibodies to AR (N-20), Integrin β1 (M-106) and Filamin A were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Rac-1 (clone 102) was from BD Biosciences (San Jose, CA, USA). Total FAK, phospho-FAK (Y397), total Src, phospho-Src (Y416) were from Cell Signaling, Inc. (Danvers, MA, USA). RNase L monoclonal antibody was kindly provided by Robert Silverman (Cleveland Clinic). Antibodies to β-actin, monoclonal and polyclonal antibodies to Flag tag, Flag-M2 agarose beads were from Sigma Aldrich. Anti-mouse IgG and anti-rabbit IgG HRP linked secondary antibodies were from Cell Signaling, Inc. (Danvers, MA, USA) and ECL reagents were from GE Healthcare (Piscataway, NJ, USA) and Boston Bioproducts (Ashland, MA, USA). Alexa 488-labeled Phalloidin, and Alexa fluor 647 donkey anti-rabbit IgG were from Life Technologies, Carlsbad, CA, USA.

Total IgG antibody levels against VSAs expressed on the surface of IEs were measured by flow cytometry as previously described [32 (link)] with slight modifications. These antibodies are believed to primarily target P. falciparum erythrocyte membrane antigen 1 (PfEMP1) [33 (link)]. In brief, 2.5 μl of patient sera (1:20 dilution) were co-incubated with IEs at 0.2% haematocrit and at approximately 7–8% parasitaemia, diluted in PBS solution with 1% HI-FBS (Heat inactivated fetal bovine serum) for 30 min. Following incubation the cells were washed 3 times with PBS/1% HI-FBS and incubated with 25 μl of 1:100 rabbit anti-human IgG (Dako, Australia) diluted in PBS/1% HI-FBS. The cells were washed again in PBS/1% HI-FBS and incubated with Alexa Fluor 647 donkey anti-rabbit IgG in 1:500 dilution (Life Technologies, Australia) and 10 μg/ml ethidium bromide (EtBr) in PBS/1% HI-FBS for 30 min. Following incubation the cells were washed in PBS/1% HI-FBS and resuspended in ice-cold 2% paraformaldehyde fixative solution (prepared in PBS). The fixed IEs were then run through a HyperCyt^® system with a plate reader adapter (Intellicyt^®, NM, USA) connected to a Cyan flow cytometer (Beckman Coulter Inc., CA, USA) where the cells were acquired. The flow cytometry data was analysed according to a previously published method [32 (link)].