0.45 m nitrocellulose membrane

Protein concentrations of cell lysates, tissue homogenates, and media samples were quantified using the BCA Kit for Protein Determination (Sigma-Aldrich, St. Louis, MO, USA). A total of 50–100 µg protein per well were loaded on 5% or 10% polyacrylamide gels and transferred to 0.45 µm nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Membranes were probed and imaged using standard immunoblotting techniques, as previously described [9 (link),10 (link),21 (link)]. Proteins were detected using a horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) and SuperSignal West Pico PLUS reagents (Thermo Fisher). Anti-STAT5A (E289; ab32043; rabbit monoclonal) and OSMRβ (AF662; goat polyclonal) antibodies were purchased from Abcam and R&D Systems, respectively.

Cells were incubated on ice for 45 min with lysis buffer containing 20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 150 mM NaCl, 10% glycerol, 1 mM Na₃VO₄, 50 mM NaF, 100 mM PMSF, and protease inhibitor mixture from Roche Molecular Biochemicals (Indianapolis, IN). Cell lysates were analyzed for protein content using BCA protein assay kit from Pierce Biotechnology (Rockford, IL). Equal amounts of denatured protein samples (60 µg) were loaded onto 10% SDS-PAGE for CD133 and ALDH1A1 analysis and equal amounts of denatured protein samples (100 µg) were loaded onto 10% SDS-PAGE for Akt, phosphorylated Akt, Oct4, and Nanog analysis before being transferred to 0.45-µm nitrocellulose membranes (Bio-Rad, Hercules, CA). Transferred membranes were blocked with medium (25 mM Tris-HCl (pH 7.5), 125 mM NaCl, and 0.05% Tween 20 (TBST)) containing 5% nonfat dry milk powder for 30 min and incubated overnight with specific primary antibodies against CD133, ALDH1A1, Akt, phosphorylated Akt (Ser473), Oct4, Nanog, α-tubulin, and β-actin. Membranes were washed three times with TBST and incubated with the following appropriate horseradish peroxidase-labeled secondary antibodies: anti-rabbit IgG or anti-mouse, for 2 h at room temperature. The immune complexes were detected by SuperSignal West Pico chemiluminescent substrate (Pierce Biotechnology) and exposed to film.

Equal amounts of bacterial whole-cell lysate were resolved in 10% SDS/PAGE gel and transferred onto 0.45-µm nitrocellulose membranes (Bio-Rad). Membranes were incubated in carbo-free blocking buffer (Vector) for 1 h. For lectin-blotting, strips were probed with the following biotinylated lectins (5 µg/ml) (Vectors) for 1 hour: Sambucus nigra (SNA), Lycopersicon esculentum (Tomato) lectin (TL), Peanut agglutinin (PNA) and Concanavalin A (ConA). For blotting with Gal-1 and Gal-3, strips were incubated for 2 hours with biotinylated recombinant Gal-1 and Gal-3 (1:1000) (Peprotech). Thereafter, strips were washed with 0.3% Tween-20 in PBS and incubated with streptavidin-HRP (1:10000) (Sigma-Aldrich) for 1 hour followed by washings with 0.3% Tween-20 in PBS. Finally, strips were treated with ECL Western blotting substrate (Promega) and visualized under Fusion FX (Vilber Lourmat).

Total protein extracts were prepared as described (35 (link)). Proteins were separated on 10% tris-glycine SDS-PAGE gels, transferred to 0.45 µm nitrocellulose membranes (Bio-Rad), and probed overnight at 4°C with mouse anti-HA (1:5,000; Sigma-Aldrich, 12CA5), rabbit anti-V5 (1:5,000; Invitrogen, MA5-15253), mouse anti-c-Myc (1:2,000–1:5,000; Sigma-Aldrich, 9E10), or rabbit anti-PSTAIR (1:5,000; Millipore-Sigma, 06-923). The rabbit polyclonal anti-H3 antibody was generated by Pocono Rabbit Farm & Laboratory and used at 1:100,000 dilution. Secondary anti-mouse and anti-rabbit antibodies conjugated to horseradish peroxidase were from Jackson ImmunoResearch (115-035-003 or 111-035-003) and used at 1:10,000 dilution for 60 min at 4°C. Immunoblots were developed with Clarity Western ECL Substrate (Bio-Rad, 170-5060) and imaged on a ChemiDoc MP multimode imager (Bio-Rad).

Protein extracts were separated by SDS-PAGE in 8% slab gels (12 mA, 30 min; 30 mA, 90 min) in a MiniProtean II Cell system (BioRad). 20 µg protein extract was loaded per well including 1 µl Benzonase Nuclease HC (Novagen, Madison, WI). Blotting onto 0.45 µm nitrocellulose membranes (BioRad) was performed at 100 V for 1 h in a Mini Trans-Blot Cell wet transfer system (BioRad). Membrane blocking was carried out with 5% skimmed milk in PBS-0.1% Tween 20 (Sigma) for 1 h and washed three times with PBS-1% Tween 20 for 15, 5 and 5 min respectively. Next, membranes were incubated with 1∶500 of rabbit anti-LACK polyclonal serum for 2 h [18] (link) or 1∶10,000 of monoclonal mouse anti-L. mexicana glycosomal GAPDH antibody kindly provided by Paul Michels (University of Edinburg) [19] (link), washed again and incubated with 1∶2,000 HRP-conjugated goat anti-rabbit IgG (DAKO, Ely, UK) for 90 min. Once the wash steps were repeated, the immunoblots were developed using the ECL detection system (GE Healthcare, Pittsburg, PA) according to the manufacturer's instructions.