Rabbit anti cd44

Cells were harvested in lysis buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% SDS, 1% NP-40, and 2 mM EDTA + protease inhibitor cocktail (Roche)) followed by sonication (5 min, medium power) and 15-min incubation on ice for cell lysis. Next, centrifugation was performed for 15 min at 14000 g and supernatant containing total cell extract (TCE) was collected for study. Protein concentration was measured by Bradford assay (Bio-Rad) with BSA as a standard. TCE was loaded on 10% acrylamide gel followed by SDS-PAGE. Rabbit anti-CD44 (1:1000; Abcam) and mouse anti–β-actin (1:2000; Cell signaling) antibodies were used with HRP-conjugated secondary antibodies for western blotting, and the blots were imaged using the Biorad Image Lab imaging system. Blots were cut to enable blotting for multiple antibodies. Densitometric analysis was performed using ImageJ and statistical analysis was performed using Prism6 software.

PFK118–310 was purchased from Sigma-Aldrich while ICG-001 was obtained from Selleck Chemicals. Alexa fluor 488 phalloidine and 4′, 6-diamidino-2-phenylindole (DAPI) were purchased from Sigma. The antibodies used for Western blot analysis and immunocytochemistry were: rabbit anti-CD44 (Abcam # 51037), rabbit anti-CD133 (Cell Signaling # 3663), mouse anti-CD166 (Abcam # 175422), rabbit anti-Lgr5 (Sigma-Aldrich # HPA012530), goat anti-actin (Santa Cruz # sc-1615), rabbit anti-beta-catenin (Cell Signaling # 8480), mouse antibodies specific for the active form of beta-catenin, dephosphorylated on Ser 37 and Thr 41 (Millipore, clone 8E7, # 05–665). Anti-rabbit IgG horseradish peroxidase-linked antibodies and anti-mouse IgG horseradish peroxidase-linked antibodies were from Cell Signaling whereas Cy3-conjugated anti-mouse secondary antibodies were obtained from Jackson ImmunoResearch Labs.
The following antibodies were used for flow cytometry analysis: phycoerythricine-conjugated mouse anti-human ABCB1 (BD Biosciences # 557003), phycoerythricine-conjugated mouse anti-human ABCG2 (BD Biosciences # 561180), mouse anti-human ABCC1 (BD Biosciences # 557594) and anti-mouse phycoerythricine-conjugated secondary antibodies (BD Biosciences # 555574).

The following antibodies were used in the experiments: allophycocyanin (APC)-conjugated mouse anti-CD44 from BD Biosciences, phycoerythrin (PE)-conjugated mouse anti-CD133 from Miltenyi Biotec, rabbit anti-CD44, rabbit anti-CD133, and mouse anti-OGT from Abcam, Alexa 647-conjugated rabbit anti-mouse from Invitrogen; rabbit anti-β-tubulin from Cell Signaling Technology (Danvers, MA, USA); Alexa 488-conjugated goat anti-rabbit from Molecular Probes, Inc., (Eugene, OR, USA), mouse anti-O-GlcNAc (RL2) from Thermo Fisher Scientific; mouse anti-GAPDH from Santa Cruz Biotechnoloigy Inc (Sta. Cruz, CA, USA).