St6gal 1

SW620 and SW837 cells were lysed in radioimmunoprecipitation assay buffer and Halt protease and phosphatase inhibitor cocktail (Fisher Scientific) 1 or 5 days after treatment. PDX model tumors were harvested and dissociated 1 week after completion of treatment. Samples then underwent electrophoresis and protein transfer to polyvinylidene fluoride membranes. Membranes were blocked in 5% nonfat dry milk in 1× Tris-buffered saline and 0.1% Tween-20. Blots were probed with 1:1000 antibodies against ST6GAL-1 (R&D Systems), (1:200) cleaved caspase 3 (Cell Signaling Technology), and (1:300) TNFR1 (Cell Signaling Technology). Protein loading was verified using 1:10,000 β-actin (Abcam). Membranes were incubated with horse antigoat IgG secondary antibody for ST6GAL-1 (R&D Systems) and biotinylated rabbit secondary for cleaved caspase 3 (Vector Labs) and rabbit monoclonal antibody for TNFR1 (Santa Cruz) and imaged using enhanced chemiluminescence.

Monoclonal or polyclonal antibodies to the following sialyltransferases were purchased from the following companies with antibodies used at recommended concentrations: β4GALT1 (Sigma) 1:100; β4GALNT2 (Abnova) 1:200; ST3GAL3 (Novus) 1:200; ST3GAL4 (Novus) 1:100; ST3GAL6 (Novus) 1:200; ST6GAL1 (R&D) 1:50; ST6GALNAC1 (Novus) 1:500, and ST6GALNAC2 (Novus) 1:1000. If antigen retrieval resulted in a clear background, citrate buffer was utilized. Detection was with either alkaline phosphatase or Vector Red as detailed previously.