Figure 6

Increased phosphorylation and altered glycosylation of members of the ErbB family of proteins in GALNT3 knockdown cells. (A) GALNT3 knockdown PDAC cells were associated with increase in phosphorylated forms of ErbB family of receptors such as EGFR, Her2 and Her3. (B) Lysates collected from scrambled control and GALNT3 knockdown cells were used for pull-down assays using biotin-conjugated VVA and PNA lectins. Immunoblotting of pulled down precipitates with biotinylated VVA and PNA lectins revealed effective pull down of Tn and T carbohydrate antigens, respectively. Further immunoblotting of pull-down lysates with anti-EGFR and Her2 antibodies demonstrated increase in Tn carbohydrate antigen association with EGFR and Her2 in GALNT3 knockdown CD18/HPAF and BxPC3 cells compared with scramble control cells. GALNT3 knockdown BxPC3 cells also displayed increased expression of T carbohydrate antigen on EGFR and Her2 as compared with scrambled control cells (IB-Immunoblotting, IP-Immunoprecipitation).