Fig. 7.

Effects of sh-ASPH knockdown on ASPH expression: U87 and A172 human GBM cells were transduced with Lenti-sh-ASPH-RNA or empty vector (Lenti-EV) as a negative control and selected with puromycin. Western blot analysis was performed with polyclonal anti-ASPH, and the monoclonal A85G6 and A85E6 antibodies to examine effects of sh-ASPH on ASPH protein expression. Polyclonal anti-ASPH and A85E6 antibodies bind to the N-terminus of ASPH. However, those antibodies also detect Humbug since its amino acid sequences are virtually identical to those in the N-terminal region of ASPH. A85G6 binds to the C-terminus of ASPH which contains a catalytic domain that is not present in Humbug. All 3 antibodies can detect cleavage products of ASPH and Humbug. Each lane corresponds to an independent experiment using different preparations of the same Lenti-sh-ASPH or Lenti-EV construct. After probing ASPH, the blots were stripped and re-probed with antibodies to the p85 subunit of PI3 Kinase as a negative control (Also see Supplementary Figure 1).