FIG. 8.
Determination of RASCAL's interaction with UL50 by co-IP. (A) Immunoblot analysis of protein extracts from HEK293T cells transiently transfected with LNCX or cotransfected with L-RASCALTB40/E and LNCX UL50-HA. Proteins were denatured in 3% SDS lysis buffer (lysate) or were subjected to co-IP with anti-HA Abs (left) or with anti-RASCAL Abs (right) prior to separation on 10% (UL50-HA) or 15% (RASCALTB40/E) SDS-PAGE gels. An aliquot corresponding to 2% of the original co-IP buffer volume was loaded onto the gels as the input control (input). Membranes were probed with anti-HA (1:500) or anti-RASCAL (1:1,000) Abs. (B) Immunoblot analysis of protein extracts from HEK293T cells transiently transfected with LNCX or cotransfected with LNCX UL50-HA and L-UL53-FLAG. Proteins were denatured in 3% SDS lysis buffer (lysate) or were subjected to co-IP with anti-HA Abs, prior to separation on 10% SDS-PAGE gels. Membranes were probed with anti-HA (1:500) or anti-FLAG (1:500) Abs. Expected molecular masses were 43.9 kDa for UL50-HA, 10.6 kDa for RASCALTB40/E, and 43.3 kDa for UL53-FLAG.