Skip to main content
. 2011 Mar;336(3):672–681. doi: 10.1124/jpet.110.173765

Fig. 2.

Fig. 2.

A, immunoblotting of immunoprecipitates (IP-SP-A and IP-TLR4) with anti-human SP-A (IB-SP-A) and TLR4 (IB-TLR4) antibodies, respectively, to confirm the immunoprecipitation of specific proteins from baboon lung. IP-SP-A, IP-TLR4, and adult baboon lung homogenate protein (40 μg) were run on 8% SDS-PAGE gel under nonreducing (no heating, no DTT) or partially reducing (+ heating, no DTT) or reducing (+ heating, + DTT) conditions. B, SYPRO-ruby-stained SDS-PAGE gel of IP-SP-A run under partially reducing (+ heating, no DTT) condition. Estimated molecular masses of major protein bands are shown within the gel image. Expected locations of SP-A, TLR4, and MD2 proteins are also marked. C, cross-immunoblotting of IP-SP-A and IP-TLR4 with anti-human-TLR4 (IB-TLR4) and SP-A (IB-SP-A) antibodies, respectively. Purified SP-A protein and lysate protein of HEK293 cells stably transfected with TLR4 (HEK293-TLR4) served as positive control. D, negative controls for immunoprecipitation reaction. Lanes 1, 2, 9, and 10: IP-SP-A and IP-TLR4 immunoblotted with nonspecific primary antibody. Lanes 3, 4, 11, and 12: IP-SP-A and IP-TLR4 without any antigen or lung tissue homogenate. Lanes 5 and 6: 1.5 and 1 μl of SP-A antibody, respectively. Lanes 13 and 14: 1.5 and 1 μl of TLR4 antibody, respectively. Lanes 7, 8, 15, and 16: IP reactions in the absence of immunoprecipitating antibodies in the columns. The numbers indicate molecular mass (kDa) of standard marker proteins.