Western Blot Analysis of Intestinal Proteins

Primary enterocyte homogenates were subjected to western blotting as described^{51 (link)} with antibodies against NPC1L1 (a gift from Dr. Bao Liang Song, Chinese Academy of Sciences), CD36 (Abcam), ATP-binding cassette transporter 8 (ABCG8; Novus Biologicals), and ABCA1 (Novus Biologicals). Homogenates of small intestine and colon tissue in PBS were used to determine levels of SPTLC2 (Proteintech Group) and SPTLC1 (BD Transduction). Colon tissue homogenate was used for western blotting for mucin2 (antibody from Novus Biologicals) and cleaved caspase-3 (antibody from Cell Signaling Technology). The activation of the MAPK pathway in colon was assessed using phospho-specific antibodies for ERK, p38, and JNK (Cell Signaling Technology, Cat. #9910S). Total protein of ERK, p38, and JNK was also quantified in colon (Cell Signaling Technology, Cat. #9926S). GAPDH was used as a loading control.