Hypothalamic Protein and RNA Quantification

Hypothalami were isolated as previously described^{14 (link)}. Tissue lysis, SDS/PAGE and Western blotting were performed as previously described^{19 (link)}. Primary antibodies in Western blots included anti-IκBα (Santa Cruz, #SC847, 1:500), anti-p-TAK1 (Cell Signaling, #4531, 1:1000), anti-p-IκBα (Cell Signaling, #2859, 1:1000), anti-RelA (Cell Signaling, #3039, 1:1000), anti-p-RelA (Cell Signaling, #4764, 1:1000), and anti-β-actin (Cell Signaling, #4967S, 1:1000), and secondary antibodies were HRP-conjugated anti-rabbit or goat antibody (Pierce, 1:2000). Quantification of Western blots was processed with Image J. Total RNA was extracted from hypothalamic tissue using TRIzol® (Invitrogen), and cDNA was synthesized using M-MLV RT System (Promega). Using SYBR® Green PCR Master Mix (Applied Biosystems), expression levels of target genes were analyzed via PCR amplification and quantification. GAPDH or TBP mRNA levels were used for normalization. CSF collection and TGFβ measurement: An anesthetized mouse was placed onto the stereotactic apparatus with the head forming an angle of about 135° with the body, and then a sagittal incision in the neck skin was made inferior to the occiput, followed by penetrating a capillary tube through the dura mater into the citerna magna to draw the CSF. Serum and CSF TGF-β content were measured using TGF-β ELISA kit (R&D Systems).