Hpa021125

For Western blot analysis, pelleted cells were lysed, protein concentration was determined, and whole cell extracts were used for SDS-Page and semi-dry Western blotting, with subsequent detection and quantification as described before in Ref. [2 (link)] Target detection was performed with antibodies against SGPL1 (HPA021125; Atlas Antibodies, Bromma, Sweden,), panAKT (C67E7), Phospho-Ser472-AKT (D9E), Phospho-Thr308-AKT (D25E6), FOXO3 (D19A7), p27kip1a (D69C12), Cyclin D1 (E3P5S) from Cell Signaling, CDK2 (E304, Abcam), and β-Actin (A5441, Sigma-Aldrich) and according secondary antibodies, anti-rabbit IgG or anti-mouse IgG (GE Healthcare, Little Chalfont, UK).

Frozen mouse liver was homogenized with a Mikro-Dismembrator S (B. Braun Biotech International) in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 50 mM NaF, 20 mM Na₄P₂O₇, 2 mM EDTA, 2 mM EGTA, 2 mM DTT, 200 µM Na₃VO₄, 40 mM β-glycerolphosphate, 10% glycerol, 1% Triton X-100), separated by SDS gel electrophoresis and blotted onto polyvinylidene difluoride membranes. SGPL1 was probed with antibody HPA021125 from Atlas Antibodies (Bromma, Sweden). Amyloid precursor protein (ab32136), fatty acid synthase (ab128856), HMG-CoA reductase (ab174830), liver X receptor (ab176323), LDL receptor (ab30532), and NPC-1 (ab134113) antibodies were from Abcam (Cambridge, UK). For PPARγ, the antibody sc-7196 from Santa Cruz Biotechnology (Dallas, TX, USA) was used. Anti-β-actin (#A5441) was from Sigma Aldrich Chemie GmbH. Horseradish peroxidase-conjugated secondary antibodies were from GE Healthcare (Freiburg, Germany), and the enhanced chemiluminescence system was from Merck Millipore (Darmstadt, Germany).