Rabbit anti p creb s133

For immunohistochemistry, the primary antibodies used were rabbit anti-cleaved caspase 3 (1:1000; Cat#9661, Cell Signaling Technology, Beverly, MA), mouse anti-Ki67 (1:200; Cat#556003, BD Pharmingen, Heidelberg, Germany), rat anti-BrdU (1:200; Cat#OBT0030G, Accurate Chemical), mouse anti-NeuN (1:500; Cat#MAB377, Chemicon). The secondary antibodies used were Alexa Fluor 555-conjugated goat anti-mouse IgG (1:1000; Cat#A21422, Molecular Probes), Alexa Fluor 488-conjugated goat anti-rat IgG (1:1000; Cat#A21208, Molecular Probe), Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:1000; Cat#4412, Cell Signaling), Alexa Fluor 555-conjugated donkey anti-goat IgG (1:1000; Cat#A21432, Molecular Probes), Alexa Fluor 647-conjugated goat anti-mouse IgG (1:1000; Cat#A21237, Molecular Probes). For western blots, the primary antibodies were, rabbit anti-p-aPKCζ/ι (T410/403)(1:500, Cat#9378, Cell Signaling), mouse anti- aPKCζ/ι (1:500; Cat#610175, BD), rabbit anti-p-CREB (S133)(1:500; Cat#9198, Cell Signaling), mouse anti- CREB (1:500; Cat#9104, Cell Signaling), rabbit anti-p300 (1:100; Cat#sc-585, Santa-Cruz), and mouse anti-p300 (1:1000, Cat#ab14984, Abcam). Secondary antibodies for western blots were HRP-conjugated goat anti-mouse or anti-rabbit (1:4000; Cat#7076 and #7074, Boehringer Mannheim).

iBAT samples were homogenized and diluted in lysis buffer. Homogenates were centrifuged and protein content of the supernatant was determined using a BCA protein assay kit (Thermo Scientific). After heating the samples (5 min, 95 °C), 20 μg of protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to a nitrocellulose membrane. Membranes were blocked with 5% milk and incubated overnight at 4 °C with the primary antibody rabbit anti-pCREB S133 (Cell Signaling; 1:1,000), goat anti-LPL (in house antibody; 1:5,000), mouse anti-β-actin (Sigma; 1:1,000), or rabbit anti-GAPDH (Santa Cruz; 1:1,000), followed by incubation for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit or anti-mouse; Promega at 1:5,000; anti-goat 1:20,000). Bands were visualized using SuperSignal Western Blot Enhancer (Thermo Scientific) and analyzed with a ChemiDoc Touch Imaging System (Bio-Rad). Protein expression of pCREB was normalized to that of the housekeeping protein β-actin and LPL to GAPDH.