Rabbit anti creb

Western bolt analysis was performed as we previously reported (18 (link)). Primary antibodies were rabbit antiphospho-CREB-ser133 (1:1,000; Cell signaling Technology); rabbit anti-CREB (1:1,000; Cell signaling Technology); rabbit anti-Bcl-2 (1:1,000; Cell Signaling Technology). Internal control was mouse anti-β-actin (1:1,000; Sigma-Aldrich). Appropriate horseradish peroxidase-linked secondary antibodies were used for detection by enhanced chemiluminescence (Pierce).

Whole-protein extracts from cells or tumors were prepared after cell scrapping or tissue homogenization, respectively, in RIPA buffer and separated on SDS-PAGE gels (NuPAGE 4–12% Bis-Tris Protein Gels, Invitrogen). Membranes were probed using the following primary antibodies: rabbit anti-CAV1 1:500 (Abcam, ab2910), rabbit anti-HER2 1:800 (Abcam, ab131490), mouse anti β-actin 1:20,000 (Sigma, A1978), rabbit anti-ubiquitin 1:1,000 (Cell Signaling Technology, 3933 S), mouse anti-ERK 1:100 (Invitrogen, 14-9108-80), rabbit anti-pERK 1:500 (Invitrogen, 700012), rabbit anti-AKT 1:1,000 (Cell Signaling Technology, 9272 S), rabbit anti-pAKT, 1:2,000 (Cell Signaling Technology, 4060 S), rabbit anti-cleaved PARP, 1:1,000 (Cell Signaling Technology, 9541 S), rabbit anti-pHER2, 1:500 (Abcam, ab53290), rabbit anti-HER3, 1:500 (Abcam, ab32121), rabbit anti-pHER3, 1:2,500 (Abcam, ab76469), rabbit anti-EGFR 1:1,000 (Abcam, ab52894), rabbit anti-pEGFR 1:500 (Abcam, ab40815), mouse anti-pTyr 0.5 μg/mL (EMD Millipore, 05-321X), rabbit anti-CREB 1:1,000 (Cell Signaling Technology, 9197 S), rabbit anti-pCREB 1:1,000 (Cell Signaling Technology, 9198 S).
The membranes were then incubated with secondary antibodies IRDye^®800CW anti-rabbit or anti-mouse IgG 1:15,000 (LI-COR Biosciences) and imaged on the Odyssey Infrared Imaging System (LI-COR Biosciences) followed by densitometric analysis.

Rats were deeply anesthetized using pentobarbital sodium and decapitated, and then the brain was harvested, and the ACC was divided into left and right sides for protein analysis, as described in our previous publication^{10 (link)}. The protein samples were separated by SDS-PAGE (Sodium dodecyl-sulfate polyacrylamide gel electrophoresis) and transferred to a polyvinylidene difluoride membrane. In most experiments, membranes were cut according to protein marker and blocked in 5% skimmed milk at room temperature for 1 h. Then, the fragments were incubated with the corresponding primary antibodies for 24 h at 4 °C: rabbit-anti-NR2B (1:500, Abcam, Cambridge, UK), rabbit-anti-PSD-95 (1:2000, Cell Signaling Technology, Danvers, MA, USA), rabbit-anti-CREB (1:1000, Cell Signaling Technology), rabbit-anti-pCREB (1:1000, Cell Signaling Technology), and mouse-anti-GAPDH (1:10,000, Proteintech, Rosemont, IL, USA). The membrane was incubated in horseradish peroxidase-conjugated secondary antibody for 2 h at 23 °C room temperature after TBST washing. The ChemiDoc MP System (Bio-Rad, Hercules, CA, USA) was used to detect immune complex bands.

Cell were lysed in RIPA buffer supplemented with cOmplete® Protease Inhibitor Cocktail (Roche) and PhosSTOP® Phosphatase Inhibitor Cocktail (Roche). The lysates were centrifuged at 20,000g at 4 °C for 15 min, and the supernatants were incubated with DTT (100 mM) and NuPAGE® LDS Sample Buffer (Invitrogen) at 70 °C for 10 min. Proteins were separated on Novex® 4–12% Tris-Glycine Mini Gels (Invitrogen) and transferred to a PVDF membrane. The membrane was incubated with 5% skim milk at room temperature for 1 hr and subsequently with primary antibodies at 4 °C for overnight. For the detection of FABP7, the step of centrifuging cell lysates was omitted, and 5% BSA was used as a blocking solution. Primary antibodies were as follows and used at 1:1000 dilution unless otherwise stated: rabbit anti-FABP7 (#13347, Cell Signaling Technology), rabbit anti-UCP1 (U6382, Sigma Aldrich), rabbit anti-PGC-1α (1:200 v/v) (sc-13067, Santa Cruz Biotechnology), rabbit anti-PRDM16 (1:500 v/v) (ab106410, Abcam), rabbit anti-CREB (#9197, Cell Signaling Technology), and rabbit anti-phospho-CREB (#9198, Cell Signaling Technology). Appropriate secondary horseradish peroxidase-linked antibodies were used (Dako, UK). Immunoreactivity was detected with ECL Prime Western Blotting Detection Reagent (Amersham) and visualized using ImageQuant LAS 4000 mini (GE Healthcare).

Total proteins from the hippocampus were homogenized on ice in 100 μl lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) plus protease inhibitors. The extract was centrifuged, and the supernatant was collected. Protein concentrations were measured by the BCA Protein Assay Kit (Beyotime Biotechnology, China). Equivalent amounts of protein were loaded in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gels. Proteins were transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with TBST containing 5% skim milk for 2 h at room temperature. Then, the membranes were treated overnight at 4°C with the different antibodies: β‐actin (1:2000; Cell Signaling), rabbit anti‐BDNF (1:500; Abcam), rabbit anti‐ERK1/2 (1:1000; Cell Signaling), rabbit anti‐pERK1/2 (1:500; Cell Signaling), rabbit anti‐AKT (1:1000; Abcam), rabbit anti‐pAKT (1:500; Abcam), rabbit anti‐CREB (1:1000; Cell Signaling), and rabbit anti‐pCREB (1:500; Cell signaling). Next, the membranes were incubated with the respective secondary antibodies for 2 h at room temperature. Then, the membrane was detected using enhanced chemiluminescence reagent (Guan et al., 2017 (link), 2021 (link)).

Proximity ligation assays (PLA) assay was performed with the mouse/rabbit red starter Duolink kit (Sigma‐Aldrich) as per manufacturer's instructions. Briefly, N2a cells were fixed with 4% PFA for 15 min. and permeabilized with 0.5% Triton PBS for 15 min. Cells were incubated in the blocking buffer at 37°C for 1 hr in a humidified chamber. Next, cells were incubated with the mouse anti‐YAP Antibody (1:100, Santa Cruz) and the rabbit anti‐CREB (1:100, Cell Signaling) at 4°C overnight. After wash, cells were then incubated with the PLA probes at 37°C for 1 hr, and then ligation was performed at 37°C for 1 hr in a humidified chamber. Cells were then incubated with the amplification mix at 37°C for 2 hrs in a darkened humidified chamber. Cells were then mounted on coverslips using the mounting media supplied with the kit and imagined using an inverted microscope (Eclipse Ti‐E Inverted Microscope).