Ab5073

The cell lysis buffer was used for the lysis of hDPCs. Protein was determined using a bicinchoninic analysis kit, separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and then transferred to a polyvinylidene fluoride or polyvinylidene difluoride membrane. Tween 20 was added to bovine serum albumin (BSA; 5%) phosphate buffer to block nonbinding sites on the membrane for 1 h. Protein was cultured at 4 °C overnight with the primary antibody (p65, ab16502, Abcam, 1:1000; STAT3, ab5073, Abcam, 1:1000; PKC, ab19031, Abcam, 1:2500; PKA, ab187515, Abcam, 1:5000; TRPV1, PA1–748, Thermofisher, 1:1000; Phospho NF-kB p65 (S536), ab86299, Abcam, 1:500; Phospho STAT3 (S727), ab30647, Abcam, 1:500; Phospho PKC (T497), ab59411, Abcam, 1:1000; Phospho PKA alpha (Ser338), PA5–64489, Thermofisher, 1:500; Phospho TRPV1 (Ser503), PA5–64860, Thermofisher, 1:200; Actin, ab8227, Abcam, 1:5000; HSP70, ab2787, Abcam, 1:1000; Lamin B1, ab65986, Abcam, 1:1000), and the secondary antibody was bound to HRP (ab205718, ab205719, Abcam). The protein bands were stained, and the gray values were measured on a C-DiGit Blot Scanner.

After 48 h transfection, cells in logarithmic growth phase were digested and total proteins were collected. 50μg isolated protein were separated by 10% polyacrylamide gel electrophoresis and transferred to PVDF membrane, and blocked with 5% non-fat milk for 1 h. Then, the membranes were incubated with primary Anti-ROR1 antibody (1:1000 dilution, catalogue number: ab5073, Abcam Co. Ltd, USA) for 4 h at 37° C and followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. Protein bands were visualized using the Pierce enhanced chemiluminescent (ECL) system.

Total protein from Arc and NTS were extracted by homogenization in ice-cold radioimmunoprecipitation assay buffer (phosphate-buffered saline, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mmol/l phenylmethylsulfonyl fluoride, aprotinin 5 μg/ml, leupeptin 10 μg/ml, pepstatin A 1 μg/ml and phosphatase inhibitors cocktail). Homogenates were centrifuged for 25 min at 14,000g, supernatants collected and extract normalized to total protein content. The protein concentration was measured by BCA protein assay reagent kit (Pierce, Rockford, IL, USA). Proteins were separated by 12% SDS-PAGE, transfer to nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) and blots was blocked for 1 h in 5% milk. Blots were incubated with antibodies to NPY (ab91262), SCOCS3 (ab16030), POMC (ab94446), ObRb (ab5593) and pSTAT3 (ab5073) (Abcam, Cambridge, UK, 1:1000) overnight at 4C°, and then incubated with their respective secondary antibody for one hour. Expression was normalized to GAPDH (CW bio CW0266A, Beijing, China, 1:1000) and protein intensities were determined and analyzed using Odyssey® Imager (LI-COR, Lincoln, NE, USA).