Aliquots containing 20 μg of protein were blotted as described previously (38, 66). In brief, protein samples were separated by 10% SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Bio-Rad). After blocking with 5% skimmed milk at room temperature for 1 hour, the blots were probed overnight at 4°C with the following primary antibodies: mouse anti-TrkB (1:500 dilution, BD Biosciences; catalog no. 610101), mouse anti-Cav3.2 (1:500, Novus Biologicals; catalog no. NBP1-22444), rabbit anti–p-AKT (1:1000 dilution, Abcam; catalog no. ab131443), rabbit anti-AKT (1:1000 dilution, Abcam; catalog no. ab179463), mouse anti–p-p38 MAPK (1:500 dilution, Cell Signaling Technology; catalog no. 9216), mouse anti-p38 MAPK (1:1000 dilution, Cell Signaling Technology; catalog no. 9228), rabbit anti–p-p44/42 MAPK (ERK1/2, 1:600 dilution, Cell Signaling Technology; catalog no. 4376), rabbit anti-ERK1/2 (1:500 dilution, Cell Signaling Technology; catalog no. 9102), rabbit anti–p-SAPK/JNK (1:2000 dilution, Cell Signaling Technology; catalog no. 4668), rabbit anti-SAPK/JNK (1:600 dilution, Cell Signaling Technology; catalog no. 9252), and rabbit anti–glyceraldehyde-3-phosphate dehydrogenase (1:5000 dilution, Sigma-Aldrich; catalog no. SAB2108266). Either anti-mouse immunoglobulin G (IgG) (catalog no. HAF007) or anti-rabbit IgG (catalog no. HAF008) horseradish peroxidase–conjugated secondary antibodies were applied (1:6000 dilution; R&D systems). Protein bands were visualized using SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific Inc.). Images were captured using a Bio-Rad ChemiDoc XRS system, and the band intensities were quantified by densitometry using Quantity One software (Bio-Rad Laboratories).

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