Rabbit anti pak4

The whole spinal cords from mice or prepared cells were lysed in RIPA lysis buffer (Beyotime Biotechnology Co., Ltd.) containing phosphatase and protease inhibitor mixture (Roche, 4906845001 and 04693132001). Lysates were centrifuged at 18630 g for 20 minutes at 4°C, and only the supernatants were analysed by Western blotting, as described previously.⁴⁷ Primary antibodies: rabbit anti‐PAK4 (1:1000, Cell Signaling Technology), rabbit anti‐pPAK4 at Ser474 (1:1000, Cell Signaling Technology), rabbit anti‐CREB (1:1000, Cell Signaling Technology), rabbit anti‐pCREB at Ser133 (1:1000, Cell Signaling Technology), mouse anti‐Bcl‐2 monoclonal antibody (1:500, Bioss Antibodies), rabbit anti‐PGC‐1a (1:1000, Abcam), rabbit anti‐total caspase3 (1:2000, Abcam), rabbit anti‐cleaved caspase3 (1:500, Abcam) and mouse anti‐β‐actin (1:1000, Beyotime). The blots were incubated with Alexa Fluor‐800‐conjugated secondary antibody (Goat anti‐mouse or anti‐rabbit, 1:10 000, Li‐COR) at room temperature for 1 hour. For signal detection, an Odyssey infrared imaging system (Li‐COR, USA) was used according to the manufacturer's instructions. The density of the blots was determined using Image J (http://imagej.nih.gov/ij/).