Rabbit anti pka

Cells were rinsed twice with PBS, lysed in RIPA lysis buffer (Beyotime, China, CAT#P0013B) supplemented with a protease inhibitor (PMSF, Beyotime, China, CAT#ST506) and Phosphatase inhibitor cocktail A (Beyotime, China, CAT#P1082), and protein concentration was measured using the BCA reagent (Beyotime, China, CAT#P0012). Electrophoresis was conducted by 10% SDS-polyacrylamide gel and transferred proteins to PVDF membranes (Millipore, Cat# IPVH00010). The following primary antibodies were used for incubation overnight at 4 °C: 1:10,000 mouse anti-beta-Tubulin (Proteintech, China Cat#66240-1-Ig), 1:1000 rabbit anti-PKA (Cell Signaling, USA Cat#5842 T), 1:5000 rabbit anti-pCREB (Abcam, UK Cat# ab32096), and 1:1000 rabbit anti-CREB (Abcam, UK Cat# ab32515). All membranes were incubated with HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (Proteintech, China Cat#SA00001-1) or HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (Proteintech, China Cat#SA00001-2) for 1 h. Immunoreactive bands were detected using an enhanced chemiluminescence (ECL) kit (Advansta, USA Cat#K-12045-D10) and quantified with a gel-image analyzing system (Fusion Optix, USA).

The peri-infarct regions of cortex were lysed in RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) and centrifuged at 12,000 g for 20 min at 4 °C. Proteins were then loaded (80 μg per lane) and subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The membranes were incubated overnight at 4 °C with the primary antibodies: rabbit anti-PKA (1:1000; Cell Signaling Technology, MA, USA), rabbit anti-phospho-CREB (Ser133) (1:1000; Cell Signaling Technology, MA, USA), rabbit anti-CREB (1:1000; Cell Signaling Technology, MA, USA), or rabbit anti-BDNF (1:500, Santa Cruz Biotechnology, Dallas, TX, USA). The membranes were processed with the respective horseradish peroxidase-labeled secondary antibody (1:2000; Beyotime Institute of Biotechnology). Bands were visualized using the ECL kit (Beyotime Institute of Biotechnology), and bands were quantified using Image Quant LAS 4000 (GE Healthcare Bio-Sciences AB, Tokyo, Japan). β-Actin (1:1000; Beyotime Institute of Biotechnology) served as the loading control.

Brain tissues were lysed in radioimmunoprecipitation assay buffer (Thermo Fisher Scientific). Proteins were then separated by SDS-PAGE and transferred to PVDF membranes (Millipore) using standard procedures [72 (link)]. The antibodies used were rabbit anti-HTR6 (1:500, Abcam #ab103016), rabbit anti-phospho-S6K (Thr389, 1:1,000, Cell Signaling Technology #9205), rabbit anti-S6K (1:1,000, Cell Signaling Technology #2708), rabbit anti-phospho-Akt (Ser473, 1:1,000, Cell Signaling Technology #9271), rabbit anti-Akt (1:1,000, Cell Signaling Technology #9272), rabbit anti-phospho-PKA (Thr197, 1:1,000, Cell Signaling Technology #4781), rabbit anti-PKA (1:1,000, Cell Signaling Technology #4782), rabbit anti-phospho-CREB (Ser133, 1:1,000, Millipore #06–519), rabbit anti-CREB (1:1,000, Millipore #AB3006), and mouse anti-α tubulin (1:10,000, GeneTex #GTX628802). Protein signals were visualized with horseradish peroxidase–conjugated secondary antibodies and ECL reagent (Thermo Fisher Scientific). Quantification of immunoblots was conducted with Image J software.