Monoclonal anti perk

Protein lysates from different brain regions and whole cells were prepared using RIPA buffer with 1X protease inhibitor and 1X phosphatase inhibitor cocktails from Sigma. Denatured protein samples were generated by boiling in 2X Laemmli buffer for 5 min. NuPAGE 4–12% Bis-Tris Midi Gel (Thermo Fisher Scientific) was used for electrophoresis. To enable the comparison of PERK knockdown efficiency in different brain regions, protein quantification was performed on protein lysates from brain tissue using Peirce BCA protein assay kit (Thermo scientific, # 23227), and 50μg protein per sample was loaded for western blot. The following primary antibodies were used in western blot analysis: monoclonal anti-PERK produced in rabbit (1:500, cell signaling, #3192), monoclonal anti-β-actin produced in mouse (1:1000, GenScript, A00702), monoclonal anti-p-PERK produced in rabbit (1:500, cell signaling, #3179), polyclonal anti-eIF2α [pS⁵²] produced in rabbit (1:1000, Invitrogen, 44728G), monoclonal anti-α-tubulin produced in mouse (1:1000, Sigma, T5168).

Western blotting was performed as previously described [31 (link)]. The antibodies used were as follows: monoclonal anti-JWA (1 : 500, contract produced by AbMax, Beijing, China); monoclonal anti-α-tubulin, anti-β-actin (loading control) (1 : 2000, Beyotime, Haimen, Jiangsu, China); monoclonal anti-P-ERK, anti-ERK, anti-P-AKT(473), anti-AKT, anti-Caspase 3, anti-EGFR, anti-HER3 (1 : 1000, Cell Signaling Technology, Danvers, MA, USA); monoclonal anti-Ub (1 : 500) and polyclonal anti-HER2 (1 : 1000) (Santa Cruz, Dallas, TX, USA); monoclonal anti-c-Cbl (1 : 2000), anti-Lamp2 and anti-HER2 (1 : 250, Abcam, USA).