Ab63638

The L4–L6 DRGs were dissected in cold RIPA buffer. Plasma membrane protein and cytosol protein were isolated with the protein extraction kit (SM-005, Invent Biotechnologies, MN, USA) following the manufacturer’s instructions. The protein samples were separated and transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories, Inc., CA, USA). The PVDF membranes were incubated in blocking buffer for 1 h at room temperature followed by incubating in a primary antibody against NOX2 (1:1000, ab80508, Abcam, Bristol, UK), PKC (1:1000, ab31, Abcam), PKCε (1:1000, ab63638, Abcam), transferrin receptor (TfR) (1:1000, QG215340, Invitrogen, USA), and β-actin (1:1000, 20536, Proteintech, IL, USA) overnight at 4 °C, and then the membranes were incubated in HRP-conjugated secondary antibody. Imaging system (iBright FL1000, Thermo Fisher Scientific, MA, USA) was used to detect the immunocomplexes by chemiluminescence (ECL) solution (Millipore Bioscience Research Reagents, USA). The immunostained bands were quantified by ImageJ software (Version: k 1.45).

A polyclonal antibody against the excitatory amino acid transporter 3 (EAAT3) was purchased from Santa Cruz Biotechnology (#sc-25658, Santa Cruz, TX, United States). Polyclonal antisera obtained from Chemicon (Temecula, CA, United States) were used for detection of glutamate-aspartate transporter EAAT1 (GLAST, #AB1782) and glial glutamate transporter EAAT2 (GLT-1, #AB1783). Protein kinase C-alpha (PKCα) was detected by a monoclonal antibody obtained from BD Biosciences (#610107, Heidelberg, Germany). A polyclonal antibody against actin was purchased from SIGMA (#A5060 St. Louis, MO, United States). Morphology of hippocampal neurons was visualized by a polyclonal antiserum against microtubule associated protein 2 (MAP2, #AB5622) and neurofilament protein of 200 kDa (#AB5256) from Chemicon International (Hofheim, Germany). An affinity purified polyclonal rabbit IgG against full length C3bot developed by our group was applied (Rohrbeck et al., 2014 (link)). To detect the phosphorylation levels of EAAT3 a polyclonal antibody directed against phosphotyrosine was purchased from Santa Cruz Biotechnology (#sc-7020). Antibodies against protein kinase C isoforms γ (monoclonal #ab71558), ε (polyclonal #ab63638), and ζ (polyclonal #ab108970) were obtained from Abcam (Cambridge, United Kingdom).