Ab108970

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).

The cells were washed twice in ice-cold PBS and lysed in 1×SDS lysis buffer supplemented with 1× protease inhibitor cocktail (Roche Applied Science, Germany) on ice for 10 min. The mixture was then collected in 1.5 ml Eppendorf tubes, sonicated for 15 s under ice bath conditions, followed by denaturation at 95°C for 10 min. The protein concentration was determined by the BCA method (BCA Protein Assay Kit, Pierce, United States). The protein samples were separated by SDS-PAGE and blotted onto polyvinylidene difluoride membranes (Millipore, United States). Each membrane was sealed with 5% skim milk or BSA at room temperature for 2 h and then incubated overnight with the primary antibody to G3BP1 (Santa Cruz Biotechnology, United States, sc-365338; 1:1000), PKCζ (Abcam, UK, ab108970; 1:3000), p-PKCζ (Abcam, UK, ab62372; 1:3000), GAPDH (Affinity, United States, AF7021; 1:3000). On the second day, after the membranes were washed with TBST to remove the unbound primary antibody, the membranes were incubated at room temperature with conjugated secondary antibody for 1 h, and protein expression was detected using ECL (Affinity Biosciences, United States).