Latrunculin a l5163

Phosphatase inhibitor cocktail IV (ab201115) and EDTA-free protease inhibitor cocktail (4693159001) were from Abcam (Cambridge, MA, USA) and Roche Diagnostics (Mannheim, Germany), respectively. [³H]-AMPA (DL-α-[5-methy-3H)) was obtained from Perkin Elmer (Boston, MA, USA; NET833250UC; 58.1 Ci/mmol). Potassium thiocyanate (KSCN; 207799), tetraethyl ammonium (TEA; T-2265) and latrunculin A (L5163) were procured from Sigma-Aldrich (St. Louis, MO, USA). Forskolin (HB1348) was from HelloBio. Rolipram (ab120029) and primary antibodies against PSD-95 (rabbit; ab18258) and GAPDH (mouse; ab8245) were from Abcam (Cambridge, MA, USA). Fluorescent secondary antibodies (IRDye^® 800 CW donkey anti-rabbit IgG and IRDye^® 680 RD goat anti-mouse IgG) were from LI-COR Biosciences (Lincoln, NE, USA). Other regents used in the study were of analytical grade and purchased either from Thermo Fisher Scientific or Sigma-Aldrich.

Bafilomycin A1 (Millipore) was resuspended in dimethyl sulphoxide (DMSO from Sigma-Aldrich) and used at a saturating concentration for autophagosome degradation of either 400 nM for 4–6 h or 200 nM left overnight. As Bafilomycin A1 is dissolved in DMSO, all conditions without BafA1 received an equivalent volume of DMSO. Latrunculin A (L5163) and Cytochalasin D (C8273) were purchased from Sigma-Aldrich, resuspended in DMSO and used at a final concentration of 1 μM in the culture media. MG132 (M8699) and Carbamazepine (C4024) were purchased from Sigma-Aldrich. Tat-Scrambled and Tat-Beclin1 were purchased from Protein Chemistry Technology Core, UT Southwestern Medical Center.