Phospho 42 44 mapk thr202 tyr204

The following primary antibodies were used: pan-LC3 (MBL, #PM036); LAMP1 (Santa-Cruz, sc-17768); phospho-Ulk1 Ser757 (Cell Signaling, #6888S); phospho-Ulk1 Ser555 (Cell Signalling, #5869); Tom20 (Santa-Cruz, sc-17764); phospho-AMPK T172 (Cell Signaling, #2535S); phospho-4E-BP1 Thr37/46 (Cell Signaling, #2855S); phospho-42/44 MAPK Thr202/Tyr204 (Cell Signaling, #4377S); mTOR (Cell Signaling, #2983S); pTSC2 (Cell Signalling, #3617), GAPDH (Cell signaling, #2118), Tubulin (Sigma, T5168). The following secondary antibodies were used: Goat-anti-Rabbit IgG (H+L) Alexa Flour 488 (Invitrogen, A11088); Rabbit-anti-Mouse IgG (H+L) Alexa Fluor 568 (Invitrogen, A11031); Goat-anti-Rabbit IgG HRP Conjugated (Sigma-Aldrich, A0545); Rabbit-anti-Mouse IgG HRP Conjugated (Sigma-Aldrich, A9044).

The following primary antibodies were used: pan-LC3 (MBL, #PM036); p62/SQSTM1 (BD Transduction, #610077); LAMP1 (Santa-Cruz, sc-17768); pan-Ras (Oncogene Science, #OP40); E1A (Santa-Cruz, sc-58658); phospho-Ulk1 Ser757 (Cell Signaling, #6888S); phospho-Ulk1 Ser555 (EMD Millipore, ABC124); phospho-AMPK T172 (Cell Signaling, #2535S); phospho-4E-BP1 Thr37/46 (Cell Signaling, #2855S); phospho-S6 Ser235/236 (Cell Signaling, #2211S); phospho-42/44 MAPK Thr202/Tyr204 (Cell Signaling, #4377S); phospho-Akt Ser473 (Cell Signaling, #4060S); phospho-p38 MAPK Thr180/Tyr182 (Cell Signalling, #9211); mTOR (Cell Signaling, #2983S). The following secondary antibodies were used: Goat-anti-Rabbit IgG (H+L) Alexa Flour 488 (Invitrogen, A11088); Rabbit-anti-Mouse IgG (H+L) Alexa Fluor 568 (Invitrogen, A11031); Goat-anti-Rabbit IgG HRP Conjugated (Sigma-Aldrich, A0545); Rabbit-anti-Mouse IgG HRP Conjugated (Sigma-Aldrich, A9044).

Hippocampal regions after perfusion were weighed and homogenized in 10 volumes of RIPA buffer (20 mM Tris-HCl, pH 7.5, 0.1% SDS, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 2 mM EDTA, and a protease inhibitor cocktail (Roche, Mannheim, Germany)). Lysates were then centrifuged at 20,000× g at 4 °C for 30 min, and supernatant solutions were collected as the protein extract. SDS-polyacrylamide gel electrophoresis was used to separate equal amounts of protein (20 µg), which were then electroblotted onto an Immuno-Blot^TM PVDF Membrane (Bio-Rad, Hercules, CA, USA) as previously described [9 (link)]. The primary antibodies used were rabbit antibodies against 44/42 ERK1/2 (Millipore), which recognize 44-kDa ERK1 and 42-kDa ERK2, and phospho-44/42 MAPK (Thr202/Tyr204; Cell Signaling, Woburn, MA, USA), which recognize phosphorylated ERK1 and ERK2. The secondary antibody was horseradish peroxidase-linked anti-rabbit IgG (Cell Signaling). Immunoreactive bands were visualized by ECL-prime (GE Healthcare, Chalfont St. Giles, UK), and band intensities were measured using a LAS-3000 imaging system (Fujifilm, Tokyo, Japan).