Anti lamp1 h4a3

Cells were seeded at 6 × 10⁴ in 24-well plates with cover glass for overnight culture and infected with GAS for 30 min. For acid indicator stain, lysotracker (red DND-99; Invitrogen) was added at a final concentration of 75 nM for 1-h incubation before GAS infection. Extracellular bacteria were killed by 100 µg/ml gentamicin. At various time points postinfection, the cells were fixed with 4% paraformaldehyde, permeabilized with 50 µg/ml digitonin, and stained with anti-LC3 (pM036; MBL) and anti-LAMP-1 (H4A3; Santa Cruz) antibodies at room temperature for 1 h. After the cells were washed with PBS, they were stained with Alexa Fluor-conjugated secondary antibodies and DAPI for 40 min, and the samples were then analyzed by confocal microscopy (FV1000; Olympus).

Antibodies used for western blotting were as follows: rabbit polyclonal anti-Rer1 (R4407, Sigma-Aldrich, St. Louis, MO, USA), anti-Hrd1 (H7915, Sigma), anti-gp78 (9590, Cell Signaling Technology, Danvers, MA, USA), a rabbit monoclonal anti-Calnexin (C5C7, Cell Signaling), goat polyclonal anti-GFP (RDI, Fitzgerald Industry, Concord, MA, USA), mouse monoclonal anti-α-actin (C4, Millipore, Billerica, MA, USA), anti-FLAG (M2, Sigma-Aldrich), and anti-GSK3β (610201, BD Transduction Laboratories, San Diego, CA, USA) antibodies. Antibodies used for immunoprecipitation were as follows: mouse monoclonal anti-GFP (3E6, Q-Biogene, Carlsbad, CA, USA), anti-α-tubulin (DM1A, Sigma-Aldrich), and anti-FLAG (M2, Sigma-Aldrich) antibodies. Antibodies used for immunocytochemistry were as follows: rabbit polyclonal anti-Rab7 (a gift from Y. Wada, Osaka University)49 (link), anti-ERGIC-53 (E1031, Sigma-Aldrich), mouse monoclonal anti-PDI (1D3, Enzo Life Sciences, Plymouth Meeting, PA, USA), and anti-Lamp1 (H4A3, Santa Cruz Biotechnology, CA, USA) antibodies. Alexa Fluor® 555 or 594-conjugated anti-mouse or anti-rabbit immunoglobulin Gs (IgGs) (Life Technologies) were used as secondary antibodies.
MG132 was purchased from the Peptide Institute (Osaka, Japan). Bafilomycin A1 was purchased from Wako Pure Chemicals (Osaka, Japan). Cycloheximide was purchased from MP Biomedicals (Solon, OH, USA).

Postmortem brain lysates were prepared from slices of individual tissues, extracts were resolved by sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by Western blotting as previously described [33] . The following antibodies were employed: a mouse monoclonal anti-LAMP2 (H4B4) (1:2000, Santa Cruz Biotechnology, sc18822), a mouse monoclonal anti-LAMP1 (H4A3) (1:2000, Santa Cruz Biotechnology, sc20011), a rabbit monoclonal anti-ACTIN (1:400, Sigma-Aldrich, A2066/030M4844), a monoclonal anti-α-Tubulin (clone DM1A, 1:10,000, Sigma-Aldrich T6199), a rabbit polyclonal Anti-Phospho-AKT473 antibody (1:500, Cell Signaling, 9271), a rabbit polyclonal Anti-AKT antibody (1:500, Cell Signaling, 9272), a rabbit polyclonal Anti-Phospho-ERK antibody (1:500, Cell Signaling, 4370) or a rabbit polyclonal anti-ERK antibody (1:500, Cell Signaling, 4695). The final detection was performed as previously described [33] .