Ab180655

Immunoblots were performed with samples containing total protein (40 μg) and 12% NuPage Bis-Tris or 7% Tris-Acetat polyacrylamide gels (LifeTechnologies). The membranes were probed with rabbit polyclonal anti-PER1 (1:500; Abcam ab136451), rabbit polyclonal anti PER2 (1:250; ab180655), rabbit monoclonal anti-PER3 (1:2500; ab177482), rabbit polyclonal anti-DBP (1:400; ab22824), rabbit polyclonal anti-Twist1 (1:400; ab49254) and rabbit polyclonal anti-TWIST2 antibodies (1μg/ml; ab66031). The secondary antibody was a horseradish peroxidase-linked goat anti-rabbit IgG (various concentrations; ab97051), and it was detected by the chemiluminescence technique using the Immobilon Western ECL system (Millipore). For a loading control, we used a mouse monoclonal anti-p84 antibody (nuclear matrix protein 84; 1:2000; Abcam ab487). The secondary antibody was a horseradish peroxidase-linked goat anti-mouse IgG (1:5000; Abcam; ab20043). Densitometric analysis was performed using the ImageJ software and the intensity of the control lanes were set as 1.

Whole brains were homogenized in RIPA buffer with protease inhibitors at 4°C. After centrifugation at 13,000 × g at 4°C for 15 min, protein amounts in the supernatant were quantified using the BCA Assay kit (Pierce Biotechnology). Proteins from whole brains of WT and calpain-1 KO mice were subjected to 10% SDS-PAGE, and proteins were transferred to a PVDF membrane with 100 V for 1 h at 4°C. After blocking for 2 h at room temperature with 3% bovine serum albumin in TBS buffer, membranes were incubated at 4°C overnight with rabbit anti-HSPA1B (1:500; PA5-28369; Thermo Fisher Scientific), anti-DNAJB1 (1:1000; 13174-1-AP; Proteintech), anti-Insulin degrading enzyme/IDE (1:1000; ab32216; abcam), anti-PLA2G4E (1:200; 18088-1-AP; Proteintech), anti-NGFI-B alpha/Nur77/NR4A1 (1:1000; NB100-56745; Novus Biologicals), anti-PER2 (1:300; ab180655; Abcam) antibodies and mouse anti-ARC (1:500; sc-17839; Santa Cruz) antibody. After incubation in primary antibodies, membranes were washed with TBST buffer and incubated for 2 h at room temperature with IRDye 680RD goat anti-rabbit (1:10,000; LI-COR Biosciences) and IRDye 800CW goat anti-mouse (1:10,000; LI-COR Biosciences). Thereafter, membranes were washed 3 times with the TBST and 1 time with TBS buffer. Immunoreactivity was detected with the LI-COR Odyssey system (LI-COR Biosciences).

Antibodies against Necdin (ab18554), SGT1 (ab30931), BMAL1 (ab3350), HSP90 (ab13492), PER1 (ab136451), PER2 (ab180655), CRY1 (ab104736) were purchased from Abcam. Antibody against AVP (sc-390723) were obtained from Santa Cruz. Antibodies against Myc-tag (2276), Flag-tag (14793), HA-tag (3724), VIP (63269) were obtained from Cell Signaling Technology. Antibody against CRY2 were obtained from Invitrogen. Antibody against GRP (20073) were obtained from ImmunoStar. 17-AAG (HY-10211) and Geldanamycin (HY-15230) were purchased from MedChemExpress.