Lc3 nb100 2220

Sertaconazole nitrate (HY‐B0736A), 3‐methyladenine (HY‐19312), Ferrostatin‐1 (HY‐100579), Z‐VAD‐FMK (HY‐16658B), and Necrostatin‐1 (HY‐15760) were obtained from Med Chem Express. MG‐132 (S2619) and CHX (S7418) were purchased from Selleck. DMSO (D2650) and chloroquine (C6628) were purchased from MilliporeSigma. Sertaconazole nitrate, Ferrostatin‐1, Z‐VAD‐FMK, and Necrostatin‐1 were dissolved in DMSO. Chloroquine and 3‐methyladenine were dissolved in PBS.
Antibodies: Caspase 3 (380189) and Cleaved‐caspase 3 (380189) were purchased from ZEN BIO. PARP (9532), Cleaved‐PARP (9532), phosphorylated (p‐)Akt (Ser473) (4060), p‐mTOR (Ser2448) (2971), p‐p70S6K (Ser371) (9208), p‐4EBP1 (Ser65) (9451), Akt (4685), mTOR (2972), p70S6K (9202), 4EBP1 (9452), Beclin 1 (3738), ATG5 (12994S), ATG7 (8558S), Bcl‐2 (15071), and ubiquitin (3936) were purchased from CST. TRADD (sc‐46635), β‐actin (sc‐1616), Ki67 (sc‐23900), HRP‐conjugated antimouse secondary antibody (sc‐2005), and HRP‐conjugated antirabbit secondary antibody (sc‐2004) were purchased from Santa Cruz Biotechnology. LC3 (NB100‐2220) was obtained from Novus. For immunofluorescence, goat antimouse Alexa Fluor 594 (A21044) and goat antirabbit Alexa Fluor 488 (A27034) were obtained from Invitrogen.

Samples were run by SDS-PAGE and transferred to low autofluorescence polyvinylidene difluoride membrane (Immobilon, FL, Millipore). Primary antibodies were used at the concentration described below overnight at 4 °C in blocking solution (1% milk powder in PBS). Secondary antibody (IR-Dye conjugation goat anti-mouse or -rabbit LI-COR Biosciences) was used at 1:50,000 dilution in blocking solution for 1 h. The membrane was scanned on the LI-COR Odyssey (LI-COR Biosciences) using the Image Studio software.
Dilution of primary antibodies. 1:250: Notch1 (ab52627, Abcam), NICD (ab8925, Abcam), Hes1(ab71559, Abcam), Dll1 (ab76655, Abcam), ATG7 (ab52472, Abcam), Numb (ab14140, Abcam)
1:1,000: LC3 (NB 100-2220, Novus), ATG16L1 (pAb PM040, MBL), Beclin (#3738S, Cell Signalling), VAMP3 (gift from A.A. Peden)
1:2,000: Actin (A2066,Sigma-Aldrich).
Notch1 on western blot shows Notch NTMD (125 kDa). It is the form which is cleaved during maturation at the plasma membrane. The NICD (activated Notch1) antibody detects VLLSRKRRRQHGQC, a sequence, which is not accessible in the uncleaved form. It is exposed after S1 cleavage. The protein is detected at 80 kDa.

Anti-microtubule associated protein 1 light chain 3 (LC3) (NB100-2220) and anti-Beclin 1 (ab55878) antibodies were purchased from Novus Biological (LLC, USA). Anti-Bcl-2-associated X protein (Bax) and anti-Bcl-2 antibodies were purchased from Abcam (Cambridge, MA, USA). Antibodies recognizing phospho-mTOR (Ser2448), mTOR, phospho-4E-BP1 (Thr37/46), 4E-BP1, phospho-p85S6K (Thr412), p85S6K, phospho-p70S6K (Thr389), and p70S6K were purchased from Cell Signaling Technology (Beverly, MA, USA). Lipofectamine 2000 (11668-019), LysoTracker Red (L7528) and MitoTracker (M7512) were purchased from Invitrogen. The β-actin (KC-5A08) and GAPDH (KC-5G5) antibodies were from Kangcheng (Shanghai, China). Melatonin and all other reagents were purchased from Sigma Chemical Co. (St. Louis, Missouri) unless otherwise indicated.

Cell lysates were prepared in 2x Laemmli sample buffer (62.5 mM Tris-HCl, pH 6.8, 25% [v/v] glycerol, 2% [w/v] SDS, 5% [v/v] β-mercaptoethanol, and 0.01% [w/v] bromophenol blue) (#161-0737, Bio-Rad, Hercules, CA). After separation in 10–12% SDS-PAGE, the proteins were transferred onto PVDF membrane (#162-0177, Bio-Rad). The membranes were then incubated with the following primary antibodies: OPA1 (#612606, BD, San Jose, CA), Drp1 (#611738, BD), ATG5 (ab54033, Abcam, Cambridge, UK), IFT88 (13967-1-AP, Proteintech, Chicago, IL), OFD1 (22851-1-AP, Proteintech), LC3 (NB100-2220, Novus Biologicals, Littleton, CO or L7543, Sigma-Aldrich), p62 (#5114, Cell Signaling Technology, Danvers, MA), AMPK (#1596, Epitomics, Burlingame, CA), phospho-AMPK (T172) (#2535, Cell Signaling Technology), cleaved caspase-3 (#9661S, Cell Signaling Technology) and actin (MAB1501, Millipore, Temecula, CA). For protein detection, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Pierce, Rockford, IL). Chemiluminescent signals were developed using Clarity Western ECL substrate (W3680-010, Bio-Rad). Densitometry was performed on scanned immunoblots using the AE-9300 Ez-Capture MG Hours Image Saver HR image capture tool (WSE-7120L, ATTO, Tokyo, Japan). Each protein expression level was normalized to that of actin.

Chemical compounds were purchased as follows: LRRK2in1 [24 (link)] from the Division of Signal Transduction Therapy, School of Life Sciences, University of Dundee, U.K.; GSK2578215A and MLi-2 from Tocris [25 (link)]; bafilomycin-A1 (B1793-2UG) and cycloheximide (01810-1G) from Sigma-Aldrich; torin-1 (CAY10997) from Cayman Chemicals. Antibodies used were as follows: LC3 (NB100-2220, Novus Biologicals); total P70S6K (sc-8418, Santa Cruz); phospho Thr389 P70S6K (sc-11759, Santa Cruz); p62 (610833, BD Transduction Labs); total ULK1 antibody (8054 and 4773, Cell Signalling); phospho Ser757 ULK1 antibody (6888 and D7O6UCell Signalling); phospho Ser555 ULK1 antibody (5869, Cell Signalling); total LRRK2 antibody (MJFF2, Abcam); phosphor Ser935 LRRK2 antibody (UDD2, Abcam) and β-actin (A1978, Sigma-Aldrich). AMPK activator (A769662) was kindly provided by Dr MPMS.